summer vacation studentship reports 2015

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Investigating Alzheimer’s disease in Down syndrome, using transgenic mouse models

Aleksandra Lasica Supervised by Frances K. Wiseman, Institute of Neurology, University College London

Introduction: Individuals with Down syndrome (DS), caused by trisomy of chromosome 21 (Hsa21), have a significantly elevated risk of developing Alzheimer’s disease (AD). Virtually all patients display the neuropathological hallmarks of Alzheimer’s disease – amyloid plaques and neurofibrillary tangles by the age of 30, and by the age of 60 up to 70% of people with DS develop dementia. This has been attributed to triplication of the amyloid precursor protein (APP) gene, present on human chromosome 21. However, recently it has been shown that genes on Hsa21, other than APP, contribute to the increased risk of AD in DS patients. The Tc1 mouse model carries an extra copy of Hsa21 but it is not functionally trisomic for the APP gene. Cross between Tc1 model and human mutant APP transgenic mouse (J20) demonstrated that trisomy of chromosome 21 is sufficient to exacerbate APP/Aβ pathology in the mouse brain (Wiseman et al., unpublished data). Currently, the main focus lies on the identification of gene or genes contributing to APP/Aβ pathology.

Aims: My project was aimed at determining which regions on Hsa 21 potentially contribute to the APP/Aβ pathology, using transgenic mouse models. In mouse genome, genes orthologous to Hsa21 genes are split over syntenic regions on chromosomes 16, 17 and 10. My project was focused on two transgenic mouse models: Ts2Yey- trisomic for Hsa21 genes from mouse chromosome 10 and Ts3Yey- trisomic for Hsa21 genes from mouse chromosome 17. Both Ts2Yey and Ts3Yey were crossed with J20 transgenic mouse. By comparing the AD phenotype in J20 mice, with the phenotype observed in Ts2Yey/Ts3Yey*J20 mice, I was able to

assess whether the extra genes on Hsa21, in the absence of APP, contribute to AD pathology. During my internship I was looking at the following phenotypic features: number of amyloid plaques (Ts2Yey) and APP levels (Ts3Yey) in transgenic mouse models.

Methods: Western blotting: Cortices from 3 month-old mice were homogenised in RIPA buffer and total protein content of the samples was determined by Bradford assay. Subsequently, samples were loaded onto NuPAGE® Novex® 4-12% Bis-Tris Protein Gels) and SDS-PAGE gel electrophoresis was performed at 150V for 75 mins in order to separate proteins. Proteins were then transferred to nitrocellulose membrane by electroblotting at 35V for 2h at RT. Membranes were then blocked in 5% milk in PBST (phosphate-buffered saline (PBS) with 0.05% Tween-20 for 1 hour at RT, to minimise non-specific binding. Primary A8717 C-Terminal APP antibody (Sigma Aldrich) (1:10,000) and primary A5441 β-actin antibody (Sigma Aldrich) (1:100,000) were applied to the membrane overnight at 4°C. Subsequently, appropriate horseradish peroxidase-linked secondary antibodies were applied. Blots were

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developed using a film processor (Xograph) and densitometric analysis of bands was performed using ImageJ.

Plaque counting: Tissue was collected from a cohort of mice at 6 months for immunohistochemical assessment of 4G8- positive Aβ plaque load. Plaque counts were recorded manually from both the cortex and the hippocampus. The data were analysed by repeat measures ANOVA using SPSS software.

Results:

Ts2Yey x J20 cross: Aβ plaque deposition I was able to determine that in accordance with previous data the presence of the APP transgene (J20) caused a significant increase in the number of plaques in the cortex (p=0.003) and hippocampus (p=0.002). Ts2Yey trisomy did not significantly influence the plaque load nor was an interaction between Ts2Yey trisomy and tgAPP observed. However, when WT and Ts2Yey groups used as controls were excluded from the analysis (based on the absence of plaque deposition), a slight trend (p=0.167) towards the decrease in the number of plaques in the hippocampus caused by Ts2Yey trisomy was recorded. This possibly protective role of Ts2Yey trisomy is further supported by the data suggesting that Ts2Yey trisomy rescues a tgAPP-induced sudden death phenotype and causes an improvement in short- term spatial memory (Pulford et al., unpublished data).

Ts3Yey x J20 cross: APP levels

Statistical analysis of my Western blot results indicated that as expected the presence of tgAPP caused a significant upregulation of APP protein levels (p=0.00016). However, no significant effect (p=0.521) of Ts3Yey trisomy on the APP levels was observed, which suggests that trisomy of Hsa21 orthologous genes on mouse chromosome 17, do not increase APP protein levels in the J20-tgAPP model system.

Figure 2: Aβ plaque deposition in the Ts2Yey *J20 cross. Hippocampus: tgAPP( F(1,27)=11.936, p=0.002), Ts2Yey (F(1,21)=2.048, p=0.167) (with control groups excluded) Cortex: tgAPP(F(1,27)=10.731,p=0.003) Label tgAPP equivalent to J20 genotype.

Figure 3: Western blot showing the levels of full-length APP (FL-APP) among all investigated genotypes. Signal intensity of the FL-APP band was calculated relative to β-actin used as a loading control.

Figure 4: Levels of FL-APP relative to β-actin. tgAPP(F(1,20)=21.538, p=0.00016) Ts3Yey(F(1,20)=0.427,p=0.521)

Figure 1: Aβ deposition in hippocampus.

tgAPP tgAPP*Ts2Yey

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Future directions: Ts2Yey*J20 cross: In order to assess possibly protective effect of Ts2Yey trisomy on the Aβ pathology, the plaque deposition could be investigated at different age time points, accompanied by the increase in the sample size. Ts3Yey*J20 cross: In order to investigate the mechanism of APP pathology, the levels of C- terminal APP fragments could be quantified using Western blotting. This would indicate the amount of APP processing that has occurred. The attempt to quantify CTFs was undertaken during my internship, however due to time constraints and problems with the primary antibody this task was not completed.

The value of the studentship to the student This studentship was a truly enjoyable experience that provided me with a greater insight into the world of few essential laboratory techniques as well as boosting my confidence in practical skills. However, my experience was not only limited to data collection. I have learned the basics of statistical analysis using SPSS software, attended group research meetings and presented my data during one of the lab meetings.

The value of the studentship to the laboratory

Aleks has made a fantastic contribution to our groups work this summer, it was delightful to have her enthusiasm in the lab and she worked very hard. She worked particularly closely with a second year PhD student producing data of publishable quality that we plan to submit for publication next year. Aleks thus will be a co-author on this paper, and we would welcome her to work with us again at any time.

Acknowledgements

I would like to thank Professor Elizabeth Fisher for accepting me into the lab and Doctor Frances Wiseman for providing me with the rounded experience of research career. I would also like to thank Karen Cleverly and Laura Pulford for teaching me the techniques that enabled me to complete my project as well as Matthew Rickman and Justin Tosh for providing their technical expertise.

Biochemical Society Studentship Report 2015

Structure-function relationship of the palmitoyl transferase DHHC5 Student: Aleksandra Tsolova; Supervisor: Dr Will Fuller, University of Dundee Background Massive endocytosis (MEND) is the major cause of injury during reperfusion of anoxic cardiac muscle. MEND occurs following dynamic surface membrane protein palmitoylation (acylation) by palmitoyl transferase DHHC5, widely recognized as one of very few cell-surface localized palmitoyl transferases. Calcium overload leading to mitochondrial stress results in transient opening of the mitochondrial permeability transition pore (MPTP) and release of coenzyme-A into the cytoplasm, where it is acylated to form a substrate for DHHC5. The enzyme then palmitoylates (reversibly attaches a 16-carbon fatty acid to a cysteine thiol via a thioester bond) surface membrane proteins, causing them to cluster together in lipid ordered domains, which leads to MEND, and thus, detrimental physiological effect on the cell. The mechanisms underlying MEND are as-yet unidentified. Importantly, MEND occurs during reperfusion of anoxic cardiac muscle, and is inhibited by interventions classically reported to reduce MPTP opening and protect against reperfusion injury. DHHC5 knockout hearts, in which MEND is essentially absent, show significantly enhanced functional recovery following anoxia-reperfusion, strongly implicating DHHC5 and the MEND pathway in reperfusion injury. Interestingly, MEND is accelerated in the presence of the Na pump regulatory subunit phospholemman (PLM), which inhibits the Na pump when palmitoylated

1, and activates the pump when phosphorylated. PLM is a substrate for DHHC5,

which is abundantly expressed in caveolar microdomains in cardiac muscle. Truncation analysis indicates interaction of PLM with and palmitoylation of PLM by DHHC5 requires a region of the DHHC5 carboxyl terminus close to the transmembrane domain (residues 218-334).

Objectives Investigate the structure-function relationship of DHHC5: 1. Narrow down the region of DHHC5 that interacts with its substrate PLM by performing additional truncations in the

region 218-334 of DHHC5. 2. Investigate the role of post-translational modifications in regulation of DHHC5 activity.

Description of the project Key DHHC5 mutants were generated before the start of the project (C236A, C237A, C236/7A, N218X, C245X, S274X, E304X)

1. Cell surface localization DHHC5 mutants All DHHC5 point mutants were expressed in HEK cells, and cell surface proteins purified by streptavidin affinity

chromatography after application of membrane-impermeable biotinylation reagents.

2. Interaction of DHHC5 & PLM. A long-term aim of the current research projects in the Fuller lab is to validate DHHC5 as a drug target for treatment of ischemia-reperfusion injury in the heart. Identifying the precise region of DHHC5 that recognises individual substrates may facilitate specific targeting of enzyme-substrate interactions. The HA tagged DHHC5 mutants were characterized alongside wild type by investigating their association with PLM in transiently transfected HEK cells that stably express PLM (described in

3). DHHC5 was immunoprecipitated via the HA tag, and co-purifying PLM detected by western blotting.

2. Post-translational modifications of DHHC5. The large carboxyl tail of DHHC5 includes multiple sites of both palmitoylation and phosphorylation, suggesting these post-translational modifications may regulate DHHC5 activity. Therefore, the palmitoyl transferase activity of DHHC5 palmitoylation and phosphorylation point mutants was evaluated. DHHC5 is phosphorylated in mouse hearts at multiple serine residues including S380, S432, and S636. Serine to alanine point mutants at these sites were generated for characterization using the Agilent QuikChange site directed mutagenesis kit. DHHC5 activity was assessed by expressing wild type and all mutant DHHC5 in a PLM-expressing DHHC5 knockout cell line recently generated in Fuller laboratory. Palmitoylated proteins were purified by resin-assisted capture, and palmitoylation of PLM assessed by western blotting.

Figure 2: DHHC5 structure showing DHHC domain, region of substrate binding, and phosphorylation sites marked in red (Source: Dr Will Fuller, University of Dundee, 2015)

Figure 1: Mitochondrial release of CoA via MPTP during reperfusion triggers massive internalisation of regions of cell surface membrane, which leads to detrimental physiological effect on the cell

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Figure 4: Co-immunoprecipitation of phospholemman and HA-tagged DHHC5 mutants with anti-HA sepharose beads. Constructs C245X, S274X and E304X exhibit signal bands, suggesting for association and interaction of phospholemman at these specific mutants.

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Figure 5: Relative palmitoylation of phospholemman in DHHC5 KO cells compared to wild-type (WT) normalized to 100%. The increasing/decreasing expression is equivalent to the instance of palmitoylation taking place at the specific expression site of the mutant. Control sample was not transfected and thus did not contain exogenous nucleic acids.

Assessment of results and outcomes of the studentship Various DHHC5 truncations were expressed in HEK cells stably transfected with PLM. All DHHC5 mutants were successfully trafficked to the cell surface (Fig. 3). Hence no truncations interfered with the normal processing of DHHC5 in the secretory pathway. Despite small variations among results, after multiple repetitions of the experiments there was a tendency of co-immunoprecipitation of the substrate PLM with DHHC5 mutants C245X, S274X, and E304X. This suggests substrate-enzyme interaction at these sites (Fig. 4). All serine to alanine point mutants S380, S432, and S636 were generated successfully and expressed in DHHC5 KO cells. After purification of palmitoylated proteins by resin-assisted capture, it was found that palmitoylation of PLM varied between constructs. The data revealed a close similarity in the level of palmitoylated protein among mutants (Fig. 5). This was regarded as insufficient to provide a conclusion for the experiment. As a solution, a repeat should be performed of the experiment to reveal a broader range of palmitoylated protein data. Due to time constrain a repetition set could not have been made during current summer placement.

Future direction The project can be taken for further characterization of the substrate-binding region. As DHHC5 substrate, PLM, is highly polar, a modification in amino acids in binding region could be made. Positively charged amino acids could be changed for negatively charged ones and substrate-enzyme interaction assessed. Following the

same principle, negatively charged amino acids could be altered into positively charged and activity of the enzyme to bind PLM assessed once again. Furthermore, as there are 6 phosphorylation sites identified, serine residues of these sites can be amended into glutamate/aspartate residues which would initiate pseudo-phosphorylation. Departures from original proposal No major departures from the original proposal. Contribution of grant to personal career aspirations and personal value of studentship Throughout my summer placement in Fuller lab I not only gained and mastered major techniques while working in the lab as well as I improved my research, writing and presenting skills, but I also understood the importance of organizing my time to be efficient and scheduling my work to fit time constraints and still be productive. I learnt that sets of trial and error underlie a successful experiment and in order to achieve good results I had to be patient and able to troubleshoot. Now, I am much more confident in working in lab environment, analyzing results and communicating in a scientific language, and I am eager to apply my knowledge again in the future. This invaluable experience will significantly aid me while I progress further in my qualifications and build my career as a scientific research leader. Value of the Studentship to the lab Aleksandra did an excellent job and produced some high quality data while working in the Fuller lab. Her work characterising DHHC5 truncations clearly showed that all the mutants she characterised trafficked correctly through the secretory pathway. Unfortunately, our DHHC5 KO cell line grows too slowly for anyone to do any meaningful work with it, but the experiments Aleksandra was able to perform showed good consistency with the co-immunoprecipitation data, which has undoubtedly helped us to map the site of interaction of PLM & DHHC5. The phosphorylation site mutants that Aleksandra produced are currently being characterised and will be a very valuable resource for this project as it moves forward. Related work and recommended literature

1. Howie J, Tulloch LB, Shattock MJ, et al. Biochem Soc Trans. 2013;41:95-100 2. Hilgemann DW, Fine M, Linder ME, et al. eLife. 2013;2:e01293 3. Fuller W, Reilly L, Fraser NJ, et al. Proc Natl Acad Sci U S A. 2014;111:17534-9

Figure 3: Representative purification of cell surface proteins by streptavidin affinity chromatography. Cell surface proteins were purified and immunoblotted as indicated: (A) DHHC5 mutants expressed in cells; (B) Cell surface fractions indicate all DHHC5 mutants are localized on the cell surface; (C) Localized cell surface Na-pump confirming success of experiment

Biochemical Society Summer Studentship Report 2015

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TIE proteins: Bacterial ester domains Angus Lovely Supervisors: Ona Kealoha Miller and Dr Uli Schwarz-Linek

Biomedical Sciences Research Complex, University of St Andrews, North Haugh, Fife, UK

1. BACKGROUND

In order to colonise their hosts, many bacteria, both commensal and pathogenic, use surface proteins, such as microbial surface components recognizing adhesive matric molecules (MSCRAMMs). Gram-positive bacteria show a diverse range of adhesin proteins. Many of these are now known to contain intramolecular cross-links between amino acid residues, which are self-generated upon folding. There are three main types of domains containing such cross-links, isopeptide, thioester and ester domains (TIE domains)1. These are found in clinically relevant bacteria such as Staphylococcus aureus and Streptococcus pneumoniae. Isopeptide domains are known to be highly stable due to their intramolecular amide bond, allowing the bacterium to have extreme resistance against mechanical stress. Thioester domains mediate the covalent binding of bacteria to a host nucleophile via a reaction through the thioester bond, resulting in a stable intermolecular bond2. The existence of the ester domains in Gram-positive surface proteins is a recent discovery, and it is not clear what the role of the domains is. Current evidence shows that they may be linked to stability3, similar to the isopeptide domain, although it is possible that the bond’s susceptibility to nucleophilic attack, similar to thioesters, makes the domain suitable to play a role in covalent interactions.

2. KEYWORDS

MSCRAMMs � TIE proteins � thioester � atomic force microscopy � ester domains � cross-links

3. GLOSSARY ;

SaED: this is the first ester domain from the Staphylococcus aureus SaTIE protein.

PnED: this is the first ester domain from the Streptococcus pneumoniae PnTIE protein.

Immobilised Metal Ion Affinity Chromatography (IMAC): this is a process of protein purification; the protein of interest has a HisTag, a histidine rich repeat that has a high affinity for a nickel ion column, which separates it from non-HisTagged molecules.

HisTag: A peptide containing six consecutive histidine residues. The His sidechain has a high affinity for nickel (and cobalt) ion resins with high affinity allowing for quick and efficient separation.

TEV protease: this is a protease isolated from the tobacco etch virus (TEV) that cleaves at a specific sequence. It is used to remove HisTags from proteins of interest in reverse IMAC. The TEV protease used is itself HisTagged, so can be removed from the protein mix along with the cleaved HisTags.

Atomic Force Microscopy (AFM): a technique used to measure the strength of protein folds – proteins are attached to a cantilever via a silicon crystal and pulled apart whilst the force is measured using a precise laser.

Site Directed Mutagenesis: a technique that introduces desired point mutations into amino acid sequences, by selectively altering codons.

Figure 1: Examples of TIE proteins from Clostridium difficile, Clostridium perfringens and Streptococcus pneumoniae.

Biochemical Society Summer Studentship Report 2015 Angus Lovely

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4. AIMS

The aim of the studentship was to target conserved residues of both a S. aureus and S. pneumoniae ED protein (SaED and PnED) by site-directed mutagenesis (SDM) in order to better the understanding of the mechanism of ester domain formation. Reconstitution experiments were also carried out using variants of the PnTIE protein: PnoED (open ester domain) and PnsED (split ester domain) using a PnET (ester tag) to observe if the ester bond forms in trans in solution. The PnET is a short C-terminal peptide from the PnED fused to a B. anthracis TIE protein thioester domain, BaTED. This experiment is being trialled as a similar reaction has been observed for a split isopeptide domain from Streptococcus pyogenes4. This reaction is so reliable that it is being used as a protein engineering technique. Finally, atomic force microscopy (AFM) constructs will be designed. This will place the gene of interest into a specially designed vector (pET3) that will express multiple copies of the protein of interest in between IgG domains. This will allow the strength of the protein fold to be measured.

5. DEPARTURES FROM THE ORIGINAL PROPOSAL

There were no departures from the original proposal.

6. MATERIALS AND METHODS

Gel Electrophoresis: All SDS-PAGE gels were precast, manufactured by BioRad® and were made up of 12% polyacrylamide. The gels were run in SDS-Glycine running buffer. Coomassie brilliant blue stain was used to visualize SDS-PAGE gels. The agarose gels were cast in-house, and were all a 1% agarose solution in TBE. The gels were visualized with SYBR Safe stain.

Side Directed Mutagenesis (SDM): This used to change single amino acids, by mutating codons in DNA. Primers are designed that are complementary to the template DNA except for the codon that is to be altered. After the initial polymerization stage, parent strand DNA will contain the mutant codon, so will be amplified. A Dpn1 digest is performed afterwards to remove any non-mutant DNA. Primers should be GC-rich at the ends to maximize primer annealing.

Miniprep, Gel Extraction and PCR Clean-up: These three DNA purification techniques were all performed using prepared kits. Minipreps and gel extraction were prepared using Sigma® kits and PCR clean-up using an Olympia® kit.

Restriction Enzymes: Four different restriction enzymes are used in this project; SpeI, BssHII, XhoI and NcoI. These are manufactured by ThermoScientific® and are used in Buffer Tango©. The alkaline phosphatase used was also ThermoScientific®.

Ligations: The DNA insert is in incubated with the plasmid vector in three different ratios, 1:1, 3:1 and 5:1. The DNA is incubated in d.H2O for annealing. A PCR machine is used to reduce the temperature from 65oC to 4oC at a rate of 1oC.min-1. The annealed DNA is then incubated overnight with T4 DNA ligase in T4 buffer.

Transformations: All transformations are carried out using pre-prepared agar plates. Ampicillin and kanamycin are the only antibiotics used, both at a concentration of 1mM. 1µL of DNA is added to 50µL of competent cells. The mixture is stored on ice for 30 min, heat shocked at 42oC for 1min15s, returned to ice for 5 min, then ~250µL of medium is added. LB is the standard medium, and SOC medium is used for ligations. For ampicillin plates the liquid medium is plated right away, and for kanamycin plates the medium is incubated first for an hour, so the cells can grow and express antibiotic resistance (as kanamycin is a bacteriocide). BL21 cells were used for expression, DH5α cells are used for DNA engineering (have no protein synthesis apparatus) and SURE2 cells are used for DNA engineering where there are lots of repeats (are missing certain replication proofreading apparatus). These are all different cell lines of E. coli.

Test Expressions: Test expressions are carried out to determine the conditions in which a protein is soluble. Cell line, medium, temperature, induction and buffer can all be altered. The cell line used in all cases for this project is BL21, and the medium is LB broth. The cells are grown up to an optimum density (OD600)=0.6, then are split into aliquots. The aliquots are induced and left at the set temperature. ITPG is used to induce the cells, by activating the lacI operon. After the set temperature, the cells are lysed by sonication, spun down, and the


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soluble fraction is analysed by SDS-PAGE to determine if protein is present, by comparing against the uninduced control. Test expressions are used to choose conditions for large-scale expression. The buffer used for test expressions should have a pH more than one pH unit away from the isoelectric point of the protein, so it remains soluble.

PCR/Colony PCR: Two different polymerase enzymes are used for PCR. Taq polymerase is isolated from Thermus aquaticus and is thermostable up to 95oC, with an optimum temperature of 75oC. Taq has no proofreading ability, but has an extension rate of 1 min.kb-1. Pfu Polymerase is from Pyrococcus furiosus. It has proofreading capability and is thermostable up to 100OC, and has an optimum temperature of 72oC. It has an extension rate of 2 min.kb-1. Taq can be used for diagnostic PCR, but Pfu should be used for DNA engineering. DNA is denatured at 95oC usually for 30s, annealing of the RNA primers occurs at 5oC below the primer melting point, also usually for 30 seconds. Extension occurs at 72oC for a time depending upon polymerase and amplicon length. DMSO can be added to the PCR mix to prevent DNA secondary structure formation. Colony PCR is used to amplify DNA directly from a plate. The PCR mix is made, a colony is transferred from one plate to another, labelled, and then vigorously shaken with the PCR mix to transfer some of the DNA.

Plasmid Vectors: Two different vectors are used in the project. The SaED and PnED proteins are both expressed in the pHisTEV vector. This vector includes a KanR gene, 6x His-tag, TEV cleavage site, lacI operon and a T7 promoter region. XhoI and NcoI are used with this vector. The AFM constructs are in the pET3 vector. This too has a T7 promoter region and a lacI operon, but instead has an AmpR gene. SpeI and BssHII are used with this vector. Protein Expression: Proteins are expressed in LB in volumes of 500mL in 2L flasks. Antibiotic is added at a concentration of 1mM, and the broth is then inoculated with 2mL cells. The flasks are then incubated overnight at a temperature determined by the test expression. The culture is then spun down, and the pellet resuspended in a lysis buffer. The solution is then passed through the cell disrupter, where the cells are lysed at 20 kpsi. The debris is then filtered from the solution and the protein is purified.

Protein Purification: All of the expressed proteins used in this project have a His-tag and a TEV cleavage site. The filtered protein mixture is passed through a Ni2+ column, which the His-tag binds to. A weak solution of imidazole, which also has an affinity for Ni2+, is passed through to prevent non-specific binding. Then, a strong solution of imidazole is used to knock the protein of interest off the column. SDS-PAGE is used to identify the eluted fractions that contain protein, which are pooled together. The imidazole is removed by dialysis, and TEV protease is then used to remove the His-tag. The protein solution is passed back through the column, and the cleaved His-tag, the His-tagged TEV protease and any leftover imidazole bind to the column and the pure protein is eluted.

7. RESULTS OF RESEARCH AND OUTCOME OF STUDENTSHIP

7.1 Staphylococcus aureus Ester Domain (SaED)

The wild type SaED DNA was transformed on kanamycin-agar, two of the colonies were cultured overnight, and DNA extracted and purified using commercial kits. This provided the entire DNA stock to be used for all mutagenesis reactions of SaED. Primers were designed to introduce two mutations into the SaED protein. Both mutations affect ester bond forming residues; Thr-7 and Gln-125. A question was if a T7C mutant would now form a thioester bond, and that the Q125E mutant would still form an ester bond.

Following the PCR to carry out the side directed mutagenesis, the PCR reaction product was cleaned up, and the DNA was sequenced to confirm the mutation had been correctly introduced.

The sequence was correct, so the two mutants were test-expressed to assess if soluble protein can be obtained. The isoelectric point (pI) of the T7C mutant (with HisTag) is 6.43, and 6.22 for the Q125E mutant (with HisTag). Therefore, PBS (pH 7.5) can be used for the test expression. After the cells’ optimum density has been achieved and they have been incubated overnight following induction, lysis was induced by sonication. Cells were sonicated for three 15-second intervals. Cells were then spun down to remove debris, and both the soluble and insoluble fractions were checked for protein content.


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The test expression showed that the protein was highly soluble, and large-scale expression was carried out. 25oC was chosen as the temperature for the overnight expression stage. Following expression, the cultures were spun down to isolate the cells. These were resuspended and lysed using a cell disruptor. The remaining solution was spun down again, this time to remove cell debris and isolate the proteins in solution. The protein was purified through IMAC, dialysed to remove the imidazole from the solution, and cleaved by TEV protease and re-purified using IMAC to remove the HisTag. Gel filtration was then used to further purify the protein eluted from this second, reverse IMAC. Alongside this protein expression, another sample of both the mutants was prepared differently. They were grown in minimal media (glucose, yeast nitrogen base and M9 salts) to incorporate 15N into the cells through 15NH4Cl as the sole source of nitrogen. This isotope labelling facilitates heteronuclear NMR spectroscopy.

The protein that was eluted was then concentrated to be analysed by various techniques. A 500µM solution was used for NMR. A well-dispersed protein fingerprint (or 1H,15N-HSQC) spectrum with the a number of resonances reflecting the protein sequence would be expected for a protein with a well-defined fold, such as one containing an ester bond crosslink. As shown in figure 2, the spectra obtained for both mutants contained many broad signals, which is an indicator that the proteins were not fully folded, indicating that ester (or thioester) bonds are not present in these proteins.

Figure 2: HSQC spectra for SaED WT, T7C and Q125E mutants. Clearly, the spectrum for the WT is better defined than for the mutants, indicative of more structural rigidity in the protein.

Another piece of evidence to suggest that the mutants did not contain cross-links comes from the mass spectrometry data. Figure 3 shows that the molecular weight observed for the two proteins is 17025.10 Da (T7C) and 17024.10 (Q125E). This is the expected molecular weight for the protein lacking the N-terminal methionine. If the thioester/ester bond was present then the molecular weight would be 17 or 18.0 Da lower, accounting for the loss of an ammonia or water molecule, respectively.


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However, a secondary species is clearly visible in both spectra; these have a molecular weight of 17006.6 Da (T7C) and 17007.7 Da (Q125E) less than the parent species. These are the correct molecular weights for the thioester/ester bond containing protein. This indicates that although the thioester/ester bond does not form as the dominant protein species, it may be capable of forming to some extent.

Crystal trials were set up for both proteins at a concentration of 30 mg/mL. This concentration was chosen as it is the same concentration as was used to solve the crystal structure of the wild type protein. After a wide crystal screen was narrowed down, the ideal conditions for crystallisation of the Q125E mutant were found to be 0.1M sodium acetate pH 5.0, 0.2M ammonium sulphate and 25% (w/v) PEG 4000. Crystal streak seeding with a cat whisker was essential for the optimisation of crystal growth. Figure 4 shows the crystal structure of this protein. An ester bond is not present, and does not form upon crystallisation of the protein. This is interesting, as the H117A mutant of the SaED protein does form the ester bond in crystal, although there is no ester bond present in solution. This difference may be due to the fact that the His-117 residue is catalytic but non-essential, and not bond forming like the Gln-125 residue. It appears that the chemical environment of the ester bond is finely tuned to only allow ester formation form an amide (Gln) but not an acid (Glu). This is in marked contrast to isopeptide domains.5

The fact that the ester domain lacking the ester bond is very similar in structure to the wildtype indicates this bond is not a determinant of structure. However, the NMR spectrum for the Q125E mutant shows the opposite in solution, as the protein fold is far less defined in

Figure 4: X-ray diffraction pattern and solved crystal structure for SaED Q125E mutant. Spacegroup is P212121 and the resolution is 2.2 Å.

Figure 3: Mass spec of SaED T7C and Q125E mutants. Molecular mass of most abundant species shown to be 17024.1 Da and 17025.1 Da respectively. There is a secondary species lacking 18 Da in both spectra.


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the non-bond containing mutant protein versus the wildtype.

7.2 Streptococcus pneumoniae Split Ester Domain (PnsED)

The second protein used for this project is PnED. The first part of the PnED project is the generation of a split ester domain (PnsED). This is the PnED protein with the 12 C-terminal amino acid residues, containing the ester-bond forming Gln, missing. The other protein used is the ester tag (PnET). This is the final 11 C-terminal amino acid residues attached to the BaTED protein (simply a choice of convenience, any other fusion protein would have been suitable).

The first step in this project is to amplify the specific oED components (oED and tag), and to include restriction enzyme sites, so that it can be inserted into an empty pHT vector and expressed. The wild type PnED DNA was transformed, cultured overnight, isolated and purified. The DNA was then treated with XhoI and NcoI. An empty pHT vector was also treated with XhoI and NcoI, as well as alkaline phosphatase, to create stick ends to allow the insert to anneal. The addition of the BaTED tag onto PnET required a 60nt long specialized primer, in order to join the two peptides. Two separate ligations were then carried out to create the PnsED protein and the PnET protein.

Once these new genes had been inserted into pHisTEV, the two separate proteins were test-expressed for solubility. Suitable conditions were found for large-scale expression, and the protein was purified. The two separate proteins were then concentrated to be of roughly equal concentration for reconstitution experiments.

Reconstitution experiments were carried out to attempt to bring together PnsED and PnET in solution. This was done at protein concentrations of 7 mg/mL and 5 mg/mL respectively, and each experiment contained 21 µg of protein in PBS. Different conditions were trailed for the reconstitution experiments, and a time course was carried out. Aliquots of the mixture were removed after 0 minutes, 15 minutes, 60 minutes and the remainder was left overnight. The different experiments were separated by SDS-PAGE (figure 5), alongside the individual parent proteins. The majority of the parent protein is seen as an intense band lower down on the gel. The fainter bands in the parent lanes (to the left) represent dimerization of the BaTED tag. There is some dimerization observed in the experimental lanes (to the right), but also a small amount of what might correspond to combined split ester domain and ester tag (highlighted in Fig. 5). However, there is also a large amount of non-reconstituted protein in solution. The conditions in which the reconstitution occurred were pH 8.0 and 9.5 incubated at 37oC for 1 hour in PBS.

The previous experiments showed that a small amount of protein was forming a cross-link in solution, although this was only a small amount. It is possible that this is due to the missing Asp-144 residue that is not present in either the PnsED or the PnET. The adjacent aspartate is known to have catalytic properties, so it is entirely possible that the lack of success is due to the missing residue. A primer was designed so as to insert an additional codon into the PnsED gene to add the additional aspartate. This DNA has been sequenced, and the correct result has been confirmed. It is currently in the test expression stage of large-scale protein expression.

7.3 Streptococcus pneumoniae Open Ester Domain (PnoED)

The open ester domain (oED) is derived from the wild type PnED protein, with a single SDM reaction carried out. It is Gln-146 close to the N-terminus that forms the ester bond in this protein. The glutamine has been removed by SDM, and replaced by a small, non-conservative alanine residue. The Q146A mutant does not form the ester bond (MS evidence). It is thought that by mixing the PnoED with the PnET used with the PnsED will result in the tag inserting itself into position to form the ester bond and disrupting the fold of the PnoED. This is because without the ester bond, the fold of the WT PnED will be weakened.

The PnoED went through a process of mutagenesis, sequencing and test expression very similar to the SaED mutants and the PnsED. Once the correct sequence had been confirmed, and soluble protein conditions had been found, the protein was expressed, purified and

Figure 5: Positive results of PnsED/PnET reconstitution


7

concentrated.

The ester bond is typically slow to form, but is very stable. Therefore, it was decided to trial high temperatures for ester bond formation with the oED. Two different temperatures were used, 50oC and 37oC at pH 7.5 and 9.5. On the gels shown in Fig. 6 there are experimental and control lanes. The controls contain the PnoED and BaTED without the ester tag. This is to ensure that any interaction between PnET and PnoED is due to the ester tag and not any region of the BaTED protein. Aliquots of the experiment were removed and examined by SDS-PAGE after 1 hour and 24 hours.

As can be seen in figure 6, the parent proteins are capable of dimerising similarly to the PnsED. In the experimental lanes (5-8) there are some bands seen that are not present in either the parent lanes (1-3) or in the control lanes (9-12). However, I believe that these bands are of too great a molecular weight to represent an ester bond-containing complex – they are more likely to represent multimers of the BaTED protein.

7.4 AFM Constructs

The starting point for the AFM project was a plasmid inserted in the pET3 vector (named AFM SRU); four repeating IgG domains are encoded with a single PnED domain in the vector after the first IgG domain. IgG is included in AFM constructs as it is a well-studied protein that has a well-defined and well-measured protein fold. It is used as a control when carrying out AFM measurements. To accurately measure the strength of a protein there must be at least two repeats of the domain in the construct.

The first step of this project was to isolate a new PnED domain to insert into the pET3 backbone (figure 7). An overnight expression culture was made from a glycerol stock, and the DNA was then isolated and purified. The DNA was then digested with SpeI and BssHII in a sequential digest. The SpeI digest was carried out first, followed by a BssHII digest. The buffer concentration was doubled between digests to maximize enzyme. The vector was also digested with the same enzymes. This removed the third IgG domain where the PnED domain is to be inserted, and also created sticky ends, so the PnED domain can anneal. The vector was also digested with alkaline phosphatase, to remove the phosphate groups, so phosphodiester bonds can form. After the annealing time, the new DNA was ligated into place.

Following the digestions and ligations, the DNA was prepared for sequencing to confirm that the ligation was successful. Following this transformation, the DNA was transformed in SURE2 cells on an ampicillin plates, and then cultured overnight in order to make glycerol stocks.

The new AFM construct (named AFM SRU-2) was then test-expressed in Tris (pH 8.5) and HEPES (pH 7.0). Large-scale expression was then carried out. Mass spec data is awaited to confirm that the construct has expressed as expected.

Figure 6: SDS-PAGE analysis of PnoED + PnET after 1 hour and 24 hours.

Figure 7: Initial AFM construct containing four IgG domains and one PnED domain, showing restriction sites.


8

8. FUTURE DIRECTIONS

8.1 SaED

Following the partial success of the attempts to observe a thioester bond in the SaED T7C mutant, further experiments will be carried out to try and find conditions that favour thioester bond formation. This will include adding DTT or BME, as the presence of a reducing agent will prevent disulphide bridge formation, therefore, the cysteine residue will not be already bonded if ester domain formation turns out to be slow. It will also involve using a higher pH to favour base-catalysed [thio]ester bond formation.

The SaED T7C mutant will undergo crystallization screening in an attempt to grow a crystal that is suitable for X-ray diffraction analysis. Therefore, this protein structure can be solved by X-ray crystallography, as with the Q125E mutant. It will be interesting to see if an ester bond forms upon crystallization as with the H117A mutant, which will be different to the Q125E mutant.

8.2 PnsED

Conditions have been discovered in which the PnET and PnsED proteins form an ester bond in solution (pH 8.0, 9.5 at 37oC in PBS). With the second split ester domain (PnsED-2) synthesised containing the additional Asp-144 residue, it is thought that further reconstitution experiments will show more promising results.

8.3 AFM Constructs

If the mass spec data for the AFM SRU-2 construct is correct then it can be used for collecting atomic force microscopy data. The next stage will be to insert mutant PnED protein into the AFM backbone. This will be done one domain at a time. First, the wild type PnED domain will be replaced, and then the third IgG domain will be replaced, using digests and ligations. Once the PnED T21A mutant is repeated in the AFM backbone, then this protein can also be expressed. Then the data collected by AFM, the strength of the ester domain in the protein can be calculated.

9. HOW GRANT HAS CONTRIBUTED TO MY CAREER ASPIRATIONS

The grant from the Biochemical Society has made me consider more seriously a career in a lab setting, as I have enjoyed working significantly longer hours in the lab than I have previously. It has also made me wish to pursue a PhD in a related field to my summer studentship. Furthermore, this studentship has made me wish to explore the molecular biology side of my biochemistry degree more, as this is a subject area that I have found most interesting. 10. VALUE OF STUDENTSHIP TO MYSELF

This studentship has taught me a wide range of technical skills well beyond the scope of a normal biochemistry degree. It has also given me a lot more confidence for the longer lab sessions that I will be starting in the honours years of my degree. This has also given me an excellent starting point for my honours project, and has also been influential in my applying for an industrial placement next year as part of the masters degree.

11. VALUE OF STUDENDSHIP TO THE LAB

This summer project made an important contribution to an emerging project in the lab. Of all cross-link containing protein domains that have been discovered over the past decade, ester domains remain the least understood, and require investigation because they may harbour important functions. The data obtained thanks to the support by the Biochemical Society, while not conclusive in all cases, will support a grant application (BBSRC) and inform more experiments. The construct made for AFM experiments will initialise a new collaboration (with David Brockwell, Astbury Centre Leeds), and will open an entirely new angle in our research. In addition to kick-starting a new project in the group and the scientific value, the summer studentship also benefitted the supervisor in the lab, a 1st year PhD student, who found the supervision experience very valuable, and rewarding.


9

12. REFERENCES

1. Schwarz-Linek U, Banfield MJ (2014) Yet more intramolecular cross-links in Gram-positive surface proteins. Proc Natl Acad Sci 111:1229-1230

2. Walden M et al. (2015) eLife 4:06638 3. Kwon H et al. (2014) Proc Natl Acad Sci U S A. 111:1367-1372 4. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M (2012)

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci 109:690-697

5. Hagan R et al. (2010) Angew Chem Int Ed Engl 49:8421-8425

13. NOTE

The X-ray diffraction pattern, and the solved crystal structure in figure 4 are unpublished data. Please do not share without permission.

The Characterisation of Calcium Response and Neuron-Specific Markers on Different Cell Lines for Parkinson’s Disease Modelling Student: Azilleo Kristo Mozihim, University College London

Supervisor: Dr. Michael Duchen, University College London

Background

Parkinson’s Disease (PD) is a debilitating neurodegenerative disease marked by the loss of dopaminergic neurons (DA) in the substantia nigra (SN) of the midbrain. Molecularly, it is marked by the pathological presence of abnormal intracellular aggregates of misfolded protein, notably α-synuclein, called Lewy bodies (LB). Clinically, it is diagnosed based on the presence of four characteristic motor symptoms: rigidity, bradykinesia, resting tremors and postural instability. PD has an estimated prevalence rate of 0.3% in the industrialised nations and about 127,000 sufferers in the UK [1]. Thus, considering the debilitating symptoms and significant number of sufferers, substantial investment has been made into PD research to develop effective treatment for it. However, before treatments can be devised, it is imperative to gain in-depth understanding of PD and one of the ways is firstly to develop an accurate disease models of PD, one of which could be a cellular disease model. This type of model allows investigators to understand a particular aspect of PD and different cellular models using different cell lines are used to investigate different aspects of PD. For my project, I investigated the suitability of three cell lines – primary neurons, SH-SY5Y and neural progenitor cells (NPC) – to be used as PD cell models for studying cellular calcium response in PD. Additionally, I also investigated the presence of neurone-specific markers –MAP2 and NeuN- to determine the extent to which SH-SY5Y and NPC are similar to primary neurons. Methods Calcium Response Assay

• A coverslip with the cell samples adhered on to it were incubated with 300 μL of calcium dye (5 μM of either Fluo-4 or Fura-2) at 37oC for 30 minutes.

• The calcium dye was removed and replaced with 500 μL of recording buffer (150mM NaCl, 4.25mM KCl, 4mM NaHCO3, 1.25mM NaH2PO4, 1.2mM MgCl2, 1.2mM CaCl2, 10mM D-glucose, 10mM HEPES, pH 7.4)

• The cell samples then were subjected to fluorescence microscopy with the settings depending on the dye used. For Fluor-4, the excitation wavelength was set at 488 nm while for the Fura-2, two channels were used with different excitation wavelength: 340 nm and 380 nm.

• After a few minutes of frame acquisition, the 100 μL of the stimulus was added with final concentration of 100μM ATP, 10μM glutamate or 50μM potassium solution.

• If the dye used was Fluor-4, 100μL of 2μM (final concentration) ionomycin was added after a few minutes of stimulus addition.

• The images were analysed using ImageJ and Microsoft Excel.

Detection of Neuron-Specific Markers

• The cell sample were fixed with the relevant antibodies, that is, either Abcam Anti-MAP2 antibody (ab11267) or Milipore Anti-NeuN Antibody (MAB377) at 1:1000 dilution and Invitrogen Alexa Fluor-4 anti-mouse IgG as the secondary antibody at 1:4000 dilution.

• The fixed sample were then subjected to fluorescence microscopy using with the excitation wavelength set at 488 nm.

Results and Discussion

Characterisation of Stimulus-Evoked Ca2+ Response in Primary Neurons, SH-SY5Y and NPCs.

For glutamate (Figure 1), the primary neurons exhibited the expected calcium response, that is, a sharp increase in intracellular calcium concentration (ICC) upon glutamate addition followed by a gradual decrease until it levels at an above-baseline concentration. The SH-SY5Y, on the other hand, did not exhibit any response upon glutamate addition and this is inconsistent with that reported by previous investigators [2]. This might due

to the difference in glutamate concentration used: previous investigators used 500 μM whereas 10 μM was used for this experiment. For the NPC, upon glutamate addition, there is a sudden decrease ICC followed by its sudden rise which then levels at a below-baseline level. Such a response is inconsistent with that reported by previous investigators and this most likely due to the differences in the NPC culturing conditions used for this experiment and those used by the previous investigators [3].

For ATP (Figure 2), the primary neurons exhibited the expected response, that is, a gradual increase of ICC followed by its gradual decrease. For the SH-SY5Y, there was a sharp increase in ICC upon addition followed by a sharp decrease which then levels at baseline level. This is a similar response obtained by previous investigators, albeit at a smaller magnitude which most likely reflects the difference in the concentration of ATP used [4]. For NPC, the calcium response is erratic (i.e. the ICC continuously decrease and increase, forming a zig-zag like pattern). Even so, it seems that the changes of the ICC is centred about a mean. This response is unlike the one reported by the previous investigators and, again, it most likely reflects the differences in the NPC culturing conditions [3].

For the high potassium solution (Figure 3), the primary neurons, the ICC increased gradually until it reaches a peak and then gradually decreases to level at an above-baseline level, the response consistent with previous investigators. For the SH-SY5Y, the ICC increased sharply and the ICC levels at that the increase level, consistent with the response obtained by previous investigators [5]. For NPC, no calcium response assay with high potassium concentration could be done due to time constraint and lack of viable cell sample.

MAP2 Antigen Detection

For primary neurons, there is clearly the presence of MAP2 which are located on the axons and cell bodies. For the NPC during the 3rd passage, there are distinct near-circular objects visualised, suggesting that these are MAP2-expressing NPC. However, for the NPC during the 11th passage, shadows of what appears to be neurone-like cells are observed. This could be due to the failure of the permeabilisation step during sample preparation, resulting in the inability of the MAP2 antibodies to enter the NPC and attach to MAP2. Even so, considering that the 3rd-passage NPC express MAP2, it is plausible that the 11th-passage NPC too express them but only a repetition of the MAP2 detection step can confirm this. For both NPC, there was significant amount of background staining which was mostly likely due to insufficient amount of washes with the phosphate buffer solution (PBS) during the sample preparation stage (Figure 4)

NeuN Antigen Detection

For primary neurones, near-circular shapes can be visualised, suggesting the expression of NeuN as expected. Similarly, the 3rd-passage NPC shows many small circular objects evenly spread, suggesting that they too express NeuN. For the 11th-passage NPC, NeuN is detected surrounding what appears to be cell bodies, indicating that these cells express NeuN as well (Figure 5).

Future Directions

Conducting the calcium response assay on NPC with high potassium solution as the stimulus could provide further information regarding the calcium response of NPC. Additionally, the NPC used could be cultured in the lab wherein the experiment is conducted instead of obtaining them from another lab to ensure proper and consistent culturing conditions. Besides that, additional experiments can be conducted to convert the fluorescence data to actual intracellular calcium concentrations. Each part of the experiment could be repeated to gain additional data and to obtain higher quality particularly for those with data that are compromised due to technical difficulties experienced. Finally, the iPSC can be subjected to the same experiments to assess its suitability as a cell model for calcium response modelling of Parkinson’s disease.

Departure from original proposal

Ideally, each of the calcium response assay should be the done with only one type of dye but two different type of dyes had to be used due to the malfunction of one of the fluorescence microscopes during the experiment. Experiments should have been done on iPSC as well but this was not possible due to inability to timely acquire the samples.

Figure 1 The Intracellular Calcium Response of the Cell Samples towards Glutamate

10 μM glutamate was added at the time indicated by the first arrow for each cell sample. In each graph, each series line represents the intracellular calcium response of a single cell. The difference in the labelling of the y-axis reflects the different type of dyes used: fura-2 was used for the first two from the top while fluor-4 was used for the bottom. For fluor-4, a second arrow indicates the addition of 2 μM ionomycin

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Figure 2 The Intracellular Calcium Response of the Cell Samples towards ATP 100 μM glutamate was added at the time indicated by the first arrow for each cell sample. In each graph, each series line represents the intracellular calcium response of a single cell. The difference in the labelling of the y-axis reflects the different type of dyes used: fura-2 was used for the one at the bottom while fluor-4 was used for first two from the top. For fluor-4, a second arrow indicates the addition of 2 μM ionomycin.

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Figure 3 The Intracellular Calcium Response of the Cell Samples towards High Potassium Solution 50 μM of potassium solution was added at the time indicated by the first arrow for each cell sample. In each graph, each series line represents the intracellular calcium response of a single cell. For both cell samples, fura-2 was used as the calcium dye.

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Figure 4 Expression of MAP2 in, from left to right, primary neurons, 3rd-passage NPC and 11th-passage NPC

Figure 5 Expression of NeuN in, from left to right, primary neurons, 3rd-passage NPC and 11th-passage NPC

References

1. de Lau, L.M. and M.M. Breteler, Epidemiology of Parkinson's disease. Lancet Neurol, 2006. 5(6): p. 525-35.

2. Naarala, J., et al., Excitatory amino acid-induced slow biphasic responses of free intracellular

calcium in human neuroblastoma cells. FEBS Lett, 1993. 330(2): p. 222-6. 3. de Groot, M.W., et al., Characterization of calcium responses and electrical activity in

differentiating mouse neural progenitor cells in vitro. Toxicol Sci, 2014. 137(2): p. 428-35. 4. Larsson, K.P., A.J. Hansen, and S. Dissing, The human SH-SY5Y neuroblastoma cell-line

expresses a functional P2X7 purinoceptor that modulates voltage-dependent Ca2+ channel function. J Neurochem, 2002. 83(2): p. 285-98.

5. Brater, M., et al., Voltage-sensitive Ca2+ channels, intracellular Ca2+ stores and Ca2+-

release-activated Ca2+ channels contribute to the ATP-induced [Ca2+]i increase in differentiated neuroblastoma x glioma NG 108-15 cells. Neurosci Lett, 1999. 264(1-3): p. 97-100.

Determining the phospho-specific interactions of 53BP1 and their role in DNA damage repair

Student: Caitlin McCarthy, University of Sussex Supervisor: Dr Felicity Watts, University of Sussex, Genome Damage and Stability Centre G4.08 Background When DNA double strand breaks occur, the cell has mechanisms to recognise and fix the damage. An MRN complex firstly finds and binds to DSBs. This complex then goes on to recruit ATM, a kinase which phosphorylates downstream proteins such as H2AX. When histone H2AX is phosphorylated, MDC1 is recruited and causes more MRN and ATM to be recruited which amplifies the signal. H2AX also causes histone modification to allow proteins to have better access to the site of damage. An important protein recruited to the DSB is 53BP1. 53BP1 is a large protein with many different domains (Fig 1). It has a BRCT domain pair at the C-terminus that has a role in repair of damage in heterochromatin. The central part of the protein has several sites which allow the protein to bind to the damaged chromatin and help form foci. The N-terminus of 53BP1 is large and contains many serine and threonine residues that can be phosphorylated and then go on to recruit other proteins to the site of damage.

53BP1 has an important role in the G1 phase of the cell cycle as it promotes canonical Non Homologous End Joining (cNHEJ) when DNA damage occurs during this phase. cNHEJ is an easy way of fixing the break as it involves simply joining the DNA ends together. It is also believed that 53BP1 may have a role in the G1 damage checkpoint to arrest cells in G1 until the damage has been repaired.

Previous work from our lab has shown that in vitro, 53BP1 co-localizes with TopBP1 at DSBs. 53BP1’s N-terminus interacts with BRCT domains of TopBP1: specifically, BRCT4,5 binds to phosphorylated Ser366 on 53BP1 and BRCT1,2 binds to phosphorylated Thr670 (data not shown). in vivo 53BP1-S366A and -T670A mutations result in an inability of 53BP1 to co-localize with TopBP1 (Fig2). In addition, it has been shown that S366 and T670 are phosphorylated in vivo when the DNA is damaged (e.g. Fig 3A). Aims To identify the kinases responsible for the phosphorylation of Ser366 and Thr670 in response to damage.

Materials and Methods Tissue cell culture, transfection, irradiation and inhibition of kinases Hela cells were grown in DMEM media. These cells were then split. Cells were reverse transfected with siCont, while others were transfected with 53BP1 siRNA. This was done by plating out 2x105 cells on dishes and adding 50µl Optimem and 5µl of either siCont or si53BP1. Cells transfected with siCont or 53BP1 siRNAs were irradiated 48 h after transfection. One h after the inhibitor was added, cells were irradiated with 8Gy IR using a 137Cs source. Kinase inhibitors were added to appropriate final concentrations (caffeine 20 mM, Roscovitine 50µM, SB203580 MAPKi 10µM and CKIIi TBB 10µM). SDS page and western blot Whole cell extracts were prepared from cells using standard protocols. Samples were boiled for 5 min and spun at 13,000 rpm for 5 min before being loaded and run on a 7.5% SDS polyacrylamide gel. The separated protein was then transferred onto a PVDF membrane. Specific antibodies to peptides containing the regions surrounding phosphorylated S366 or phosphorylated T670, 53BP1 (to check knockdown) and tubulin (loading control) were then added in 4% milk to each membrane and left on a shaker overnight. The primary antibody was washed off and the

DAPI HA-53BP1 TopBP1 CENPF Merge

WT

S366A

T670A

Fig 1 Organisation of 53BP1

Fig 2 53BP1 S366A and T670A mutations affect colocalisation of 53BP1 and TopBP1. U2OS cells were reverse transfected with 53BP1 siRNA, 48 h later cells were transfected with an siRNA resistant construct containing wt 53BP1 or S366A or T670A mutants. 16 h later cells were exposed to 8 Gy IR. Cells were left to recover for 4 h before staining for HA, TopBP1 and CENPF (to identify G1 cells that don’t stain with CENPF). Cells were visualised using a Deltavision spectris microscope.

secondary antibody conjugated to HRP was then added to each membrane. Once the secondary antibody has been left for an hour, unbound antibody was washed off and the membranes were soaked in light and dark solution ECL reagent and exposed to X-ray film.

Results and Conclusions Western analysis with phospho-specific antibodies against pS366 and pT670 peptides of extracts from cells +/- siRNA 53BP1 and +/- IR indicate that 53BP1 is phosphorylated in vivo in response to IR (Fig 3 compare lanes A1, B1 with A3, B3). We wished to identify the kinases responsible for phosphorylation of S366 and T670. Computer analysis showed that cdks, MAPK and CK2 as the most likely candidates. Cdks were first to be tested using Roscovitine which inhibits most cdks. Comparison of Fig 3A,B lanes 3 and 5 indicate phosphorylation of S366 and T670 even when cdks are being inhibited. Thus cdks do not appear to be the responsible kinases. In Fig 4A,B lanes 3, caffeine was added as a control as it inhibits all kinase activity. A decrease in pS366 and pT670 can be observed in these lanes compared to lanes 1. In lanes 5, there is phosphorylation of S366 and T670 even when MAPK is being inhibited, suggesting MAPK is also not the responsible kinase. However, in this experiment DMSO, the solvent for the inhibitors, appears to affect phosphorylation as no bands are visible in lanes 4, thus this experiment needs to be repeated. Fig 5 shows in lane 5 that CK2 inhibition also has no effect on phosphorylation of Ser366. Therefore neither cdks, MAPK or CK2 are responsible for the phosphorylation of Ser366, and cdks and MAPK are not the kinases for Thr670.

Future Directions Future work is to continue looking for the kinase responsible for the phosphorylation of S366 and T670 in response to damage. Once the kinase has been determined, the role of the phosphorylation-mediated interaction with TopBP1 will be determined, e.g. the effect on repair of DSBs in G1 and G1 damage checkpoint.

Departures from the Original Proposal The original project was to probe the role of the phosphopeptide binding site of the BRCT domain of 53BP1. Since submitting the application this work was carried out by another member of the lab. I therefore undertook a different project, but analysed the same protein (53BP1). In this new project I learnt a range a techniques such as being able to split and transfect HeLa cells, visualisation of transfected cells using the spinning disk microscope and was able to perfect my skills in SDS PAGE and western blotting. I have consolidated and refined my knowledge in molecular genetics, cell biology and the DNA damage response and now feel much more confident in the lab.

Fig 3 CDKs do not phosphorylate S366 or T670. Hela cells were transfected with either siRNA targeting 53B1 or a non-targeting control, followed by treatment with Roscovitine (CDK inhibitor). Cells were irradiated with 8Gy using a 137Cs source 4h before lysis. Cell extracts were separated by SDS PAGE and western blotted with anti-sera against pS366, pT670, 53BP1 and tubulin

Fig 4 MAPK does not phosphorylate S366 or T670. Hela cells were transfected with either siRNA targeting 53B1 or a non-targeting control, followed by treatment with caffeine (inhibits all kinase activity), DMSO (solvent for inhibitors) or MAPK inhibitor. Cells were irradiated and processed as in Fig 3.

Fig 5 CK2 does not phosphorylate S366. Hela cells were transfected with either siRNA targeting 53B1 or a non-targeting control, followed by treatment with caffeine, DMSO or CK2 inhibitor. Cells were irradiated and processed as in Fig 3.

Characterising mutants of β-phosphoglucomutase Biochemistry Society Summer Vacation Studentship

Carly Wynne

Supervisor: Angus Robertson - Biophysics, Department of Molecular Biology

and Biotechnology, University of Sheffield

β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl-transfer enzyme that has been the

focus of intense research for over a decade. βPGM catalyses the interconversion of β-glucose-1-phosphate

(G1P) to β-glucose-6-phosphate (G6P), lying strongly in favour of the formation of G6P with an equilibrium

constant of 28 (2). The enzyme is a phosphomutase and catalyses both phosphatase and kinase reactions. βPGM

has a mass of 25kDa and can exist in both open and closed states, which

alternate depending upon whether the forward or reverse reaction is

proceeding (3). The active site is located between a core and a cap

domain and is freely accessible to solvent when in the open conformation.

A catalytic Asp8 residue is found in the active site, with Asp10, Thr16,

Ser114, Lys145, Glu169 and Asp 170 all found within a 6Å radius of this.

The Mg2+

cofactor is octahedrally coordinated by the backbone carbonyl

of Asp10, the side chains of Asp8, Glu169 and Asp170, one oxygen from

the Asp8 phosphoryl group and one water molecule. The phosphate

bound to Asp8 also interacts with the side chains of Ser114 and Lys145

(4) (Fig. 1.) The coordination of this Mg2+

cofactor is crucial to the

functionality of the enzyme, by

balancing the negative charges of

several active site residues. The mechanism of PGM has continued to cause

fierce debate, especially regarding the structure of the transition state. This

debate intensified when it was claimed that a pentavalent phosphorane

intermediate was directly observed using high intensity x-ray crystallography

(2.4Å resolution) and electron density maps, however this was later proved to

be an MgF3- molecule. Baxter et al determined that the electron density

observed in crystal structures of PGM were from an MgF3-

molecule. The

group then used difference Fourier maps to explain the electron density

patterns observed with MgF3-

compared to a PO3-

molecule. The initial

proposal of a pentacovalent intermediate showed large difference peaks when

mapped in this format (Fig. 2A), however these could be resolved when the

PO3- is replaced with MgF3

- (Fig. 2B). MgF3

- is isoelectronic with a PO3

-

molecule, with both molecules possessing 24 valence electrons, a net negative

charge, and the ability to form pentacoordinate geometry, and therefore

provides a way to observe a PGM transition-state analogue (TSA) (5). Due to

the timescale of this reaction, it is not possible to directly observe the structure

as the reaction processes. Therefore, molecules that mimic the PO3- of the

substrate are used to observe the reaction at a certain point, including BeF3- and the previously mentioned MgF3

-.

Due to the geometry of Beryllium (strictly tetrahedral and cannot form a pentacovalent structure), when added

to βPGM, the Be(OH)3 and NaF form a covalent group that assembles on D8, forming a ground-state analogue

(GSA) of the enzyme. The electronic configuration of the BeF3-

moiety prevents nucleophilic attack, thus

limiting the complex to a GSA and providing an elegant example of the importance of stereoelectronic effects,

originally highlighted by Blackburn et al (6).

A minor set of peaks are observed in the wild type fluorine NMR spectra, corresponding to a change in

coordination geometry of the transition state mimic Mg2+

, via the addition of a carbonyl ligand from the protein

backbone (Fig.1). The rotation of this carbonyl is implicated in a gating mechanism, whereby the enzyme

prevents back reaction of the transferring phosphoryl group. K145A is a mutant that displays similar fluorine

chemical shifts and thus was a suitable candidate for further study.

Fig. 1 Diagram showing the Mg2+ octahedral

geometry in the active site of PGM

Fig.2 Diagram showing the

difference Fourier maps for a

pentavalent phosphorane

intermediate (A) and an MgF3-

transition-state analogue (B) in

the active site of PGM (Baxter et

al, 2010)

Our Research

We will focus on characterising several mutants in an attempt to shed light on the minor conformation, which is

likely to be involved in intermediate release and enzyme catalysis, with the aim to determine the driving force

behind the inter conversion of the two species. Asp10 has been observed to move in and out of the active site

throughout the reaction, however D10 importance beyond the role as a general base is unknown. Lys117 is a

residue that assists the closing of the two domains when the substrate is bound in the active site. We aimed to

create two mutations, D10A and K117A, and observe the structure and function of the enzyme with these

changes. However, due to complications with equipment and sourcing the D10A/K117A mutants, our focus

shifted to Pro138 and K145 mutants of PGM.

Pro138 occupies two different environments in the apo structure, indicating cis-trans isomerism, which may be

responsible for the two species viewed in previous NMR experiments. Therefore, a P138A mutant was created,

which removes the capacity of the residue to occupy these two environments, and consequently may

validate/invalidate the hypothesis that isomerism is the driver behind the two species observed. Preliminary

NMR data has suggested that this mutant did not affect the ratio of the two species in the apo enzyme, however

the crystal structure will give more indication of how, if at all, this mutation changes the ratio of the two species.

We will characterise the mutant as an apo enzyme, and as a TSA using an MgF3- molecule. A crystal structure

of the wild type (WT) apo enzyme shows that this proline residue is within Van der Waals contact distance with

the carbonyl that rotates in and out in the minor/major transition, and therefore may be having an effect on the

ratio of the two species. Therefore, we aim to create crystals of the P138A TSA to see if this is the case, as in

WT crystals the minor conformation has not previously been observed.

Another mutant that we will focus on is K145A, a mutant that has previously been observed to exist in the

closed minor form as a crystal structure, when saturated with MgF3-. However, further characterisation using

NMR determined that, in solution, the mutant adopts a more open conformation and requires high

concentrations of sugar and metal fluorides to saturate the complex. Consequently, as aluminium has a higher

affinity for the enzyme-sugar than magnesium, AlF4- is used to form a TSA complex as opposed to MgF3

-, as

energetic costs of an equilibrium between MgF3- and MgF2 push the complex away from forming a TSA. Our

aim is to form this TSA with AlF4- to analyse using NMR, and to obtain a crystal structure, as NMR does not

tolerate the high levels of MgF3- used to form the original crystal. Our final aim is to characterise the double

mutant K145A D10N. D10 may be involved in an activation step, as it is a general base that moves into the

active site to mediate catalysis. Previous analysis shows that D10N retains some catalytic activity before

forming a bisphosphate intermediate and inactivating itself. A crystal structure has already been obtained for a

D10N bisphosphate GSA, in the presence of the catalytic Mg2+

, however, the NMR data showed significant

chemical shift changes between the Mg2+

bound and unbound states. Therefore, we need a crystal structure of

the D10N Mg2+

unbound state.

Methods & Materials

Competent Cell Preparation

Four E.coli cell lines (XL-gold, XL-blue, XL-blue SC and BL21-DE3) were plated on to LB plates, which were

incubated overnight at 37˚C. Two colonies from each of the original cell line plates were selected and inoculated

in 5 ml LB broth, and incubated overnight at 37˚C. A solution of LB was created and autoclaved along with four

conical flasks, ready for next step. 0.5ml of the cells were added to 50ml LB broth and incubated at 37˚C until

the OD reached 0.6 (exponential growth phase). The cells were then made competent in several steps. Cells

were centrifuged and resuspended in 10ml ice cold CaCl2, and incubated on ice for 10 minutes. Cells were then

centrifuged once more, and re-suspended in 2ml CaCl2 with 70µl DMSO. Ice for 10 minutes, and further 70µl

DMSO was added, and 200µl aliquots were dispensed into freezer Eppendorf tubes. Stored at -80˚C. Check

competency of the four cell lines with a transformation of the WT plasmid. 2µl of plasmid added to the cells,

and incubated on ice for 15-20 minutes. Heat shocked at 42˚C for 45 seconds, and then placed on ice for 2

minutes. 800µl of LB (no amp) added and incubated at 37˚C for one hour. Plated onto LB agarose + amp plates

and those that have up-taken the WT plasmid formed colonies.

Mutagenesis

The D10A and K117A mutations were created by using mismatched forward and reverse primers to the wild

type plasmid. Solution of 70µl prepared: 5µl forward primer, 5µl reverse primer, 5µl WT plasmid, 5µl 10x

reaction buffer, 1µl PfuUltra HF DNA polymerase, 1µl dNTP mix, and topped up with 28µl H2O. These

plasmids encoded the mutation and an ampicillin resistance marker, allowing for selection when the PCR

products are plated. After 18-20 PCR cycles, WT methylated plasmids were digested with DPN1. The products

were plated on LB agarose + amp plates.

Transformation & Mini-Prep

Using the XL-blue SC and BL21-DE3 strains, a mini prep was prepared, by adding wild type plasmids to each

solution and plating. These were then incubated at 37˚C. Two colonies from the mini prep plates were selected

and inoculated in 10ml LB broth to maximise DNA mass. These plates were incubated at 37˚C overnight. The

QIAprep Spin Miniprep kit. 850µl of mini prep solution was added to the column, and several buffers added

(including lysis and neutralisation buffers), in accordance with the kit instructions, before eluting the DNA using

sterile water. Both solutions were transferred to microfuge tubes and stored at -20˚C until required. WT and

K145A D10N plasmids were transformed into XL-Blue SC cells, and mini-prepped using the same kit and

protocol as previously listed.

X-ray Crystallography X-ray crystallography experiments were set up to try and visualise mutants of PGM that have already been

isolated. A motherliquor of 700µl was dispensed in the well, including PEG 4000 as the precipitant, 200mM

NaAcetate as the salt, and 100mM Tris pH 7.5 buffer. This was topped up with an appropriate volume of

sterilised water. Concentrations of 24%, 26%, 28%, 30% and 32% PEG 4000 were used. 2µl of this

motherliquor was pipetted onto a cover slip, along with 2µl of isolated protein. 4 mutants/complexes were used:

wild type (WT) apoenzyme, K145A-BeF3, K145A-BeF3-G6P and K145A-BeF3-G6P (half saturated and

saturated conditions). The crystals were viewed under a microscope, and one drop of the apo WT was selected,

and one drop of the apo K145A line. These crystals were used for a seeding experiment of the same 4

mutants/complexes, where they act as nucleation sites for further crystal development. The motherliquor was

prepared in the same way, however the crystallisation solution was made up of: 1µl protein, 0.3µl seeding

solution and 0.7µl motherliquor. A further 7 mutants/complexes were laid: WT-Ammonium Sulfate, P138A

apo, P138A-BeF3, P138A-BeF3-G6P, P138A-MgF3-G6P, K145A apo, K145A-BeF3-G6P and a second stock of

K145A-BeF3-G6P (with 10mM G6P). These trials were carried out under the same conditions as previously

stated. Any needles/crystals that formed were extracted and used for seeding further experiments. A needle

pipette was used, which partially breaks up the crystals, and this solution was transferred into a microfuge tube.

The microfuge tube was vortexed for ~10 seconds. A serial dilution was performed, and the 100,000 fold

dilution used for seeding. K145A apo, P138A-BeF3 and P138A-BeF3-G6P mutants were relayed using seeding

solutions (same method as previously stated). K145A-AlF4--G6P was trialled under the same conditions as

previously stated, however, no crystals were observed. Buffer exchange of K145A-AlF4--G6P was performed

using 50mM BisTris pH 6.0 (5mM MgCl2 and 2mM NaN3), so the complex was now at pH 6.0. D10N MgF3-

trialled using conditions where crystals previously observed: PEG 4000 24-34%, NaAc 200mM and pH 7.5 Tris

buffer. This complex also trialled with seeding stock P138A BeF3- 10,000 fold.

NMR

NMR spectroscopy was performed across two institutions: University of Sheffield and University of

Manchester. Measure rate of turnover of PGM + 1M (NH₄)₂SO₄ using NMR. Total solution of 500µl including

50µl PGM (1200nM), 14.1µl AcP (20mM), 5µl TSP, 45µl D2O and topped up with 364µl H2O (16.67µl 10mM

G1P added later). Initially, achieve 1D spectra with peaks for PGM, AcP and PPi. Enzyme now primed. Add

G1P and run NMR. Fluorine, proton and 2D TROSY NMR were performed on four complexes - K145A-AlF₄⁻-G6P, K145A D10N MgF3-G6P, P138A-MgF3-G6P and P138L-MgF3-G6P.

Results

Competent Cell Preparation

Analysis of the transformant plates showed that XL-Blue and XL-Blue SC had a high transformation efficiency,

whereas BL21-DE3 and XL-Gold showed slightly lower transformation efficiency with fewer colony growths.

This may be due to the plates reaching too high an O.D. (1.03 and 1.20 respectively), which results in cells

passing the exponential phase and dying after being transformed. Therefore, competent cell preparation was

repeated for XL-Gold and BL21-DE3.

Mutagenesis

The PCR product transformations plates had no colonies. This could be due to many factors, including the PCR

kit itself, incorrect primer sequences or missing out a certain reagent. The reason for lack of colonies will have

to be identified and the experiment repeated. The mutagenesis experiment is repeated with a different kit. The

PCR products are transformed into the competent cells, and plated onto LB + amp plates. This mutagenesis did

not work and two control mutagenesis reactions were performed (P138A and control solutions). These also did

not work and therefore this suggests that there may be an issue with the PCR kit not working correctly.

Transformation & Mini-Prep

There was no visible change in cell density in the starter culture for WT and K145A D10N transformants,

therefore this protocol is repeated using XL-Blue cells to determine whether the other cells were not efficiently

transforming. These new starter cultures produced WT DNA of approximately 100ng/µl and K145A D10N

DNA of approximately 100ng/µl.

X-ray Crystallography

The initial trials produced needles

for only K145A apo, P138A BeF3

and P138A BeF3 G6P, therefore

these are used to create seeding

solutions. Those

mutants/complexes that produced

no needles/crystals were seeded

with seeding solutions of the same

mutant class. The seeding of the 4

mutants that initially produced no

crystals, yielded useable crystals

for P138A apo (Fig.3A) and P138A

MgF3 G6P and therefore seeding

solutions were created from these

crystals. However, despite seeding,

there were still no crystals for WT-Ammonium Sulfate (1M) and K145A BeF3 G6P. The WT-Ammonium

Sulfate (1M) will be tested using the robot to survey a range of conditions. The crystals were shot using the

Diamond synchrotron in Oxford. Electron density maps were formed with a resolution of ~1.7Å and the

molecular replacement completed for the apo structure (Fig. 3B). By the end of my project, these were yet to be

analysed.

NMR

When PGM + (NH₄)₂SO₄ was observed as a crystal

structure, only one species was isolated. This led to the

hypothesis that the (NH₄)₂SO₄ may reduce the lag phase,

which was previously believed to be the enzyme being

primed, by having the enzyme already in the activated

state. However, a lag phase was still observed, suggesting

that the lag phase may not be accredited to a change in

enzyme conformation of the enzyme. Fluorine NMR for

the K145A-AlF₄⁻-G6P complex was run at a pH range of

6.5-8 (Fig.4). The movement of the peaks highlighted

suggests that one species of this complex is pH dependant.

In this spectra, species are identified by four fluorine

peaks, attributed to the 4 fluorine atoms of the AlF₄⁻ bound to the active site. Currently, there is no indication of

the structures of these two species are, and this would

require further analysis.

The 2D TROSY experiments of P138A-MgF3-G6P (Fig. 5A)

and P138L-MgF3-G6P (Fig. 5B), in comparison to wild type spectra, have significant overlay, suggesting that

there is no change in folding state and that the TSA is very similar to the WT. However, it can be noted that the

overlay for the P138L TSA is not as good, which suggests that this mutation has a greater effect than P138A.

When these 2D TROSY spectra are overlaid, it is clear that there are little discrepancies in the overlap of the

peaks, with the tryptophan mutants in identical environments (Fig. 6A), however P138L has a smaller

Fig.3 P138A apo crystals (A) under conditions – PEG 4000 23-32%, 200mM NaAc and 100mM Tris

buffer pH 7.5 and the electron density map of crystals observed at a resolution of ~1.7Å

A B

Fig.4 Fluorine NMR spectra of K145A-AlF₄⁻-G6P at pH 6.5

(green), 7.0 (blue) and 8.0 (red)

A B

A B

tryptophan peak, suggesting that the complex is not saturated and there is a slow exchange between apo and the

TSA. Despite the differences in the 2D TROSY spectra, there is very little difference between the P138A-MgF3-

G6P and P138L-MgF3-G6P fluorine spectra (Fig. 6B). The Fa peak of P138a is downshifted, suggesting the

mutation has caused this fluorine to experience an increased magnetic field due to a decrease in shielding by

neighbouring atoms, most likely caused by an increase in the amount/strength of the hydrogen bonds it is

involved in. This has most likely had effect on Fb and Fc, and has drawn electron density away from these

atoms, resulting in a slight up field shift for both peaks.

The double mutant K145A D10N MgF3-G6P was run 3 times, with a range of G6P concentrations from 0mM –

35mM (Fig. 7A). The 2D TROSY spectra overlay of these 3 experiments shows that upon ligand binding there

is no real conformational changes, with only a few chemical shift differences. This suggests that this mutant

may possess an open complex, with a sugar phosphate molecule bound. The fluorine spectra (Fig. 7B) is

interesting as there is no peak for an MgF3 TSA, and this complex is therefore not forming a TSA. Using both

the 2D TROSY and fluorine spectra, it has been hypothesised that there is something different forming upon

ligand binding, and may be a bound bisphosphate. This has previously been observed in the single D10N mutant

active site, and eventually renders the enzyme inactive.

Fig.5 2D TROSY spectra for P138A-MgF3-G6P (black) and WT (red) overlay (A) and P138L-MgF3-G6P (black) and WT (red) overlay (B)

A B

Fig. 6 2D TROSY P138A-MgF3-G6P (red) and P138L-MgF3-G6P (black) overlay (A). Fluorine spectra for the P138A-MgF3-G6P (blue)

and P138L-MgF3-G6P (red) overlay (B)

Fig. 7 2D TROSY overlay of K145A D10N apo (black), K145A D10N 17mM G6P, 10mM NaF, 5mM MgCl₂ (red) and K145A D10N

35mM G6P, 10mM NaF, 5mM MgCl₂ (blue) (A). Fluorine spectrum for K145A D10N MgF3-G6P (B)

A B

Future Directions

Perform 1D phosphate NMR to determine what is affecting the structure of the double mutant K145A D10N,

and whether it is in fact a bound bisphosphate. Molecular replacement of the electron density maps of the

crystals, with the known WT structure. This will be analysed together with the NMR data to attempt to

determine the structural changes caused by these mutants. Attempt to crystallise D10N without magnesium

bound, as it has only been previously done in the presence of magnesium. When the D10A plasmid has arrived,

characterise this mutant in the same way as above to determine the differing effects of an asparagine and alanine

mutant of the general base.

Value of the Studentship

Student

The studentship has been a valuable and unique experience, which has enhanced my laboratory experience and

analytical skills. Over 8 weeks, I have had the opportunity to use different scientific methods to determine

structural properties, interpret complex data sets, and work with experts in this field of study. I have gained an

insight into the day-to-day operation of a research lab, which is completely different to any previous laboratory

experience. Before I began this project, I was unsure about a career in research and whether I had the mind-set

to do so, and I believe that without this studentship, I would still not have the self-belief to be strongly

considering completing a PhD in the future.

Supervisor

The studentship has been of great value to our laboratory and Carly has managed to achieve excellent

crystallography data, in some cases to a resolution of 1.06Å. Through working in a biophysics laboratory, she

has gained a huge variety of skills, from good laboratory practice, to working with new equipment and

interpreting complex data. Her work ethic has helped to maintain the fast pace of the project and it was a

pleasure to have her working in the laboratory.

1. Zhang, G., Dai, J., Wang, L., Dunaway-Mariano, D., Tremblay, L. W., & Allen, K. N. (2005). Catalytic cycling in

beta-phosphoglucomutase: a kinetic and structural analysis. Biochemistry, 44(27), 9404–9416.

2. Baxter, N. J., Bowler, M. W., Alizadeh, T., Cliff, M. J., Hounslow, A. M., Wu, B., … Waltho, J. P. (2010). Atomic

details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF-3 rather than by

phosphoranes. Proceedings of the National Academy of Sciences of the United States of America, 107(10), 4555–

4560.

3. Dia, J.B, Liu, Y., Ray W.J. Jr, Konno, M. (1992). The crystal structure of muscle phosphoglutamase refined at 2.7

– angstrom resolution. Journal of Biological Chemistry, 267(9):6322-6337

4. Lahiri, S. D., Zhang, G., Dunaway-Mariano, D., & Allen, K. N. (2002). Caught in the Act: The Structure of

Phosphorylated b-Phosphoglucomutase from Lactococcus lactis. Biochemistry, 41(26), 8351–8359.

5. Blackburn, G. M. (2003). Comment on “The Pentacovalent Phosphorus Intermediate of a Phosphoryl Transfer

Reaction.” Science, 301(5637), 1184c–1184.

6. Griffin, J. L., Bowler, M. W., Baxter, N. J., Leigh, K. N., Dannatt, H. R. W., Hounslow, a. M., … Waltho, J. P.

(2012). Near attack conformers dominate -phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate. Proceedings of the National Academy of Sciences, 109(18), 6910–6915.

Connor Sampson - University of Kent - Summer 2015

1

Biochemical Society Summer Studentship Report: The Effects of Varying Translational Accuracy

on Protein Behaviour and Cell Phenotype in S. Cerevisiae.

Introduction

It is well established that while multiple codons may represent a single amino acid, not all codons

are translated with the same degree of accuracy. [1] While this accuracy is affected by a number of

factors, such as relative aatRNA abundance and decoding times, [1] the principle may be used to design

genes which produce proteins of a desired fidelity. Here two versions of GST were created, one using

high accuracy codons (GST_Max) and the other low accuracy codons (GST_Min), in order to study the

effect of these errors in a controlled manner.

Aims

The original aims were to extract both GST_Min and GST_Max from transformed strains of S.

cerevisiae and subject them to mechanochemical analysis to study the functional manifestations of these

errors. During the project, however, difficulties extracting the products in a non-denatured state caused

the studentship, and it's limited timescale, to focus instead upon the in vivo effects of these variants

Experiments

Plasmids containing the desired genes were transformed into a standard BY4741 and a triple

protease mutant (ΔProt_3; lacking pre1 [Proteasome catalytic subunit] [2], prb1 [Vacuolar protease] [3],

and pep4 [vacuolar protease] [4]) haploid cell lines.

The use of a standard GST affinity column was decided against as it would have acted

selectively, binding only well formed proteins. The use of a N-terminal His-tag was instead used for both

constructs, however this was only accessible under heavily denaturing conditions (8M urea).

As the Mw of GST was known, SDS-PAGEs of purified GSTs were run, the relevant gel slices

excised and destained, and placed into the wells of IEF gels in order to compare charges.

Further to this, new transformants were constructed for strains deficient in particular degradation

pathways (Table 1) in order to study possible in vivo breakdown mechanisms of highly erroneous

proteins, and the associated effects on cell function. Growth curves were constructed for these

transformants, and western blots were carried out to observe the comparative GST levels.


The IEF preparation, involving destaining, renaturing, and the loading of gel slices, was only

partially successful, producing IEF protein "smears". Optimisation is therefore required.

Western blots for BY4741 and ΔProt_3 colony extracts (Fig. 1) showed greater expression of

GST_Max verses GST_Min in all strains with a PGK1 loading control. (For the purposes of this project,

PGK1 abundance is assumed constant in all mutants.) This was to be expected from codon usage, as

rapidly translated codons are associated with greater accuracy, allowing greater levels to be produced by

the cells. [1] It did not show a clear difference in relative GST abundance between the two strains.

It may be noted that all proteins in ΔProt_3 showed a decreased molecular weight, although the

exact causes of this are unclear.

The growth curve data showed no significant change in maximum growth rate in the tested

knockout strains for the insertion of either GST_Min or GST_Max. (fig. 2)

Due to time constraints it was not possible to carry out knockout strain western blots in replicate

and as such significant differences cannot be claimed from the data (fig.3), although expression was

confirmed to follow standard patterns, with greater GST_Max than GST_Min abundance in all strains.

Future Directions

Insertion of a penta-Gly linker before the His tag may improve accessibility in the native protein.


2

The IEF could be optimised through the application of a more controlled loading medium, such as

a homogenised gel slice suspension, or even a solution of extracted protein.

The knockout strain western blots may be carried out in replicate in order to properly examine for

significant differences in strain GST abundance.

Career Aspirations

I previously had difficulty deciding between a career in research and a career in science

communication, fearing that lab work may not be sufficiently engaging. This studentship has done much

to dismiss these fears and I am now significantly more likely to pursue laboratory work.

Value to Myself

The studentship itself represented a highly valuable episode in my academic career, providing

both experience and a greater understanding of laboratory work. I had carried out experiments previously

as part of my degree, however it quickly became apparent that these were very different to actual lab

work where I was now able to pursue a larger project, make decisions based on actual data, and work in a

far more flexible environment. The studentship, while being enjoyable, has provided both the

encouragement and some of the skills to pursue a future career in research.

Value to the lab

"Connor's summer placement provided valuable help and extended an ongoing project, allowing

us to pursue an avenue of exploration which otherwise would have remained unexplored. Connor's

assessment of how properties of DNA sequences which should give rise to identical proteins nevertheless

lead to subtly different behaviour of the expression host enriched ongoing work characterising minor

protein sequence variants of recombinantly expressed proteins. Moreover, Connor road-tested novel

electrophoresis-based procedures which we are now starting to use for the characterisation of

heterogeneity in various gene products. Overall, we value Connor's contribution to the project and the

general life in the lab very highly, and we thank the Biochemical Society for enabling this contribution by

providing Summer Vacation Studentship funding."

Bibliography

[1] D. Tarrant and T. v. d. Haar, “Synonymous codons, ribosome speed, and eukaryotic gene,” Cellular and Molecular Life Sciences, vol. 71, no.

21, pp. 4195-4206, 2014.

[2] M. C. Morris, P. Kaiser, S. Rudyak, C. Baskerville, M. H. Watson and S. I. Reed, “Cks1-dependent proteasome recruitment and activation of

CDC20 transcription in budding yeast,” Nature, vol. 423, pp. 1009-1013, 2003.

[3] C. M. MOEHLE, R. TIZARD, S. K. LEMMON, J. SMART and E. W. JONES, “Protease B of the Lysosomelike Vacuole of the Yeast

Saccharomyces cerevisiae Is Homologous to the Subtilisin Family of Serine Proteases,” MOLECULAR AND CELLULAR BIOLOGY, vol. 7,

no. 12, pp. 4390-4399, 1987.

[4] G. AMMERER, C. P. HUNTER, J. H. ROTHMAN, G. C. SAARI, L. A. VALLS and T. H. STEVENS, “PEP4 Gene of Saccharomyces

cerevisiae Encodes Proteinase A Vacuolar Enzyme Required for Processing of Vacuolar Precursors,” MOLECULAR AND CELLULAR

BIOLOGY, vol. 6, no. 7, pp. 2490-2499, 1986.

[5] H. H. Lee, Y.-S. Kim, K. H. Kim, I. Heo, S. K. Kim, O. Kim, H. K. Kim, J. Y. Yoon, H. S. Kim, D. J. Kim, S. J. Lee, H. J. Yoon, S. J. Kim,

B. G. Lee, H. K. Song, N. Kim, C.-M. Park and S. W. Suh, “Structural and Functional Insights into Dom34, a Key Component of No-Go

mRNA Decay,” Molecular Cell, vol. 21, pp. 938-950, 2007.

[6] N. Gallastegui and M. Groll, “The 26S proteasome: assembly and function of a destructive machine,” Trends in Biochemical Sciences, vol.

35, no. 11, pp. 634-642, 2010.

[7] O. Brandman, J. Stewart-Ornstein, D. Wong, A. Larson, Christopher, C. Williams, G.-W. Li, S. Zhou, D. King, P. S. Shen, J. Weibezahn, J.

G. Dunn, S. Rouskin, T. Inada, A. Frost and J. S. Weissman, “A ribosome-bound quality control complex triggers degradation of nascent

peptides and signals translation stress.,” Cell, vol. 151, no. 5, pp. 1042-1054, 2012.


3

Tables and Figures:

Knockout Strain Deficient Protein Protein function Phenotype

Δdom34 Dom34 Endonuclease involved in

ribosome release and mRNA

breakdown in No Go Decay

pathway. [5]

No Go Decay pathway non-

functional.

Δpep4 Saccharopepsin Vacuolar protease, responsible for

activation of multiple zymogens.

[4]

Vacuolar protein degradation

heavily compromised.

Δump1 Proteasome maturation

factor UMP1

Chaperone for proteasome 26S

subunit formation. [6]

Greatly reduced levels of

functional proteasomes.

Δrqc1 Ribosome quality

control complex

subunit 1

Complex binds stalled ribosomes,

facilitating peptide excision and

ubiquitination. [7]

No degradation of stalled

polypeptides.

Table 1: Knockout strains used in the latter experiments.

Figure 1. Comparative western blots for BY4741 and delta Prot 3 showing the greater expression

of GST_Max, and the decreased Mw of all proteins from the protease deficient strain. GST_Min and

GST_Max levels vary proportionally in all stains.

Figure 2. Maximum exponential growth

rates for the knockout strains, with the

95% confidence interval shown. No

significant difference is seen, indicating

that no version of GST effects maximum

growth rate.

Figure 3. Western blots for the knockout strains in Table 1, showing apparently standard ratios of

GST_Min to GST_Max.

0

0.05

0.1

0.15

0.2

Δrqc1 Δump1 Δpep4 Δdom34

Max

imu

m D

elt

a

Knockout Strain

Control

GST_Min

GST_Max

Biochemical Society Summer Vacation Studentship Report 2015

Development of a new photoactivable green fluorescent protein for photoactivation localisation microscopy

Student: Dahlia Eldosoky

Supervisors: Dr Colin Rickman1 and Dr Amy Davies1

1 Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University

Background & Aims

The onset of super-resolution microscopy has allowed the detailed description and observation of dynamic processes in living cells, by surpassing the resolution limit of traditional fluorescence microscopy, which is ca. 250 nm (1). Targeted labeling of specific cellular components using fluorophores that have the ability to emit at different wavelengths, allows the examination of cell structures in a live sample. However, the advances in super resolution microscopy have allowed 3D imaging, observation of cell dynamics and imaging molecular interactions to be possible (2). One technique in particular uses photoactivatable proteins that allow single protein molecules – with distances of a few nanometers apart - to be resolved by using photoactivation localisation microscopy (PALM) (3). In order for this technique be successful, the fluorescent protein’s photoactivatable behaviour must be exhibited and sufficiently prominent for there to be detection at the molecular level. PALM at present, is only practically achievable using photoactivatable proteins that are detected in the red region.

A fluorescent protein from Haeckelia beehleri, called HbGFP1, is a new photoactivatable green fluorescent protein (4) that has shown fluorescent properties but in spite of this, is not yet optimal for PALM studies due to the long carbonyl

and amino termini.

The aim of the project was therefore to: Clone and express the truncated versions of HbGFP1 Spectroscopically analyse ensemble switching dynamics

And look at single molecule microscopy of individual purified proteins

Work Carried Out & Results Clone and express the truncated versions of HbGFP1: During the 8 weeks, 3 truncated versions of HbGFP1 were made and the proteins of each version were expressed in bacteria and then purified using several methods. A plasmid DNA backbone with regions encoding StrepTag and HisTag along with four different primers – two used for 5’ end and other 2 used for 3’) was initially designed prior to cloning. Truncation of HbGFP1 at 5’, 3’ and 3’ end of the truncated 5’ plasmid (5’3’) were achieved using a Q5 Site-Directed Mutagenesis Kit. XL10-Gold ultracompetent cells were transformed with the plasmids and bacteria were grown up in larger quantities, which then the plasmid DNA was purified via miniprep and maxiprep. The sequencing results after plasmid purification are shown in Figure 1, showing that 39bp were deleted in total 3’, 12bp for 5’ and 51 bp for 5’3’.

Figure 1. DNA sequencing results of 3’ (A), 5’ (B) and 5’3’ (C), showing comparison between truncated versions and original HbGFP1. Gap indicates deletion.

B

A

C

Protein expression was carried out using 5’, 3’ and 5’3’ plasmids using BL21 ultracompetent cells along with IPTG to induce protein expression. The proteins have HisTag on 5’ end and StrepTag on 3’ end, which allowed two forms of protein purification via StrepTrap and HisTrap column purification using ÄKTAPurifier. Protein fractions that were eluted from columns were then analysed using SDSPAGE, which the results are shown in Figure 2.

5’ purification was successful with protein fractions obtained at under 37kD range but just above 25kD for which HbGFP1 is 34.6kD. A further purification step was carried for 5’ using a spin concentrator and a dialysis cassette but this also resulted in protein aggregation that formed 2 layers in an eppendorf tube. 3’ and 5’3’ protein gels on the other hand, showed smearing which suggests that the protein aggregated due to lack of stability of protein conformation.

Overall, 3 truncated versions of purified plasmid DNA of HbGFP were obtained but once protein was expressed, there was much difficulty in purifying the proteins. In terms of future directions, 3’ and 5’3’ truncations are evidently not possible and so further optimisation – for example buffer composition, pH and temperature control - for purification of 5’ truncation is required in order for spectroscopic analysis to be carried out, and, to ultimately look at single molecule microscopy of individual purified proteins.

Value of Studentship From student: The studentship has given me an opportunity to spend time in a research laboratory, allowing me to gain an insight of both the theoretical and practical aspect of a research environment. I was able to master the techniques of cloning and protein expression, both of which I was unfamiliar with - in a practical sense - at the start of the project. As a result, it has extended my practical ability, along with gaining the confidence of being able to apply my academic theory in something I had no experience in. Ultimately, the whole experience was very valuable; it has boosted my confidence for when I come to tackle my Masters project and has confirmed my interest in hopefully obtaining a PhD in the future. I would like to thank the Biochemical Society for giving me this chance to broaden my scope and to the lab for their constant support and assistance throughout the 8 weeks.

From supervisor: Dahlia has been a great help over the 8 week summer project. She has made excellent progress and quickly learned the new skills required to perfrom this project. This project is being continued by Amy Davies (who supervised Dahlia on a daily basis) and any resulting publication will include some of the work performed by Dahlia this summer.

References (1) J. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New York, 2006. (2) M. Fernández-Suárez, A. Y. Ting, Nat. Rev. Molecular Biology, 9, 931-932 (2008) (3) E. Betzig, G.H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, Science, 313, 1643 (2006) (4) S. H. D. Haddock, N. Mastroianni, L. M. Christianson, Proc. R. Soc., 277, 1155 (2010)*

Figure 2. 5’ StrepTrap gel (A) showed a band of contaminants from Strep aridin, but this was later removed after HisTrap purification step as shown on HisTrap gel (A’). 3’ StrepTrap gel (B) showed smearing, indicating no bands and hence no protein which was later confirmed by HisTrap gel (B’). 5’3’ StrepTrap gel (C) also showed no protein was obtained.

25kD

A

A’

B

B’

C

* After completion of this project the initial article was retracted. However, this was merely due to incorrect identification of the host species and the DNA sequence, protein sequence, spectroscopic properties and all other findings in the original publication and in this project remain valid.

CONSTRUCTION OF BILE-ACID REPORTER IN BACTERIAL COMMENSAL STRAIN

Daniel de la Torre - University College London - Computational Systems and Synthetic Biology Lab (Prof. Geraint Thomas)

BACKGROUND AND AIMS

Ongoing studies like the Human Microbiome Project and

MetaHIT are slowly unravelling the physiological importance

of the symbiotic relationship between humans and microbes

and providing us with new insights into the role of

microbiota in human health and disease1,2

. The commensal

nature of the microbiota and its constituents provide a

number of well-tolerated microorganisms that could

potentially exploit the intestines as an entry point and act as

vectors for therapeutically relevant synthetic genetic

circuits. An example of this is the use of commensal E. coli

strain NISSLE1917 for targeting Vibriocholerae infection3,

diabetes4 and obesity

5.

The primary and secondary bile acid axis in the intestines is

thought to be implicated in microbial pathogenesis6,

degenerative diseases7 and gastric cancers

8. Hence, there is

a growing need for tools to monitor and investigate the

complex pathways and networks involved in bile acid

metabolism. Recently, the bbr_0838 gene in Bifidobacterium

breve was found to encode a bile-inducible membrane

protein which provides the bacteria with tolerance against

bile acids9. The promoter region of bbr_0838 was shown to

activate in the presence of bile acids using a gusA reporter

assay. The aim of this project was to investigate whether

this promoter region could be used for the creation of a new

investigative tool for monitoring the bile acids axis in the

intestines, by cloning into a bacterial fluorescent reporter

plasmid and transformation into commensal strain

NISSLE1917. In addition, construction of a simple model for

this construct could help determine whether this reporter

plasmid could be used to monitor physiological levels of bile

acids in vivo.

DESCRIPTION OF WORK

Construction of bile-acid reporter strain

The BBr_0838 promoter region was synthesised with

appropriate restriction sites on either end, for cloning into

the high-copy OG241 plasmid from Oxford Genetics,

upstream of a GFP. The size of the fragment was 304 bp.

Digestion with adequate restriction enzymes was carried out

in both the insert and plasmid, followed by gel-extraction

and DNA purification, for posterior ligation of the BBr_0838

promoter into the OG241 backbone, resulting in the

BBr0838-OG241 reporter plasmid. This plasmid was

transformed in E. coli DH5α, and colonies were picked and

grown overnight for further plasmid purification. The

BBr0838-OG241 reporter plasmid was then transformed into

E. coli NISSLE 1917, and its identity confirmed by Sanger

sequencing (service was provided by Source BioScience).

Fluorescence analysis

In order to analyse the inducibility of the system, DH5α cells

bearing the BBr0838-OG241 plasmid were grown in 1ml LB

medium shaking at 37 ºC. Plasmid-free DH5α and DH5α cells

carrying insert-free OG241 were used as a negative controls.

When mid-exponential phase was reached, cultures were

induced with either 50μM deoxycholic acid or 0.025%

cholate. They were then incubated for another 7 hours

shaking at 37 ºC. 200 μL of each culture were then

centrifuged at 10000rpm for 2 minutes, and the supernatant

discarded. The cells were then resuspended in 400 μL PBS.

The fluorescence intensity of the samples was measured in

an AccuriTM

C6 flow cytometer. The fluorescence data from

this assay was processed using the R programming language.

RESULTS AND DISCUSSION

Construction of both DH5α and NISSLE1917 bile-acid

reporting strains was successful, as was verified by Sanger

sequencing. However, the inducibility assay yielded

unexpected results. While in the presence of deoxycholic

acid and cholate the construct shows strong expression, in

the order of 10-fold, this is also the case in the absence of

bile acids. The construct presented constitutive expression

and hence lacked the bile-acid sensing capacity (Figure 1).

Previous characterisation of this promoter by Ruiz et al. used

a similar genetic construct, but employed B. breve (the

species that originally hosts this promoter in its genome) as

a chassis instead of E. coli. The constitutive nature of the

promoter led us to think that our system might be missing a

repressor which was present in the native strain.

Bioinformatic analysis of the region upstream of the

promoter revealed the presence of a hypothetical protein

BBr_0834 (ABE95519.1), which has not been characterised

but is predicted to contain an AAA+ ATPase domain. This

type of protein has been previously shown to be involved in

switching between different states of gene expression in

yeast10

. BBr_0834 could potentially be involved in re-

structuring of the chromatin, with a resulting halt in

expression, or in inhibition of another activator. The precise

mechanisms of regulation by the BBr_0838 promoter have

not yet been fully characterised, and further research would

be required for determining whether this protein effectively

plays a role in bile acid induction and how bile acids would

contribute to relieving the repression. A simple ODE model

was constructed in Matlab to confirm that the presence of a

repressor would indeed make the system inducible.

DEVIATIONS FROM ORIGINAL PROPOSAL

The unexpected results with regards to the bile acid sensor,

and the unfeasibility of troubleshooting the system in terms

of available time due to the wider scope of this new project,

drove me to start working on a different acid-sensor which

was of interest to the group. This sensor, constituted by the

cadBA promoter region, is induced in conditions of acidic

pH, and its strength is enhanced by anaerobiosis11

. The

fragment was amplified by PCR from genomic DNA, and

cloned into OG241 using the techniques described above.

DH5α cells were grown in LB and diluted in fresh minimal

medium, previously adjusted to pH 5.8 or pH 7.6 and

appropriately buffered. Unfortunately, fluorescence analysis

did not show induction of gene expression in acid stress

conditions and hence this reporter requires further

troubleshooting.

FUTURE DIRECTIONS

The precise molecular mechanisms of bile-acid induction of

the BBr_0838 promoter remain to be elucidated, and the

potential effect of hypothetical repressor BBr_0834 on gene

expression could constitute a starting point for this

investigation. If the inclusion of this protein in the system

resulted in a functional bile acid inducible reporter

construct, more data would need to be gathered towards

estimation of the crucial parameter values for the

mathematical model. Ultimately, the bile-acid sensing

NISSLE1917 strain could be employed in in-vivo studies of

bile-acid reporting in mice, as a step towards its

implementation in humans for diagnostic and therapeutic

purposes.

VALUE OF THE STUDENTSHIP

To the student:

This has been a great opportunity for me to get more

familiar not only with molecular biology techniques, but also

with the design and planning of a project from beginning to

end. It was also confirmation that in science things don’t

always go as expected, and that the paths towards testing

and confirming a hypothesis are often more intricate and

tortuous than they might seem in the first place. But

perhaps most importantly, and although this is not

extensively reflected in the results, it has introduced me to

the field of computational modelling and systems biology,

which seem like invaluable tools for making biology more

reliable, predictable and reproducible, all mandatory

requisites in the construction of new synthetic genetic

devices and systems. After this experience it is clearer to me

that I would like to pursue a career in this area, and play an

active part in the synthetic biology era.

To the supervisor:

Daniel has done very useful work in his initial

characterisation of a potential bile acid reporter construct

for eventual use in in vivo experiments. While it is

unfortunate that the reporter system didn’t work first time,

Daniel's excellent and meticulous work has given us useful

additional insights into the complexities in the problem and

suggested potential avenues for further investigation and

possible solutions. In addition Daniel has helped to further

establish our techniques and procedures for working

successfully in commensal strains of E.coli suitable for our

future investigations.

REFERENCES

1. Consortium, T. H. M. P. Structure, function and diversity of the

healthy human microbiome. Nature 486, 207–214 (2012).

2. Qin, J. et al. A human gut microbial gene catalogue established by

metagenomic sequencing. Nature 464, 59–65 (2010).

3. Duan, F. & March, J. C. Engineered bacterial communication

prevents Vibrio cholerae virulence in an infant mouse model. Proc.

Natl. Acad. Sci. 107, 11260–11264 (2010).

4. Duan, F., Curtis, K. L. & March, J. C. Secretion of Insulinotropic

Proteins by Commensal Bacteria: Rewiring the Gut To Treat

Diabetes. Appl. Environ. Microbiol. 74, 7437–7438 (2008).

5. Chen, Z. et al. Incorporation of therapeutically modified bacteria

into gut microbiota inhibits obesity. J. Clin. Invest. (2014).

doi:10.1172/JCI72517

6. Buffie, C. G. et al. Precision microbiome reconstitution restores bile

acid mediated resistance to Clostridium difficile. Nature 517, 205–208

(2015).7. Jones, M. L., Martoni, C. J., Ganopolsky, J. G., Labbé, A. &

Prakash, S. The human microbiome and bile acid metabolism:

dysbiosis, dysmetabolism, disease and intervention. Expert Opin.

Biol. Ther. 14, 467–482 (2014).

8. Bernstein, H., Bernstein, C., Payne, C. M., Dvorakova, K. &

Garewal, H. Bile acids as carcinogens in human gastrointestinal

cancers. Mutat. Res. 589, 47–65 (2005).

9. Ruiz, L. et al. A bile-inducible membrane protein mediates

bifidobacterial bile resistance. Microb. Biotechnol. 5, 523–535 (2012)

10. Wilcox, A. J., Laney, J. D. A ubiquitin-selective AAA-ATPase

mediates transcriptional switching by remodelling a repressor-

promoter DNA complex. Nat. Cell. Biol. 11, 1481-86 (2009).

Figure 1. Constitutive GFP expression in the BBr0838-

OG241 construct. For both 0.025% cholate (A) and 50

uM deoxycholate (B), the top plot represents the no-

promoter control, and the bottom plot represents the

construct including the BBr0838 promoter. The plots

were obtained using the R programming language; the

code extracts medians for the values and then

calculates means and standard deviations, fits the

parameters to a Hill function and then plots the density

on the ‘y’ axis against the fluorescence on the ‘x’ axis.

11. Cüper, C., Jung, K. CadC-mediated activation of the cadBA

promoter in Escherichia coli. Journal of Molecular Microbiology and

Biotechnology 10, 26-39 (2006)

Developing a method to detect cysteine oxidation of the glucose-sensor enzyme glucokinase Name: Daniel O. Fajonyomi.

Supervisor: Dr Catherine Arden, Institute of Cellular Medicine, Newcastle University.

Glucokinase (GK) catalyses the first step of glucose metabolism, i ts phosphorylation to glucose 6-phosphate, and

serves as the glucose sensor for glucose-stimulated insulin secretion in tissues with a major role in blood glucose

homeostasis including the pancreatic beta-cells. The activity, s tability and/or expression of GK is regulated by a number of

post-translational modifications including oxidation of its cysteine residues which inhibits enzyme activity. Susceptibility of

GK to oxidation is a predisposing factor to the instability of several GK-MODY mutants that cause diabetes in man and

endogenous GK is inactivated by elevated oxidative s tress, this supports a model whereby endogenous GK activity i s

inhibited by thiol oxidation in conditions of anti-oxidant depletion and/or reactive oxygen species production which is

particularly relevant given that increased oxidative stress is assumed to be a critical component in the impairment of beta -cel l function during the development of type 2 diabetes. Whether modulation of GK activi ty/stability by changes in its thiol

s tatus plays a role in the development of type 2 diabetes remains to be determined.

The aim of this project:

The a im of this research project was to develop the Purification of Reversibly Oxidised Proteins ( PROP) method

to detect oxidation of glucokinase (GK) in pancreatic beta-cell extracts, in order to achieve this the research project

involved three sets of experiments which are: I) Optimisation of the PROP technique with GK IP using recombinant proteins

II) Application of the PROP technique to beta-cell extracts treated with oxidising agents III) Application of the PROP

technique to beta-cells exposed to increased oxidative s tress.

A description of the work carried out (Methods & Results):

The PROP method involves four major steps outlined below:

1. Samples treated with, and without, alloxan (which oxidises cysteine residues on glucokinase) are denatured and

treated with NEM which irreversibly blocks reduced thiols on glucokinase.

2. NEM is removed by protein precipitation and DTT is then used to reduce the thiols oxidised by a lloxan.

3. DTT is removed and the newly-reduced thiols are then tagged with biotin.

4. Total Glucokinase protein is immunoprecipitated using a GK antibody and the unbound and boun d (IP) fractions

are resolved using western blotting.

Figure 1: Outl ine of PROP method.

Figure 2: Outl ine of GK immunoprecipitation technique.

Typica lly two gels are prepared for SDS-PAGE resolution of both the control and alloxan samples, and both samples

are ran on each gel in order to obtain two separate PVDF blots. One blot i s blocked in 5% mi lk and then incubated with a

primary antibody and then Rabbit-HRP (secondary antibody) to determine the total amount of glucokinase in each sample,

whi le the other blot is blocked in 1% BSA and then incubated with Streptavidin-HRP which binds to biotin a llowing

visualisation of the level of oxidation in both samples.

Optimisation of the PROP technique with GK IP using recombinant proteins: In these experiments recombinant GST tagged

glucokinase and cleaved glucokinase (GK without the GST-tag), both of which are isolated from E.coli, were used as the

glucokinase sources. In the first e xperiment recombinant GST-GK protein samples were treated with, and without (control sample), alloxan and the PROP method (without immunoprecipitation) was used to detect oxidation. Using this approach, I

was able to demonstrate that treatment of GST-GK with alloxan caused oxidation of glucokinase as evident from an

increase in the HRP-streptavidin signal, indicating an increase in biotin-tagging and thus oxidation.

I then repeated this experiment using recombinant glucokinase protein from which the GST-tag had been removed (cleaved GK) and achieved similar results which confirmed that the increase in the Strep-HRP signal from previous

experiments was due to the oxidation of glucokinase and not the GST-tag.

Since the aim of the project is to develop a method to detect the oxidation of glucokinase within cell lysates, i t is

essential to isolate GK from other unwanted proteins using immunoprecipitation. We therefore confirmed that the IP

protocol did not alter the biotin-tagging by incorporating GK immunoprecipitation into the protocol using recombinant GK

sources. I was able to show that total GK could be immunoprecipitated successfully with the current protocol and produce

results similar to those obtained without the IP protocol.

Appl ication of the PROP technique to beta-cell extracts treated with oxidising agents: The second set of experiments aimed

to successfully apply the PROP method to the detection of GK oxidation in cell extracts from INS1E beta-cell lines treated

with a lloxan, two experiments were done towards this aim. First we confirmed that we could IP GK from cell lysates by

carrying out the IP protocol as described above using cell extracts and were able to show that total GK can be

immunoprecipitated from beta-cell extracts successfully.

After successfully immunoprecipitating total GK from cell extracts, we determined whether we could detect

oxidation of glucokinase from cell lysates using PROP followed by IP. However, when PROP (with alloxan oxidation) and IP

were included in the protocol, the IP and unbound fractions could n ot be visualised on the total GK and Streptavidin-HRP blots. It is believed that this was due to a loss of protein during the protein precipitation s teps in the PROP method and

therefore optimisation of the protein precipitation steps would be required to achieve a successful experiment.

Deviations from original proposal:

Due to time constraints it was not possible to begin the last phase of experiments (Application of the PROP

technique to beta-cells exposed to increased oxidative s tress).

Future directions in which the project can be taken:

As indicated previously, there is a need to optimise protein precipitation to ensure that sufficient protein is

reta ined to a llow for immunoprecipitation. Once the technique is able to detect glucokinase oxidation in this artificial

system, the technique can then be used to determine whether glucokinase is oxidised under conditions related to type 2 diabetes which will include increased oxidative s tress and a lso nutrient excess (glucolipotoxicity). This will eventually lead

to the application of the technique to detect glucokinase oxidation in animal models of diabetes and potentially human

tissue from type 2 diabetic patients.

Value of the studentship to the student:

This research placement with Dr Arden and her team have truly been invaluable, during this 8-week period I feel I

have not only gained insight and appreciation for research but greater interest in the work being done in diabetes research

through seminars and presentations I was given the opportunity to attend and interactions with other researchers in the

lab. In addition I have also had the opportunity to learn new lab techniques, use techniques I was a lready familiar to new

problems and gain lab skills which I believe would be incredibly useful during my final year lab-based research project. All

of this was made possible by Dr Arden and her team and the grant provided by the biochemical society.

Value of the studentship to the lab: It was a pleasure to have Daniel in the lab. He quickly adjusted to the laboratory environment and interacted well

with s tudents and research staff. During his time, he gained experience in techniques such as immunoprecipitation, cell

lys is and western blotting. The data he has generated will be used for future grant applications to expand on this project.

Figure 1: Cas1 homodimeric structure showing canonical

nuclease active site

UNIVERSITY OF ST ANDREWS

BIOMEDICAL SCIENCES RESEARCH COMPLEX

BIOCHEMICAL STUDENTSHIP 2015 ELYSE FISCHER

CASPOSON CAS1 – PROBING THE BASIS FOR CRISPR ADAPTIVE IMMUNITY

Supervisor: Professor Malcolm White

Daily Supervision: Shirley Graham and Clare Rollie

Background CRISPR-Cas is a prokaryotic adaptive immune system which provides protection against invading genetic elements through homology-directed detection and destruction. The CRISPR loci comprises of a leader sequence followed by an array of short palindromic repeats interspersed by small “spacer” sequences. As well as a variety of CRISPR associated (Cas) genes. Spacers, captured from viruses, are integrated in the host genome and provide a “memory” of past infections. Upon reinfection, the CRISPR locus is transcribed generating pre-CRISPR RNA (pre-crRNA). This transcript, subsequently processed and loaded into “Interference” or “Effector” ribonucleoprotein complexes, utilizes the crRNA to detect and degrade previously encountered foreign entities (1). This method of capturing foreign DNA and integrating it into the host genome is known as Adaptation, and requires Cas1 and Cas2 (2). Cas1, a homodimer, is believed to act as a metal-dependent nuclease (figure 1), cleaving the CRISPR locus specifically at the integration site to allow spacer addition (3). Cas1 is reported to preferentially cleave branched DNA substrates, particularly ones which model replication forks (4). Recently, a group of Cas1 orthologues called “Casposons” has been identified from a family of transposons (5). These Casposons are believe to play a role in transposon integration and excision and may underlie the evolution of the CRISPR system.

Project Aims The project aimed to express and purify a Casposon-derived Cas1 using a commercial synthetic gene. Furthermore, to determine specificity and reaction mechanism of the Casposon Cas1 against model DNA substrates through biochemical assays.

Experimental Procedure and Results Cloning and Expression of Casposon Cas1

Initial cloning experiments and subsequent troubleshooting failed to insert the MmaCas1 synthetic gene into a standard pHisTEV expression vector. Five new vectors were trialled and successful ligation was achieved in three separate vectors. All three clones were transformed into C43 expression cells, and induce with Isopropyl β-D-1-thiogalacto-pyranoside (IPTG). Optimal temperature and growth time for Casposon Cas1 expression was determined using mini expression trials in a BioSprint machine and analyzed via SDS-page (Figure 2A). Two expression conditions were chosen with promising protein solubility, Cas1-pET17b induced at 16C overnight, and Cas1-pHisTEV induced at 37C for 4 hours. However, expression did not follow mini trials when large sale cultures were induced and a large percentage of the total protein was insoluble. Despite this, purification was still continued.

Figure 2: Top Left: Polyacrylamide gel showing Cas1-pET17b mini expression trials. IPTG induction at 16C, 25C, 37C and at 4hours and overnight. Top right: HisTrap Column FF chromatograph of Cas1-pET17b induced IPTG at 16C overnight. Bottom left: Subsequent S200 gel filtration chromatograph.

Bottom right: SDS-page analysis of Cas1-pET17b sample post gel filtration. First well following the protein ladder contains pre-gel filtration sample, remaining wells contain fractions taken from post-gel filtration.

Purification of Casposon Cas1

Protein purification of Cas1-pET17b was completed using a TEV-protease cleavable polyhistidine tag on the C-terminus of the protein. Supernatant prepped in Tris was passed through a HisTrap FF column for metal affinity chromatography using an AKTA purification system and eluted using a high concentration of competitive imidazole (Figure 2B). The polyhistidine was then cleaved off using TEV protease and a second nickel column was run. A final polishing step of size exclusion chromatography using gel filtration was completed (Figure2C). SDS-page was used to analyze fractions from all steps (Figure2D). Several complications, including Cas1 precipitation after TEV cleavage and concentrating, resulted in only 400ul at .535mg/ml of pure protein being obtained. A second round of Casposon Cas1 purification was completed. Two different vectors were tested under different expression and purification conditions in an effort to reduce the amount of protein precipitation and aggregation. Most notably, the polyhistidine tag was not cleaved by TEV protease and purification buffers with a lower pH of 6.5 were used. Despite the changes made, final results did not fare much better. Nevertheless plenty of protein was obtained in the three batches to test Casposon Cas1 activity in biochemical assays.

Substrate Binding and Cleavage Assays

Four DNA oligonucleotide substrates were designed (Figure4) to test the ability of Casposon Cas1 to catalyse trans-esterification on branched DNA substrates. Polyacrylamide electro-mobility shift assays (EMSA) were used for analysis. The four substrates included ssDNA, dsDNA, ssDNA flap, and a 3-way-junction. Substrates were assembled from oligonucleotides by hybridization and gel purification, and fluorescently labelled with 6-flourescein amidite (FAM) for visualization. Casposon Cas1 was found to strongly bind all four substrates, with the strongest preference for the ssDNA and ssDNA flap substrates. This corresponds with results seen for the E.coli Cas1 previously tested in the lab. However, cleavage assays showed no activity for Casposon Cas1 in any of the substrates, compared to the E.coli Cas1 control, which showed non-specific cleavage of ssDNA, and specific cleavage of the 5’-ssDNA flap substrate at the branch site.

Future Directions Future work is needed to acquire more pure protein, especially in order to achieve enough for crystallization trials. This would include optimizing expression conditions to obtain better solubility and refining purification conditions to limit the amount of protein precipitation which occurs. Furthermore, much of the mechanism and activity of Casposon Cas1 still eludes us, therefore further biochemical assays could be completed on a larger variety of substrates. Casposon Cas1 was not found to contain any cleavage activity in my project. I plan to return later in the semester to complete a trypsin digest of Casposon Cas1. The hope is that this will clarify whether the protein was properly folded despite containing an extra helix-turn-helix domain, and possibly allow removal of the extra domain, in an effort to increase its cleavage activity.

Departures from Original Proposal Due to time constraints caused by complications in cloning and purification further biochemical activity assays were not completed. Only four of 10-12 originally designed substrates were tested.

Value of Studentship My lab experience this summer was absolutely phenomenal in all aspects. By my own choice, I prolonged my project for an extra 4 weeks because I did not want to stop. It cemented my resolve to pursue a PhD following my undergraduate studies and increased my confidence and perseverance as a scientist. I learned and refined invaluable experimental techniques including cell transformation, protein expression and purification, DNA substrate construction, and fluorescent imaging. All which will be extremely helpful in preparing me for not only my graduate studies but also my dissertation research starting this October, completing structure studies on the MscL mechanosensitive channel with Professor James Naismith. My experience was very cooperative but also exceedingly independent. In some ways I am glad many steps of my project did not work on first try. Obtaining disappointing results not only forced me to troubleshoot and gain more experience redoing steps, but also taught me to never be disheartened when things don’t go how you plan. My work was also highly beneficial to my lab. Cas1 Casposon had never been worked on before so my project built a foundation of knowledge to which others may use to carry on further work in the area.

References 1) Reeks, et al. (2013) Biochem. J., 453, 155-166. 2) Makarova, et al. (2006) Biol Direct, 1, 7. 3) Wiedenheft, et al. (2009) Structure, 17, 904-912.

4) Babu, et al. (2011) Mol. Microbiol., 79, 484-502. 5) Krupovic et al. (2014) BMC Biol. 12:36

Figure 3: Hybridized DNA oligonucleotide substrates used to test Casposon Cas1 activity. From left to right: ssDNA,

dsDNA, ssDNA flap, 3-way junction. The X50 blue strand was fluorescently labelled with FAM (yellow dot).

A Role of SH3BP1 in Spindle Assembly Frankie Butera

My placement took place in Dr Chris Bakal’s Dynamic Cell Systems laboratory at the Institute of Cancer

Research (ICR), under the direct supervision of Dr Alexis Barr. Over eight weeks I investigated the assembly of bipolar spindles, which are critical for the correct segregation of chromosomes during mitosis. chTOG is a gene with a major role in spindle pole organisation and microtubule stabilisation (Gergely et al., 2003). Depletion of chTOG results in a multipolar spindle phenotype in a number of cell lines, including HeLa cells (Barr & Bakal, 2015). In an image-based double RNAi screen of a RhoGEF/GAP library, the gene SH3BP1 was identified to genetically interact with chTOG (unpublished data). SH3BP1 has GAP activity and employs this to regulate a number of cellular processes, including activation of Cdc42 in junction assembly (Elbediwy et al., 2012) and inactivation of Rac1 at the front of cells to drive motility (Parrini et al., 2011). This project aimed to validate the screen result using independent siRNAs and ultimately determine if SH3BP1 has a role in spindle assembly. My original proposal had involved looking at p21 dynamics during the G1/S transition in human cells, however, this had already been achieved before I began my placement.

Procedures and Results Throughout the studentship, I maintained two HeLa cell lines at 37°C in DMEM. One cell line had chTOG

shRNA that was created using a Doxycycline-inducible shRNA system, whereby addition of Doxycycline stimulates the expression of chTOG shRNA and RFP mRNA. RFP expression was later used to select cells expressing the shRNA. The other cell line was created using the same system but with non-silencing (NS) shRNA.

I investigated whether co-depletion of chTOG and SH3BP1 has an effect on spindle morphology by carrying out a reverse transfection of the two cell lines with SH3BP1 siRNA. As a negative control, non-targeting siRNA was used. Co-depletion of chTOG and TUBG1 is known to rescue a bipolar phenotype, and so TUBG1 siRNA was used as a positive control to verify that the reverse transfection worked. Four different SH3BP1 siRNAs were used independently to ensure that the phenotype observed was not due to off-target effects, and a pool of these four SH3BP1 siRNAs was also used. Prior to reverse transfection, I carried out a real-time PCR to check that the siRNAs effectively deplete the SH3BP1 mRNA. Figure 1 shows that the siRNAs have varying efficiencies, with the SH3BP1 pool, SH3BP1 10 and SH3BP1 12 having significant reduction in SH3BP1 mRNA.

For the reverse transfection, the control and SH3BP1 siRNAs were plated onto a 384-well plate and Lipofectamine RNAiMAX (a transfection reagent), 2000 cells, and Doxycycline were added to each well. Prior to fixing, MG132 was added to each well in order to arrest the cells in metaphase. The cells were fixed with formaldehyde and permeabilised with TritonX-100. I stained with rat anti-alpha-tubulin to visualise the mitotic spindles and rabbit anti-PHH3 to help mark mitotic cells, as serine-10 and serine-28 of histone H3 is only phosphorylated during mitosis. I also stained with Hoescht (DNA stain) to select and segment all nuclei. Images were then taken of each well using the

Figure 1: fold-changes (2-ΔΔCt) from quantitative

PCR, normalised to a housekeeping gene (GAPDH) and adjusted to the control cDNA (TOG con) set to

1.0.

Perkin Elmer Opera high-throughput confocal microscope, and the images were uploaded to Columbus, an image analysis system for automated phenotypic screening. I customised and ran a script on Columbus to select the mitotic nuclei based on cell roundness, tubulin intensity, and PHH3 intensity. The script also removed any nuclei on the border of an image in case these had extra poles outside of the image. The final steps of the script counted the number of spindle poles in each cell using a spot finder function, and classified the spindles with more than two poles as ‘multipolar’.

Figure 2: reverse transfection of HeLa cells results in a rescue of bipolar spindles in cells. (a) cells with typical bipolar spindles, (b)

cells with typical multipolar spindles (images from Perkin Elmer Opera with tubulin staining visualised). (c) mean % cells with multipolar spindles, calculated from six technical repeats for each experiment, for each shRNA and siRNA combination (calculated

using Columbus software). Each square represents one biological repeat.

This experiment was repeated twice and the results from image analysis are shown in Figure 2c. When chTOG is depleted, the percentage of cells with multipolar spindles increases. When comparing the control siRNA treatment and the SH3BP1 siRNA treatments, there is a reduction in the percentage of multipolar spindles when chTOG and SH3BP1 are co-depleted, thus rescuing a bipolar spindle phenotype. This suggests that SH3BP1 has a genetic interaction with chTOG and plays a role in spindle assembly.

Future of this project Depletion of SH3BP1 protein by siRNA will be checked using a Western blot. Furthermore, we will verify

that SH3BP1 depletion is specifically causing this rescue of bipolar spindles in chTOG-depleted cells by co-transfecting the SH3BP1 cDNA that is resistant to siRNA into these depleted cells and seeing if this prevents the rescue in phenotype. We can also check if the GAP activity of SH3BP1 is relevant by adding SH3BP1 cDNA with defect GAP activity.

Value of the Studentship This studentship has enabled me to explore biology from a perspective beyond my undergraduate degree.

I have been able to gain an honest experience of laboratory research and, as a result, am more prepared and determined for a career in cell biology research. Moreover, I have learnt many protocols key for this kind of research, including transfection, quantitative PCR and Western blot, and I now understand how to use these experiments to test original hypotheses about cellular mechanisms. Finally, during my time at the ICR I have discovered areas of cancer biology that I may decide to specialise within in the future, such as cancer and metabolism, and this will be invaluable when applying for a PhD project.

References Barr, A. R. & Bakal, C. (2015) A sensitised RNAi screen reveals a ch -TOG genetic interaction network required for spindle assembly. Scientific Reports. 5.

Elbediwy, A., Zihni, C., Terry, S. J., Clark, P., Matter, K. & Balda, M. S. (2012) Epithelial junction formation requires con finement of

Cdc42 activity by a novel SH3BP1 complex. The Journal of Cell Biology. 198 (4), 677-693.

Gergely, F., Draviam, V. M. & Raff, J. W. (2003) The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells. Genes & Development. 17 (3), 336-341.

Parrini, M. C., Sadou-Dubourgnoux, A., Aoki, K., Kunida, K., Biondini, M., Hatzoglou, A., Poullet, P., Formstecher, E., Yeaman, C. & Matsuda, M. (2011) SH3BP1, an exocyst-associated RhoGAP, inactivates Rac1 at the front to drive cell motility. Molecular Cell. 42 (5), 650-661.

1

Investigating plant calcium response in plant-aphid

interactions

Student: James Canham

Supervisor: Dr Tony Miller

John Innes Centre, Norwich Research Park, Norwich, NR4 7UH. UK.

Background and Aims

The phloem-feeding green peach aphid, Myzus persicae, is a prolific pest to a number of agriculturally

important crops and is responsible for substantial crop losses globally. The plant-aphid interaction remains

poorly understood and the reasons behind the success of M. persicae are not fully elucidated1. During an

incompatible interaction between plant and aphid, pattern recognition receptors (PRRs) specifically bind

herbivore-associated molecular patterns (HAMPs) triggering cell signalling and plant defence responses2. The

early dynamics of this signal is unknown but Ca2+

and reactive oxygen species (ROS) bursts have been

implicated. M. persicae can supress plant signalling, overcoming the plant defence response using effector

molecules such as Mp103.

E Ca2+

-interacting vidence for a central role of a kinase, CIPK3.2 in the plant-aphid interaction was identified

knock out (KO) during RNA-seq experiments. This led to the requirement for CIPK3 mutants in Arabidopsis for

An aim of this project was determine whether the CIPK3 phenotype is Arabidopsis further investigation. KO

ecotype specific by replicating published results in the Ws mutant (cipk3-1) and screening new CIPK3 T-DNA

insertion lines in the Col background. M. persicae feeds and successfully reproduces on Col-0 and Ws

Arabidopsis ecotypes.

To measure the plant response during an aphid interaction, a Ca2+

reporter, GCaMP6 has been genetically

engineered in A. thaliana plants. The potential role of the vacuolar two pore channel 1 (TPC1) was investigated

and the aphid effector Mp10 was knocked-down using RNAi technology to discover its effect on Ca2+

bursts

during the interaction.

Work carried out

Extraction and purification of plant and aphid DNA and RNA was carried out in order to perform analytical PCR

and RT-PCR techniques. Germination assays were performed to test for cipk3-1 KO osmotic stress germination

phenotype using 150 mM NaCl ¼ strength MS media and stereo microscopy to screen the seedlings.

Fluorescence stereo microscopy was used to image Ca2+

real-time in GCaMP6 reporter plants during the

Arabidopsis-M. persicae interaction. Image frames were analysed for their fluorescence intensity using ImageJ

and data was investigated using the statistical package, GenStat. Col-0 and TPC1 KO plants (tpc1-2) were

imaged and compared to assess the plant dependence on the vacuolar Ca2+

amplifier TPC1 during the

interaction. Aphid populations were cultured on dsMp10 plants in order to knockdown the putative effector,

Mp10, using RNAi technology. The aphids were tested for the knocked-down effector by qPCR. The Mp10

knockdown aphids were exposed to Col-0 reporter plants to test the effect of Mp10 on the Ca2+

burst during

the interaction.

Results and discussion

CIPK3 KO osmotic stress germination phenotype is Arabidopsis ecotype dependent

2

Successful replication of the CIPK3 osmotic stress germination phenotype was achieved in the Ws background,

but CIPK3 KO lines in the Col background failed to exhibit a similar phenotype

(Fig. 1)4.

Figure 1. Germination of CIPK3 KO seed is

sensitive to osmotic stress conditions in the Ws

background but not in the Col background.

These data show that the Arabidopsis CIPK3 KO germination phenotype is significantly reduced in 150mM

NaCl relative to Ws-0, but the same osmotic stress phenotype was not replicated in the Col-0 background.

M. persicae induces a localised but not systemic Ca2+

burst in Arabidopsis during a plant-aphid

interaction which is dependent on TPC1

GCaM6 Ca2+

reporter plants were imaged during an aphid interaction and frames were analysed for changes in

fluorescence intensity using ImageJ (Fig. 2).

Figure 2. Fluorescence intensity within a specified

region of interest (ROI) of imaged GCaMP6 Col-0

and tpc1-2 reporter plants. (A) Col-0 aphid vs. no

aphid samples at a systemic ROI. (B) Col-0 aphid

vs. no aphid samples at the feeding ROI. (C) Col-0

vs. tpc1-2 control samples at feeding ROI. (D) Col-0

vs. tpc1-2 aphid exposed samples at feeding ROI.

The marker line on the x-axis (t=0) represents aphid

settling to feed. Shaded region represents a

significant different between treatments (p<0.05).

A significant Ca2+

burst was recorded between 2-24 min in Arabidopsis Col-0 reporter plants at a localised site

around the area of aphid feeding during the plant-aphid interaction. No changes in fluorescence intensity

between aphid interaction and control leaves at the systemic site suggests the Ca2+

burst is not systemic in

nature. Ca2+

bursts in TPC1 KO reporter plants appears to be reduced relative to the Col-0 control.

Mp10 knockdown M. persicae may be less capable of supressing the Ca2+

burst in Arabidopsis

M. persicae raised on dsMp10 containing Arabidopsis show a 30-60% knockdown in the aphid effector Mp10.

During the aphid-plant interaction, Mp10 knockdown M. persicae show reduced suppression of the Ca2+

burst

relative to control, dsGFP raised M. persicae (Fig. 3).

Figure 3. dsMp10 knockdown M. persicae feeding on Arabidopsis GCaMP Ca2+

reporter plants. dsGFP raised M.

persicae are control. Shaded areas represent significant increases in fluorescence intensity (p<0.05).

0 min 1 min 2 min 3 min

4 min 6 min 5 min 7 min

3

Ca2+

bursts are induced in Arabidopsis in response to aphid interactions. This signal is limited to the site of

stylet penetration and is not systemically propagated. The Ca2+

response may be TPC1 dependent and further

evidence that Mp10 acts to supress Ca2+

is presented.

Future directions

Repetition of imaging data is necessary and further investigation into candidate receptors is required to

confirm the aphid role in inducing Ca2+

bursts. Comparisons between Ca2+

signals during non-compatible and

compatible interactions can be assessed using non-host aphid species.

References [1] Bass, C., Puinean, A. M., Zimmer, C. T., Denholm, I., Field, L. M., Foster, S. P., Gutbrod, O., Nauen, R., Slater, R. & Williamson, M. S.,

(2014). The evolution of insecticide resistance in the peach potato aphid, Myzus persicae. Insect. Biochem. & Mol. Biol., 51, 41–51

[2] Hogenhout, S. A. & Bos, J. I. B., (2011). Effector proteins that modulate plant-aphid interactions. Cur. Opin. in Plant Biol., 14, 422–42.

[3] Pitino, M. & Hogenhout, S. A., (2013). Aphid protein effectors promote aphid colonization in a plant species-specific manner. MPMI,

26(1) 130–139.

[4] Kyung-Nam Kim, K., Cheong, Y. H., Grant, J. J. & Pandley, G. K., (2003). CIPK3, a Calcium Sensor–Associated Protein Kinase That

Regulates Abscisic Acid and Cold Signal Transduction in Arabidopsis. Plant Cell, 13, 411-423.

Grant impact (self)

The positive impact of the Biochemistry society summer studentship on my enthusiasm for research and

ambition to pursue a career in scientific research has been immeasurable. I feel extremely privileged to have

had the opportunity to conduct such work this summer and I am determined to build on this experience.

Value of the studentship (Dr Tony Miller)

The important data obtained by James has helped to uncover the role of several important components of the

plant-aphid Ca2+

signalling system. His work has demonstrated the role of aphid effector molecule, Mp10 in

suppressing plant defence responses. On the plant side, he has helped to demonstrate a clear role for the

vacuolar channel TPC1 in aphid-elicited Ca2+

signals. The role of the plant Ca2+

-interacting kinase, CIPK3 is

these responses is less clear. His work has provided underpinning information for a grant proposal to

investigate these components in more detail.

http://www.sciencedirect.com/science/article/pii/S0965174814000794










Background: Duchenne muscular dystrophy (DMD) is part of the muscular dystrophies group of inherited disorders, which are characterised by progressive muscle tissue wasting and its replacement with fibrotic tissue. In DMD, this leads to the gradual loss of strength and mobility, leading to severe disability and eventual death from cardiac and respiratory failure. There are currently several therapeutic approaches in trial to address the genetic basis of DMD. However, fibrosis is a major challenge for these strategies because the development of fibrotic tissue precludes a complete restoration of muscular function. As a result, an additional strategy must be developed to address the fibrotic problem.

Fibrogenesis involves different cellular and molecular elements. One is connective tissue growth factor (CTGF), which acts downstream of transforming growth factor type-β 1 (TGFβ1), which is associated with the induction and progression of virtually all fibrotic remodelling in different biological systems, including skeletal muscle. CTGF is thus a potential target for strategies seeking to address fibrosis in DMD. It has been demonstrated that treatment with an anti-CTGF monoclonal antibody can reduce fibrosis in the mdx mouse model of DMD (Morales et al. 2013). In addition, antisense oligonucleotides (AOs) have been used to inhibit CTGF expression and limit fibrosis in other biological systems (Sisco et al. 2008). However, the potential of AOs to address fibrosis in muscle has not as yet been investigated.

Aim: To investigate the potential of AOs to knockdown CTGF expression in myoblasts by disrupting the CTGF transcript reading frame via the induction of exon skipping. To evaluate this in vitro in cultured murine C2C12 myoblasts at the transcript and protein level through nested RT-PCR and Western blot analyses.

Description of work: Human and mouse CTGF transcript sequences were interrogated in silico to identify exonic splicing enhancer (ESE) sequence motifs within out-of-frame exons. A series of 25mer AOs with phosphorodiamidate morpholino oligomer (PMO) chemistry with high theoretical binding energies and potential invasive nature were designed to target relevant ESEs. Designed AOs were tested in vitro in murine C2C12 myoblasts grown at 37°C, 5% CO2 in DMEM supplemented with GlutaMax, 4.5g/L D glucose, pyruvate, 10% fetal calf serum and 1% penicillin/streptomycin.

The effect of TGFβ1 on CTGF transcription over time (0, 1, 3, 6, 9 and 24 hours) and at different concentrations (0, 5, 10, 15 or 20 ng/ml TGFβ) was evaluated by RT PCR analysis of harvested RNA. To assess the effectiveness of designed AOs to knockdown CTGF expression at the transcript level, a dose response (2, 5 or 10 μM) was carried out, with and without TGFβ1 (10 ng/ml). RNA was harvested at 24 hours or at indicated timepoints using QIAshredder and RNeasy mini kits from QIAGEN. 500 ng of purified RNA from each sample was amplified using the GeneScript RT-PCR System (Quantig). 2 μl of first round product was further amplified using Taq polymerase mastermix (Quantig). 10 μl of second round product was run on 2% agarose gels against Hyperladder IV (Bioline) with 1x SYBRSafe DNA gel stain in TBE. Densitometric analysis of band intensity on gel images was undertaken using GeneTools software from Syngene.

The capability of the optimal AO (at 10 µM) to knockdown CTGF protein expression both with and without TGFβ1 (10 ng/ml) was assessed. Protein was harvested following 24 hours incubation and the protein concentration of the cell lysate determined by DC assay. Following denaturation, 50 μg of protein from each sample was run on a 3-8% Tris Acetate NuPage gel and then transferred to nitrocellulose membrane. The membrane was blocked with 4% BSA in 1x PBS-T and then incubated overnight with 1:10000 mouse anti-vinculin (Sigma SAB4200080) and 1:1000 rabbit anti-CTGF (abcam ab6992) primary antibodies. After repeated washing in 1x PBS-T, the membrane was incubated with 1:10000 goat anti-mouse IRDye800CW and donkey anti-rabbit IRDye680LT (Licor) for 1 hour. Antibodies were diluted using 4% BSA in 1x PBS-T. Following washing, the membrane was analysed with Odyssey software. Bands for vinculin and CTGF were quantified using ImageJ software and CTGF was normalised to vinculin.

Results: Results of nested RT-PCR analysis of the effect of TGFβ1 on CTGF transcription over time showed that transcription levels increased from 3 to 9 hours, but decreased 24 hours after treatment, suggesting that the optimum incubation time for inducing CTGF expression was 6-9 hours. Nevertheless, a 24 hour incubation was used in subsequent experiments since this is minimum required for AOs to have an apparent effect in vitro. Results showed that 5-15 ng/ml TGFβ1 induced similar levels of CTGF transcription. 10 ng/ml was selected for subsequent experiments. However, the method used could not accurately determine levels of CTGF transcription. The signal of bands analysed on gels reached a maximum level for some treatments and differences in expression levels could not be distinguished.

An investigation into the potential of antisense oligonucleotides

to reverse skeletal muscle fibrosis.

Student: James March Supervisor: Professor George Dickson Daily Supervision: Dr. Linda Popplewell

Of the four AOs tested, only AO1 showed evidence of exon skipping when harvested RNA was analysed by RT-PCR and thus potential effectiveness at knocking down CTGF transcript levels in C2C12 cells (Fig. 1). When treated with 2 μM PMO1 without TGFβ1, the observed skipping was 7.2%. This increased to 38.4% when treated with 5 μM PMO1 and 44.1% with 10 μM. However, the degree of skipping was lower when cells were also treated with TGFβ1: at 2 μM PMO1

no skipping was observed, at 5 μM there was 6.9% skipping and at 10 μM 17.2% skipping. These results indicate that PMO1 is effective at inducing exon skipping at higher doses, which are particularly required in the presence of TGFβ1.

Western blot analysis showed that 10 ng/ml TGFβ1 induced CTGF expression by 67.1% in C2C12 cells (Fig. 2). The blot appears to show knockdown in expression by 10 μM AO1 without TGFβ1. However, once normalised to vinculin, no

knockdown of endogenous and TGFβ1-induced CTGF expression by AO1 was observed. This suggests that AO1 was not effective at knocking down CTGF expression after this incubation time at this concentration. However, it is possible that knockdown may be evident upon analysis of CTGF levels in culture media since CTGF is a secreted protein.

Future directions: Quantitative PCR could be used to quantify more accurately the effect of TGFβ1 on CTGF transcription over time and at different concentrations, as well as the degree of exon skipping observed with AO1. To assess more accurately the ability of AO1 to knockdown CTGF protein expression, the abundance of CTGF present in growth media should be assessed by Western blot analysis in addition to cell lysates. A further dose response

experiment could be carried out to assess what concentration is required to induce significant skipping in the presence of TGFβ1. Longer AOs could also be examined as these will have higher binding energies and hence enhanced efficacy.

Departures from original proposal: Before transfection of C2C12s with AOs, the effect of TGFβ1 on transcription levels over time and the effect of different concentrations of TGFβ1 were assessed. Endoporter was used to deliver AOs to cultured cells instead of nucleofection. Due to time constraints, designed AOs were not tested in human RD myoblasts.

Value of the studentship to the student: The Studentship has been of immense value to me. It has afforded me opportunity to gain experience of working in a research laboratory and allowed me to develop my laboratory skills, grow in confidence and learn new skills. I have been able to learn from experienced researchers and discuss with them their current projects, broadening my horizons. The experience provided by the grant has confirmed to me that my career aspiration to work as a research scientist in the field of genetic therapy is indeed what I want to do.

Value of the studentship to the lab: This studentship has enabled the securing of important preliminary data that will strengthen planned applications for research funding. The need for effective targeting of skeletal muscle fibrosis is becoming more and more vital. With the necessary development and further study, the work performed within the studentship has the potential to provide an anti-fibrotic therapy. New avenues of research for the lab have been investigated with this studentship and it has therefore been extremely fruitful and worthwhile.

Bibliography: 1. Morales, M.G., Gutierrez, J., Cabello-Verrugio, C., Cabrera, D., Lipson, K., Goldschmeding, R. & Brandan, E. 2013, "Reducing CTGF/CCN2 slows

down mdx muscle dystrophy and improves cell therapy", Human molecular genetics; Hum.Mol.Genet., vol. 22, no. 24, pp. 4938-4951. 2. Sisco, M., Kryger, Z.B., O'Shaughnessy, K.,D., Kim, P.S., Schultz, G.S., Ding, X., Roy, N.K., Dean, N.M. & Mustoe, T.A. 2008, "Antisense inhibition of

connective tissue growth factor ( CTGF/ CCN2) mRNA limits hypertrophic scarring without affecting wound healing in vivo", Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, vol. 16, no. 5, pp. 661.

Student: Jamie Hanna

Supervisors: Dr Maelíosa McCrudden and Aaron Courtenay

Investigating dissolving microneedle arrays as a vaccine delivery platform

Background:

In today’s society, the emergence of microneedle (MN) drug delivery systems has had a major

impact on the potential administration of various drugs and vaccines painlessly and in a minimally

invasive manner (Quinn et al. 2014). MN delivery offers an alternative to the utilisation of

conventional parenteral delivery devices such as the hypodermic needle and syringe, which

ultimately results in nociceptive stimulation upon administration leading to much discomfort and

potential distress in numerous patients (Quinn et al. 2014). Extensive research being conducted in

this area has led to the development of five main MN subtypes; solid, hollow, coated, dissolving and

hydrogel (super-swelling), with the latter two forms being of major focus throughout the project

described herein. Delving deeper into the functionality of firstly the dissolving MN platform, the

drug/vaccine to be delivered is incorporated into the polymeric matrix of a dissolving MN

formulation, composed of either Gantrez S-97 (Polymethylvinylether/maleic acid or PMVE/MA) or

polyvinylpyrrolidone (PVP) (McCrudden et al. 2014). The heights of the needles on MN patches can

range between 50 – 900 µm and function by imbibing interstitial fluid from the skin upon delivery

and this results in fluidisation of the polymeric chains and hence dissociation of the needles,

resulting in release of the drug across the stratum corneum, the

outermost layer of the skin and that which is most difficult to traverse

(Quinn et al. 2014).

The second MN form previously mentioned, that was majorly utilised

throughout this project, was the hydrogel subtype. Making use of

Gantrez S-97 as the main polymeric network for the MN formulation;

the formulation also contains polyethylene glycol 10,000 (PEG 10,000)

which forms cross-links with the PMVE/MA and enables the array to

swell as a result (Donnelly et al. 2014). Therefore, on insertion of a

hydrogel MN array into the skin of an individual, this results in

swelling of the array upon absorption of fluid from the skin and

thus allows for diffusion of drug from a reservoir (a lyophilised

wafer containing Ibuprofen-Sodium, for example) through the

hydrogel system, and across the stratum corneum, along a

concentration gradient (Quinn et al. 2014). The two MN types

described in detail were utilised extensively throughout this project.

Departures from the original proposal:

It was agreed with my supervisor, Dr. McCrudden that I would be heavily involved in the day-to-day

experimental work on two distinct projects, due to the fact that the vaccine delivery project involved

some substantial lag times (e.g. time between prime and boost vaccinations etc.). To this end, my

eight week project in the lab involved two themes, namely:

Vaccine delivery via MN

Comparing and contrasting MN arrays manufactured using two different mechanisms

Figure 1. Image of personally

produced hydrogel MN, using light

microscope

Vaccine Delivery Project:

To deliver an efficacious dose of a novel vaccine, which is used to induce immunisation

status to the common bacterium, Haemophilus influenzae NCTC8467 , utilising a dissolving

MN platform, via the ears of CRL Nude Hrhr elite mice, as described previously by (Zaric et al.

2015).

To determine immunoglobulin titres subsequently quantified from the mice serum (post-

secondary vaccine boost) which are then compared with titres obtained when the mice

received an IM injection of the same vaccine, to determine if delivery was successful, and to

what extent.

Comparing and contrasting the production quality for both hydrogel and dissolving MN platforms

between two different manufacturers:

To obtain release data, utilising a modified Franz cell set-up, for two small molecular

therapeutics (Ibuprofen-sodium and Caffeine) through two different model membranes,

namely neonatal porcine skin and Aquacel® (both of which successfully mimic the stratum

corneum), using dissolving and hydrogel MN manufactured by two different organisations

(Queen’s University Belfast (QUB) and a pharmaceutical partner company).

To make use of the relevant analytical techniques such as Enzyme-linked Immunosorbant

assay (ELISA) and High Performance Liquid Chromatography (HPLC) as determinant methods

for quantification of sample concentrations, which allowed for calculation of ‘amount of

drug/protein released’ and subsequently the ‘Percentage (%) release’ of the relevant

substances, in order to gain knowledge on whether the MNs manufactured by the different

companies will portray different release curves, as a result of differences in manufacture

technique.

To also determine if there is any significant difference in the release of pharmaceuticals/

biomolecules through the two different MN platforms being utilised (Dissolving and

Hydrogel) through comparison of release data produced for each MN type, for a given

substance.

Methods:

Hydrogel MN arrays were formulated by preparing aqueous polymer blends, casting into silicone

moulds, and thermal polymer cross linking procedure as described by (Donnelly et al. 2014).

Dissolving MN arrays were formulated by mixing aqueous polymer blends, followed by casting into

silicone moulds, and drying at room temperature, as described by (McCrudden et al. 2014).

Ibuprofen-sodium lyophilised wafers were manufactured according to a previously documented

method briefly, excipients were incorporated into a homogenous liquid, cast into moulds and

lyophilised as per protocol (McCrudden et al. 2015).

In vitro drug permeation experiments were carried out

as previously described by (Ng et al. 2010) with some

variations, including the use of two model membranes,

namely Aquacel® and neonatal porcine skin.

IgG ELISA protocol, for quantifying the proportions of

immunoglobulin G levels, was conducted in accordance

with the kit provided (Mouse Anti-Hib-PRP IgG

(Haemophilus influenzae type b Polysaccharide,

PRP), ELISA Kit Cat. 980-120-PMG, ALPHA

DIAGNOSTIC INTERNATIONAL, 6203 Woodlake

Centre Drive. San Antonio. Texas 78244. USA.).

Results and Discussion:

Vaccine Delivery Project:

Following collection of serum samples from the test mice, analysis of IgG antibody titre was

conducted using a commercially available ELISA. Concentrations of detectable IgG antibody titres are

represented in the scatter plots figure 3, below:

Contr

ol

1ug IM

2ug IM

Indust

ry P

artn

er S

-97

MN

QUB S

-97

MN

QUB P

VP M

N

0

200

400

600

IgG

An

tib

od

y t

itre

U/m

l

Figure 3. Scatter plot representation of nude mice IgG antibody titre vaccinated via IM injection and

MN application of Industry partner S-97 MN, QUB S-97 MN, and QUB PVP MN containing a

Haemophilus influenzae vaccine.

Mice exposed to no treatments were kept as absolute negative controls and did not show any

detectable IgG antibody titre. The data obtained for the 10 mice subjects, whom received a 1µg dose

Figure 2. A representation of a modified

Franz cell setup using a lyophilised wafer

and a hydrogel MN system, designed by

(Donnelly et al. 2014).

of IM vaccine, showed that 9 of them displayed appreciable titres for the present of IgG antibodies,

which the highest and lowest titres being 237.2 and 7.9 U/ml, respectively, with an average titre of

72.9 U/ml. 3 mice were treated with 2µg IM, in which 1 of them showed a positive IgG titre at 140.4

U/ml. The 4 mice which were treated with QUB PVP MN arrays, only one of them showed a positive

antibody level of 387.3 U/ml. A further 4 mice were treated with QUB S-97 MNs containing the

vaccine, in which all of them displayed positive IgG antibody titres with the highest and lowest titres

being >500 and 6.8 U/ml. the average titre was found to be 137.6 U/ml. Upon treating 4 of the mice

with MN arrays produced by the partner company containing the vaccine, none of the mice

produced detectable IgG antibody levels.

Investigating results further, the partner pharmaceutical company formulations displayed no

detectable response, which when compared to QUB MN arrays containing the vaccine, all of the

mice attained appreciable antibody titres. Upon completion of an in vitro dissolution test of all the

MN arrays utilised during the test, it became obvious that the partner company’s MN arrays swelled

when immersed in 30mls phosphate-buffer saline, whereas all other arrays dissolved fully. The MN

manufacturing protocols of each of the organisations was then reviewed, and it appeared that the

partner company utilised a heating step, of 50 ˚C for 15 minutes, during their drying stage of

manufacture, whereas QUB MNs were dried for approximately 48 hrs at room temperature. The

implications of the heating step, using high thermal energies, could result in overcoming various

activation energies of components within the formulation, resulting in the Gantrez S-97 potentially

crosslinking with the active vaccine component, and therefore trapping, and preventing release of

the vaccine from the MN array. This would ultimately result in the mice not developing a humoral

response to the vaccine, which is one theory that has been drawn from the data collected and

ultimately requires a more in depth investigation.

Comparing and contrasting the production quality for both hydrogel and dissolving MN platforms

between two different manufacturers:

Upon analysis of the caffeine permeation from both types of dissolving MNs through an

Aquacel®/tinfoil model membrane (Figure 4.), QUB dissolving MNs showed percentage release of

64.3 ± 10.9 % caffeine over 24 hours when compared to the partner pharmaceutical company’s

permeation data, which showed 50.1 ± 16.0 % caffeine permeation after 24 hours.

Statistically, on completion of the Man Whittney U test, there is no significant difference (p>0.05)

between the permeation data obtained for either company. Similar release is displayed, indicating

that there is little difference in caffeine permeation across the stratum corneum (modelled in this

study by Aquacel® and tinfoil, using a modified Franz cell setup) between the MNs from the two

organisations. Therefore this indicates that the manufacturing method utilised by QUB, could be

translated to industrial manufacture for dissolving MN containing caffeine.

Figure 4. Percentage caffeine permeation from LTS caffeine dissolving and QUB caffeine dissolving

MN through Aquacel®/aluminium foil synthetic skin model on a modified Franz cell apparatus over

24 hours. n = 3 ± SEM.

Assessing the data obtained from the QUB vs. the large pharmaceutical partner company

permeation study (Figure 5.), utilising hydrogel MNs formulated by each company and Ibuprofen

wafers through neonatal porcine skin, there was approximately 50.1 ± 23.0 % ibuprofen permeation

through the porcine skin using QUB MN arrays over a time period of 24 hours, compared to the

partner pharmaceutical company’s MN arrays which demonstrated that 52.5 ± 13.3 % Ibuprofen

permeation was achieved over the same period of time.

Upon further analysis of the results obtained, Mann Whitney U analysis (p>0.05)showed no

significant difference between the percentage of Ibuprofen released from either the QUB hydrogel

MN formulation or the partner pharmaceutical company’s MN formulation. This indicates that the

QUB formulation could therefore be translated to industrial manufacture, to allow for up-scaled

production of these particular MN arrays. Advantages of industrial manufacture include mass

production capabilities, cost efficiencies, and ultimately advances in further MN development

processes.

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Figure 5. Percentage Ibuprofen permeation from LTS manufactured super swelling MN and QUB

manufactured super swelling MN through neonatal porcine skin on a modified Franz cell apparatus

over 24 hours. n = 3 ± SEM.

Conclusion:

The antibody titre values obtained from the vaccination of mice with various MN formulations

containing the vaccine, indicated that the QUB S-97 and QUB PVP may be suitable for MN

manufacture as all mice responding with antibody production for QUB S-97 and one mouse

responding in the QUB PVP cohort, whereas the partner company’s formulation of the vaccine-

containing MN arrays (S-97), there was no detectable levels of IgG antibody. This indicates that

further formulation work is required to optimise the industrial manufacturing process.

The ibuprofen permeation study through porcine skin, utilising both the QUB and a partner

pharmaceutical company’s MN arrays, indicated that hydrogel arrays were developed that allowed

50.1 ± 23.0 % and 52.5 ± 13.3 % caffeine permeation, respectively, over a period of 24 hours when

combined with a lyophilised wafer. The Caffeine permeation study through an Aquacel®/Tinfoil

model membrane, utilising both QUB and the partner company’s caffeine dissolving MN arrays,

indicated that the dissolving arrays developed by QUB and the pharmaceutical company lead to 64.3

± 10.9 % and 50.1 ± 16.0 % caffeine permeation, respectively. This data obtained suggests that the

QUB MN systems could be successfully transcribed and utilised in order to allow for large scale

production of both the hydrogel and dissolving MN platforms by the partner pharmaceutical

company.

Future directions of the project:

Work currently being carried out in utilising the dissolving MN platform to deliver an efficacious dose

of a novel vaccine could potentially lead to a vast number of further in vivo tests being conducted,

such as quantifying antibody levels in Guinea pigs or Rabbits, for example, on administration of

vaccine-containing dissolving MNs. If the results look promising, and this drug delivery methodology

is proving to show the development of an immune response in in vivo studies, then Phase I clinical

trials could, in time, be carried out, to test the viability of this concept in human subjects.

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Value of the Studentship:

I believe that in obtaining this studentship, it has proven to be a most invaluable experience and I

have gained more from the 8 week placement than I could have hoped for. I had the opportunity to

gain continuous lab experience, which under normal circumstances throughout the normal academic

year due to scheduled lectures alongside lab classes, would not be realistically achievable. My

confidence and independence has grown drastically over the course of the placement and I now feel

more comfortable conducting experiments and analysing data sets. I’ve developed my skills on a

variety of different laboratory and analytical techniques that will no doubt aid me in practical classes

during the next two years of my degree, for example, how to carry out permeation studies utilising

modified Franz cells, how to carry out ELISA, characterisation of MN arrays, how to manufacture

both dissolving and hydrogel type MN and also how to analyse HPLC data obtained from collected

samples. I have also undoubtedly gained new knowledge on topics at the forefront of ground-

breaking research which I found will benefit me during the remaining course of my degree.

On completion of the 8 week summer placement, I have definitely gained a greater insight into the

field of scientific research and into what it could potentially be like to be studying for a PhD. I have

immensely enjoyed my experience here in the lab and I definitely will likely endeavour to undertake

a PhD post-graduation, potentially in an industry-related setting, as it will, of no doubt, be a very

rewarding experience and will benefit in my future career progression.

From the supervisor: Dr. Maelíosa Mc Crudden I firmly believe that undergraduate vacation studentships are a very valuable experience for both the student who undertakes the research project but also the research team in which the work is undertaken. With this in mind and as explained by Jamie previously, this studentship has offered him a wonderful opportunity to carry out ground breaking research that simply cannot be covered in routine undergraduate practical classes. Jamie has been an excellent addition to the team in our laboratory and has been a very conscientious and enthusiastic student. He was very motivated and after undergoing training in various methodologies, he became proficient and independent in his approach to his work. I am confident that Jamie will excel as he continues his MPharm studies and I would like to personally thank the Biochemical Society for funding Jamie’s work. I believe that it was a worthy investment in this talented young student.

References:

Donnelly, R.F. et al., 2014. Hydrogel-forming microneedles prepared from “super swelling” polymers combined with lyophilised wafers for transdermal drug delivery. PloS one, 9(10), p.e111547. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4216095&tool=pmcentrez&rendertype=abstract [Accessed August 16, 2015].

McCrudden, M.T.C. et al., 2015. Considerations in the sterile manufacture of polymeric microneedle arrays. Drug delivery and translational research, 5(1), pp.3–14. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25787335 [Accessed August 18, 2015].

McCrudden, M.T.C. et al., 2014. Design and physicochemical characterisation of novel dissolving polymeric microneedle arrays for transdermal delivery of high dose, low molecular weight drugs. Journal of controlled release : official journal of the Controlled Release Society, 180, pp.71–80. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4034161&tool=pmcentrez&rendertype=abstract [Accessed July 5, 2015].

Ng, S.-F. et al., 2010. Validation of a static Franz diffusion cell system for in vitro permeation studies. AAPS PharmSciTech, 11(3), pp.1432–41. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2974154&tool=pmcentrez&rendertype=abstract [Accessed August 14, 2015].

Quinn, H.L. et al., 2014. The role of microneedles for drug and vaccine delivery. Expert opinion on drug delivery, 11(11), pp.1769–80. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25020088 [Accessed August 16, 2015].

Zaric, M. et al., 2015. Dissolving microneedle delivery of nanoparticle-encapsulated antigen elicits efficient cross-priming and Th1 immune responses by murine Langerhans cells. The Journal of investigative dermatology, 135(2), pp.425–34. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25243789 [Accessed August 16, 2015].

Student: Jennifer Shoesmith

Supervisor: Professor Alison Baker and Dr David Carrier

Gaining structural insights into the plant ABCD transporter COMATOSE through Cysteine

Probing

Introduction

Comatose (CTS) is a plant peroxisomal membrane

protein (From Arabidopsis thaliana) and is an ABC

transporter (1). CTS functions in many plant

developmental and metabolic processes. It has

been shown that CTS transports long chain fatty

acids and some hormone precursors into the

peroxisome to undergo β-oxidation (1, 2). CTS

appears to transport Acyl-CoAs and has a thio-

esterase activity (3). CTS is a large hydrophobic

membrane protein (4), and therefore traditional

structural techniques like X-ray crystallography are

challenging for determining its structure. CTS

contains 10 cysteines in total (5), and their

accessibility can be probed with Cys reactive

reagents e.g. maelimide biotin (6) allowing

information to be gained about the accessibility of

particular cysteines (7). By selectively mutating

individual cysteines and combining results with

information from a homology model structutal

information can be obtained. To ensure mutants are

functional they are tested for the ability to

complement S.cerevisiae ∆Pxa1/∆Pxa2 for growth

on oleate (8).

Aims

To test the importance of individual cys in

COMATOSE with a view to creating a cysless

mutant and reintroducing at various sites for

structural studies.

To assess the functionality of cys mutants in yeast.

To use cysteine mutants in maelimide binding

experiments to elucidate structural information

using mass spectrometry.

Methods

Cysteine mutants were generated using single

primer reactions in parallel mutagenesis (9) and

traditional site directed mutagenesis(10). Plasmids

were maintained in Omnimax cells grown on LB

ampicillin and prepared using Promega wizard kits.

Plasmids were transformed into yeast cells by

treatment with lithium acetate and heat shock for

30mins at 42̊c and spread on SD-agar with

appropriate drop out amino acids for selection (11).

Oleate growth assays performed according to

methods in (12).

To correct deletions in mutated plasmids overlap

extension PCR was used (13).

All sequencing was performed by Beckmann

Coulter Genomics.

HA and His tagged halves of the CTS protein were

also transformed into ∆Pxa1/∆Pxa2 yeast cells,

from these cells a light mitochondrial fraction was

produced containing peroxisomes (14). Coomassie

protein gels and a western blot were used to

determine the presence of CTS in the fraction.

Results and conclusions

Mutants C608S, C1118S and C1180A were

successfully generated using mutagenesis,

confirmed by sequencing. These mutants were then

transformed into yeast cells containing the other

(wild type) half of the protein for functional studies.

Other mutants that had already been generated

were tested for function by growth on oleate.

According to the growth assay fig 1, C55S, C156S,

C245S, C1118F, and C1278S all grew on the oleate

plates therefore function has not been compromised

by the mutation. C608G did not grow in the oleate

assay therefore function has most likely been

compromised by this mutation. Tagged halves of

the CTS protein were transformed into yeast. A light

mitochondrial fraction was taken from these cells

and run on SDS and probed by western blot. There

was a signal for Anti-HA antibody as well as Anti-

His antibody suggesting that both halves are

present in the fraction and not in cytosolic

supernatant Fig 2.

Discussion and future directions

The cys mutants that had not yet been produced

were C608S, C768S, C1118S and C1180A.

Mutagenesis was performed with appropriate

primers and plasmids were propagated and

sequenced. The first set of colonies which was

sequenced all were WT. The mutagenesis

experiments were repeated using the single primer

method and traditional mutagenesis. The traditional

mutagenesis experiment gave a better level of

plasmid amplification so these reactions were used

to transform cells. These plasmids were also sent

for sequencing. According to

Fig 1, oleate growth assay. Yeast mutants were grown on SD-low glucose to acclimatise to using a different carbon

source. Cells were grown overnight in YPO. Cells were collected by centrifugation and re-suspended in sterile water.

Cells were diluted to the same OD600 and streaked on oleate plates. A, negative control, growth is due to

contamination no yeast present. B, positive control many yeast cells is some contamination. C, C55S some growth

observed. D, C156S some growth. E, C245S large amount of growth. F, C608G no growth. G, C1118F some growth. H,

C1278S large amount of growth.

agarose gels the Dpn1 digest to remove parent

plasmid was successful which was backed up by a

transformation with Dpn1 digested plasmid where

no colonies were observed. Further colonies from

original mutagenesis were grown and the plasmids

sent for sequencing.

C768S still needs to be generated as all sequencing

came back as wild type.

C631S previously generated by another student

also contained a secondary mutation A412V,

primers were designed to remove this mutation and

mutagenesis was performed. The plasmid was

propagated in E.coli and the plasmid sequenced.

However this mutagenesis came back still

containing A412V and will require repeating.

C1115-1p had a 45bp deletion upstream of the

desired mutation, to correct this overlap extension

PCR was performed. The first step PCR

amplification of the correct sequence was

successful, however the second step correction had

no amplification of the plasmid.

Mutants of C608S, C1118S and C1180A were

successfully generated, these plasmids were then

Fig 2. SDS Page and western blot of light mitochondrial

fraction. A, coomassie stained gel. 1 molecular weight ladder, 2

supernatant 5µL, 3 pellet 5µL, 4 supernatant 10µL, 5 Pellet

10µL, 6 supernatant 15µL, 7 pellet 15 µL, 8 pellet 20 µL,9

supernatant 20 µLB western blot of light mitochondrial fraction

to detect pseudo-halves of CTS. 1 and 4, molecular weight

ladder. 2 supernatant with Anti-HA no protein present. 3 pellet

with Anti-HA Small band between 46kDa and 35kDa N-terminal

half is present in the light mitochondrial fraction as expected

although not at the expected size. Full length CTS is 149,576,

and the half size ~70kDa. 5 supernatant with Anti-His no

protein present. 6, pellet with Anti-His 2 bands present

therefore C-terminus is also present in the light mitochondrial

fraction, although bands are also not at the expected length.

used to transform yeast cells containing the other

respect half of the protein for functional assay.

Mutants yeast cells were grown on oleate as a

functional test. Most of the existing mutants

displayed some growth on the oleate media as seen

in fig 3. C55S, C156S, C245S, C1118F, and

C1278S were all able to grow on the oleate media

meaning these mutants have retained function to

transport fatty acyl-CoA across the peroxisomal

membrane. C608G had no growth on the oleate

plates suggesting that the function of this mutant

has been disrupted and is unable to transport fatty

acyl CoA’s across the peroxisomal membrane.

However this experiment is only a qualitative

measure of function. A better test of function would

be to directly measure the levels of β-oxidation in

mutant cell peroxisomes. The other mutants

generated also require testing for function C608S,

C1118S and C1180A.

For structural studies tagged halves of CTS were

transformed into ∆Pxa1/∆Pxa2 yeast. These cells

were grown in low glucose to acclimatise to the

oleate media without glucose. This culture was

used to inoculate 1L of YPO in which the cells were

grown overnight. This culture was used to generate

a light mitochondrial fraction containing

peroxisomes. This fraction was run on a SDS PAGE

and blotted with anti-His and anti-HA antibodies to

detect each half of the protein. Figure 2 shows that

both halves of the protein are present in the light

mitochondrial fraction and not the cytosolic fraction.

The protein is correctly localised. However bands

are not observed at the expected length for CTS full

or half size. This may be due to degradation during

the purification process which CTS is susceptible to.

To test if the bands contain degraded CTS they can

be cut out and used in mass spec. These cells

expressing tagged halves of CTS can now be used

for purification of CTS for structural studies using

mass spec.

Departures from original proposal.

Due to time constraints and issues with yeast

transformations the maelimide biotin experiments

were not performed. However the basis for these

experiments in having the tagged halves of CTS in

yeast cells is a good starting point for purification

and mass spec. The mutants that were generated

were also not tested for function due to time

constraints.

Value of the studentship to the student

The studentship has allowed me to learn many

techniques that I have not had the chance to learn

in my undergraduate degree such as yeast culture

and manipulation and how to purify organelles from

yeast. The studentship has allowed me to practise

many existing skills like mutagenes, E.coli culture

and manipulation, DNA gels, protein gels and

western blots. I have learnt how to troubleshoot

various experiments when they are not working.

The studentship has furthered my aspirations to

continue my study to PhD level and undertake

further research.

Value of the Studentship for the lab.

The studentship has allowed us to obtain 3 new

mutants and re-test some existing ones. This is a

good outcome for only 8 weeks. It has also allowed

us to start developing the methodology for probing

the accessibility of the cys residues, something we

intend to take forward in future. Jenny has been a

great asset to the lab and the techniques she has

learned will help her in her M.Biol project with us in

the upcoming academic year.

References

1. Theodoulou, F.L. et al. Peroxisomal ABC transporters. FEBS Lett. 2006, 580(4), pp.1139-55.

2. Theodoulou, F.L. et al. Jasmonic acid levels are reduced in COMATOSE ATP-binding cassette transporter mutants. Implications for transport of jasmonate precursors into peroxisomes. Plant Physiol. 2005, 137(3), pp.835-40.

3. De Marcos Lousa, C. et al. Intrinsic acyl-CoA thioesterase activity of a peroxisomal ATP binding cassette transporter is required for transport and metabolism of fatty acids. Proc Natl Acad Sci U S A. 2013, 110(4), pp.1279-84.

4. Rea, P.A. Plant ATP-binding cassette transporters. Annu Rev Plant Biol. 2007, 58, pp.347-75.

5. Hayashi, M. et al. Ped3p is a peroxisomal ATP-binding cassette transporter that might supply substrates for fatty acid beta-oxidation. Plant Cell Physiol. 2002, 43(1), pp.1-11.

6. Hansen, R.E. and Winther, J.R. An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations. Anal Biochem. 2009, 394(2), pp.147-58.

7. Kim, Y. et al. Efficient site-specific labeling of proteins via cysteines. Bioconjug Chem. 2008, 19(3), pp.786-91.

8. Nyathi, Y. et al. The Arabidopsis peroxisomal ABC transporter, comatose, complements the Saccharomyces cerevisiae pxa1 pxa2Delta mutant for metabolism of long-chain fatty acids and exhibits fatty acyl-CoA-stimulated ATPase activity. J Biol Chem. 2010, 285(39), pp.29892-902.

9. Edelheit, O. et al. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnol. 2009, 9, p.61.

10. Liu, H. and Naismith, J.H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 2008, 8, p.91.

11. McDonald, P.N. Two-hybrid systems. Methods and protocols. Introduction. Methods Mol Biol. 2001, 177, pp.v-viii.

12. Marchi, E. and Cavalieri, D. Yeast as a model to investigate the mitochondrial role in adaptation

to dietary fat and calorie surplus. Genes Nutr. 2008, 3(3-4), pp.159-66.

13. Bryksin, A.V. and Matsumura, I. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques. 2010, 48(6), pp.463-5.

14. Distel, B. and Kragt, A. Purification of yeast peroxisomes. Methods Mol Biol. 2006, 313, pp.21-6.

Biochemical Society Studentship Report Jennifer Wood

PROJECT AIM: The project aim was to culture A549 cells, extract proteins using a dounce homogenizer, add CO2 (C12 and C13 labelled) to the sample, transfer the sample into a pH stat (pH 7.4), add (trimethylsilyl)diazomethane to enable CO2 binding to protein amine groups for carbamate formation, dialyse the sample, and digest the sample proteins into peptides using heat, dithiothreitol, iodoethane, and trypsin. The sample peptides were then pre-fractionated using stagetips, fractionated using a mass spectrometer to identify peptides bound to CO2, analysed on databases to reveal the original proteins, and investigated for the role these CO2-binding proteins had in physiological and pathophysiological processes.

PROJECT REPORT (DEVIATIONS INCLDUED): Introduction: CO2 is a vital molecule in mammalian, bacterial, and plant cells but its protein-binding mechanisms and roles are poorly understood due to limited appropriate laboratory equipment/techniques; our team developed a protocol able to investigate CO2 molecular interactions. This project used the protocol for CO2-binding protein identification in A549 adenocarcinoma human alveolar basal epithelial cells, hoping to reveal CO2-binding proteins and their role in physiological and pathophysiological states for therapeutic purposes. Method: The protocol was used to generate three C12 and C13 mammalian A549 CO2-trapped peptide samples. The steps included cell growth (1), splitting (2), harvesting (3), lysis (4), CO2-

trapping (5), dialysis (6), digestion (7), and mass spectrometry (8).

1. Transfer 10mL DEMM media into 15mL falcon tube with A549 live stock cells, centrifuge (1200rpm/3minutes), discard supernatant, add 10mL media and resuspend pellet, transfer to T25 flask, and incubate (37C/~3days).

2. Remove media, wash in sterile PBS x 2, add trypsin and incubate (37C/3minutes), remove trypsin, wait until mobile (view under microscope), add 10mL of DEMM and resuspend cells, add 4mL into T75 flask x 2, add fresh DEMM into T25, T75, and T75 flasks, incubate (37C/~3 days), remove media, wash in PBS x 2, add 10mL of PBS and scrap cells into buffer, and centrifuge cells (1200rpm/3minutes).

3. Centrifuge cells in 25mL PBS x 3 (1000g 4C/10minutes), resuspend in 1mL hypotonic lysis

buffer, incubate for 10 minutes on ice, dounce homogenise cells (40 strokes) and view lysis under microscope, centrifuge sample (4C 1500g/15minutes), resuspend pellet in 2mL reaction buffer, and read peptide concentration using nanodrop.

4. Weigh out 6.8mg of C12 or C13 CO2 and add to reaction, weigh out 3x80mg TEO into

separate vials, add reaction to pH stat, and add each TEO reagent with 300uL water.

5. Microwave dialysis tubing for 5 minutes, add clips and reaction to tubing, add to 1L beaker water, and stir overnight.

6. Collect dialysed samples in 15mL falcon tubes, centrifuge (5500g for 5 minutes), remove

supernatant, resuspend in 1mL 100mM ammonium bicarbonate buffer, transfer 100uL into Eppendorf for digest, add 1uL 10 % SDS, add 2uL DTT, incubate at 80 °C for 15 minute, add 5uL iodoethane, incubate at room temperature in dark for 30minutes, heat trypsin aliquots from freezer at 37 °C, add trypsin to each digest, incubate at 37 °C/20 hours, and take to technician and perform mass spectrometry.

Results & Discussion: Unfortunately, the A549 cells did not yield peptide results following mass spectrometry. It was hypothesised that cellular lysis may be responsible and thus a new douncing method using a hypotonic cellular buffer, incubation on ice, and centrifugation was used. The sample protein concentration was also estimated using a nanodrop. The nanodrop revealed that the problem was more likely due to the TEO CO2-trapping agent causing massive protein aggregation. During the digestion process, addition of ammonium bicarbonate helps trypsin function, SDS aids dissolving protein (thus removing precipitate), DTT binds cysteine residues for disulphide bridge breakage, iodoethane maintains this bond breakage, 80C and 32C incubations denature protein, and trypsin digests the protein; consequently, protein aggregation should not have been an issue. Therefore, it seems the problem lay prior to digestion. For instance, when removing the sample from the pH stat into test tubes, the aggregates frequently became lodged on the side of the pH stat, thin glass pipette, or plastic pipette tips and were consequently lost. Thus, only a small amount of aggregated protein (if any) remained prior to dialysis. Furthermore, digestion was performed using only a relatively small amount on the dialysis sample. Therefore, the very low amount of protein in the sample prior to dialysis, small amount of sample used for digestion, and potential peptide loss in pre-fractionation methods prior to mass spectrometry must have caused complete peptide absence from the mammalian samples before mass spectrometry. This protocol was also performed on E.coli to identify the issue, which did yield peptides following digestion. It seems likely that there were many more bacterial cells and thus proteins at the start of the experiment, which is thus likely to maintain protein within the samples even following TEO-induced aggregation.

FUTURE PROJECT DIRECTION AND PROJECT ACHIEVEMENTS: It is hypothesised that the peptide absence during the project was due to the CO2-trapping agent (TEO) causing massive protein aggregation and thus precipitant formation that is subsequently lost due to handling difficulties in combination with the small volume of the sample used. E.coli and plant cells yielded peptides using the same protocol. Therefore, it seems likely that the protocol is effective for CO2-binding protein identification, however, alternations must be made when performing it on the A549 cells. A higher quantity of mammalian cells used per experiment and methods that improve sample transfer during protocol steps will both help to prevent protein loss. This will enable mammalian CO2-binding protein identification by mass spectrometry that may potentially have abundant therapeutic applications. I have undertaken numerous practical sessions for my Biomedical Science course, however, these short, pre-planned, outlined practical sessions did not seem to reflect most applied laboratory research. Currently, my main interest involves carcinogenesis and cancer metastasis, understanding mechanisms and signalling pathways that enable mutated cells to form a primary tumour and migrate to distinct locations. Working on A549 CO2-binding proteins in attempt to investigate their cellular pathways has given me insight as to how on going novel research is approached, ultimately inspiring me to continue the pursuit of scientific-related research. Furthermore, I have gained many skills throughout this internship. Applying for the Biochemical Society Studentship helped me learn about funding application processes that will aid for future masters or PhD grants. Furthermore, I obtained many transferable research skills such as flexibility and adaptability. Unforeseen issues arose during the project and thus adjusting, learning new techniques, and attempting new and improved methods was vital for project continuation. This also strengthened my time management skills. The project involved generating numerous samples, which had numerous steps requiring 24 hours to complete. Therefore, deviating from the project plan required time considerations that involved generating multiple samples simultaneously without rushing the project. Another important achievement made was gaining confidence for interpersonal communication. This project required working in several laboratories with various individuals to use specific scientific equipment. I learnt how to confidently create a professional relationship with peers and professors, asking questions when necessary. The project improved my general communication skills; effectively explaining the project protocol and procedures, issues, and attempts to overcome these issues. In addition, the most important achievement was having discussions and proactive debates with others to hypothesise the peptide absence causation, which led to E.coli experiments that helped narrow down the potential error origin. Finally, I became familiar with numerous important scientific techniques that will have use in many different research environments.

Membranes, Amyloid-Beta and Human Cystatin C

Background:

Alzheimer’s disease, a form of dementia, is a complex neurodegenerative disorder characterised by the

extracellular deposition of insoluble plaques of amyloid-beta, the presence of intracellular fibrillary tangles

and neuronal atrophy. The Alzheimer’s Society estimate that around 850,000 UK residents are currently

suffering from dementiai, with the cost of care at a substantial £26 billion a year. As a disease predominantly

affecting the elderly, a continued increase in life expectancy makes our mission to understand its

pathogenesis, and potentially develop treatments to avert it, all the more important.

Toxic, soluble assemblies of Amyloid-beta1-42 are believed to be key in the pathogenicity of Alzheimer’s

disease. Importantly, a cerebrospinal-fluid protein, cystatin C, exhibits a protective effectii; preventing fibre

formation and regulating the toxic effect of oligomersiii

. The nature of this interaction is of great interest and

is arguably most important at the membrane surface where binding is much tighter than in bulk solution.

Additionally, both peptide-membrane interactions, and the local environment at a membrane surface have

been implicated in facilitating assembly of Aβ oligomers. A review by Relini et al.iv

explores current

research into these facets of membrane surfaces, considering the two-dimensional lipid-water interface and

key membrane components such as GM1 and cholesterol to be instrumental in creating ‘nucleation sites’

and thus promoting the formation of higher order structures. Considering this, an investigation of the

interaction between Aβ and hCC on a model membrane will likely provide a detailed insight into the

modulating effect of hCC.

Aims:

To use atomic force microscopy to characterise the behaviour of the Aβ peptide at a membrane surface and

to investigate the ability of hCC to modulate this behaviour.

Work Carried Out:

Human Cystatin C Preparation:

BL21 cells were transformed with an IPTG inducible hCC plasmid, with the hCC protein targeted to the

periplasm. These cells were later used to inoculate 5l of M9 minimal media-Amp for growth in baffled

flasks. Cells were grown to an OD of ~0.6 and hCC expression was induced by addition of IPTG. Cells were

lysed by resuspension in 28ml EDTA following centrifugation and resuspension in 20% sucrose solution.

After another round of centrifugation the supernatant was collected and dialysed in 5l cold sodium

phosphate buffer pH7.4

Anion exchange chromatography, and size exclusion chromatography were used to obtain a suitably pure

hCC sample, as determined using SDS-PAGE analysis and size-exclusion hplc (see figure 1).

0 2 0 4 0 6 0 8 0

0 .5

0 .6

0 .7

0 .8

0 .9

1 .0

A n a ly s is o f P u r if ie d h C C u s in g H P L C

T im e (m in s )

Ab

s

F ib B u ffe r

h C C

Figure 1: High performance liquid

chromatography readout for

our purified human cystatin C

protein. Aβ fibrillisation buffer

was run down the column to

provide the baseline shown in

pink. The orange line indicates

our hCC sample. The cystatin C,

as expected, was eluted after

38 minutes and the single sharp

peak confirms the absence of

contaminants and therefore a

good purification of the

protein.

Vesicle Preparation for Dye-leakage Assay:

Stock lipids, solubilised in chloroform, were mixed to relevant concentrations in a round-bottomed flask and

the chloroform was evaporated off under a gentle nitrogen stream. The resulting lipid film was flash frozen

in liquid nitrogen and lyophilized to remove any remaining solvent. The film was then resuspended in 1ml

5-(6)-Carboxyfluorescein by vigorous vortexing for at least 15 minutes to create Carboxyfluorescein loaded

multilamellar vesicles. These were extruded through an Avanti mini-extruder containing a 200nm membrane

to create large unilamellar vesicles and run down a PD-10 size exclusion column to remove excess

carboxyfluorescein.

Aβ Oligomer Preparation:

A dry 0.1mg stock of Aβ was resuspended by sonicating in 140µl of 10mM NaOH for 30mins. This is then

diluted twice in 10mM HEPES buffer and the pH was adjusted to 7.4 by the addition of HCl. The resulting

suspension was then refrigerated for 24 hours. Before use, the preparations were centrifuged to remove

preformed fibres.

Transmission Electron Microscopy:

Carbon coated grids were glow discharged to create a hydrophilic surface. These were then coated in 10μl

samples of oligomer preparations and negatively stained using urinyl-formate. The grids were imaged using

TEM to confirm successful oligomer formation.

Atomic Force Microscopy:

GM1, Cholesterol and DMPC were spin-coated onto a silicon wafer at 1000-1500rpm. Elipsometry was

used to characterise the thickness of the coating and showed a consistent value of ~5nm, indicating the

presence of a bilayer.

Oligomer preparations were buffer exchanged into water and 50μl added to the bilayer. The sample was

allowed to sit for ~5mins before the surface was blow-dried with nitrogen. The same preparation was used to

produce samples in the absence of a bilayer on mica. The samples imaged included bilayers, oligomers on

mica and oligomers on a bilayer.

Samples were imaged using tapping mode AFM in air on a Bruker Dimension 3100 NSIV and an Asylum

MFP 3D microscope. [The tips used had a resonant frequency 320kHz and a spring constant of 42Nm-1

.]

Departures from the Original Proposal:

The unforeseen breakdown of the Atomic force microscope left us behind schedule as both me and my

supervisor had to be retrained on a different machine. However, this was beneficial for me as I now have

experience with two different AFM machines and consequently two types of imaging software.

Due to numerous challenges encountered during AFM sample preparation we were unable to confidently

identify Aβ oligomers occupying the surface of a lipid bilayer. However, as shown in the results, we were

able to image these on mica.

The dye-leakage assay proved an interesting addition to the project, especially as we encountered numerous

complications and the results obtained were unexpected. As a result I was faced with real-world laboratory

problems which challenged me in a way that undergraduate teaching labs could not.

Results and Discussion:

The preparation of Aβ oligomers for use in both the dye-leakage assay and AFM was consistently

successful, with a variety of oligomeric species being produced (see figure 2).

The initial preparation of the model membrane on silicon wafers was also reliable and a characteristic result

is shown in figure 3. The width of the constructed bilayer was consistently within 5-6nm.

Unfortunately it was not possible to fully maintain the

integrity of the bilayer once the oligomer preparation

was added. Numerous approaches to the sample

preparation were tested but none were particularly

successful. As a result, we were unable to

conclusively identify the presence of oligomers on

our model membrane. The oligomers were instead

imaged on mica (see Figure 4) giving a result

comparable to our EM image, with the same globular

oligomers easily identifiable. The extended worm-

like oligomers seen in the EM image are also present

although they are less easily distinguishable.

Figure 2 A (left) and B (right):

Transmission electron microscopy images of the Aβ oligomer preparation. Where A= 11500X magnification and B= 21000X

magnification. The grid was densely populated with various oligomeric species; predominantly globular oligomers and

extended worm-like oligomers. The extended oligomers are consistent with the appearance of amyloid-beta derived

diffusible ligands seen in the literature.

Figure 3:

Model bilayer composed of DMPC,

Cholesterol and GM1. A 5 micron height

retrace of the model bilayer, imaged using

atomic force microscopy. The purple region

indicates good bilayer coverage whilst the

darker regions indicate patches of bare

silicon. Small areas coloured orange are

due to the stacking of two bilayers.

Figure 4: Aβ oligomers on mica. A phase retrace of

oligomers on mica showing similar species to those

observed using transmission electron microscopy

The dye-leakage assay was unsuccessful due to problems with osmotic balance and an as yet unexplained

quenching of the fluorescence signal observed upon the addition of Aβ oligomers to the carboxyfluorescein-

loaded vesicles. The protocol was subject to numerous amendments throughout the placement but we were

unable to completely eliminate dye-leakage in the negative control (vesicles on their own) or prevent the

quenching effect that was being observed.

Future Directions:

Once the dye-leakage assay is optimised, it can be used as a reliable test for the toxicity of oligomer

preparations prior to AFM imaging.

Imaging in water is a natural progressive step for the AFM work and will likely be key in further

investigation of the effects of hCC on Aβ assemblies at the membrane. There is also an alternative to spin-

coating that will be trialled, involving the adsorption, rupturing and rolling out of vesicles to give a bilayer

for use as a model membrane. In this case it will be possible to add oligomeric Aβ to the vesicles prior to

bilayer formation, thereby eliminating the problematic step of adding the oligomers as a wet sample to an

already intact, although both will be attempted.

Both circular dichroism and NMR spectroscopy are expected to feature heavily in further investigation of

the interaction between Aβ and hCC.

Outcomes of the Studentship:

This project has been an invaluable experience and has introduced me to a huge range of techniques and

equipment that I would otherwise have not encountered during my degree course. The first-hand research

experience has given me confidence in my decision to pursue a PhD, whilst attending weekly lab meetings

introduced me to fields of research that have inspired specific interests and will help guide my choice of

PhD project. I would like to thank the Biochemical Society for this opportunity and everyone in the NMR

group for being so welcoming and supportive throughout my placement.

Summary of Results:

We produced recombinant human cystatin c protein from a genetically engineered bacterial culture with the

intention of examining the influence of this protein on the behaviour of amyloid-beta at a model cell

membrane. The model membrane was composed of lipids in a ratio representative of the natural cell

membrane and a solution of these lipids was spin-coated onto a silicon wafer; resulting in the formation of a

membrane on the silicon surface. Amyloid-beta was incubated in conditions that allowed small clusters

called oligomers to form and it is these that were added to the membranes. The oligomers were left to

interact with the cell membrane before the sample was dried for imaging with atomic force microscopy in

air. Unfortunately we were unable to optimise our sample preparation and as a result we didn’t quite get

around to adding the human cystatin c. However, I am confident that the research work carried out in my 8

weeks will allow this very shortly.

i Alzheimer’s Society (2014). Financial Cost of Dementia. http://www.alzheimers.org.uk/site/scripts/documents_info.php?documentID=418 Accessed 22

nd August 2015

ii Kaur, G. and Levy, E. (2012). Cystatin C in Alzheimer’s disease. Frontiers in Molecular Neuroscience. 5: 79 iii Tizon, B. et al. (2010). Cystatin C Protects Neuronal Cells from Amyloid-β-induced Toxicity. Journal of Alzheimer’s Disease. 19,

885-894 iv Relini, A., Marano, N., Gliozzi, A. (2013). Probing the interplay between amyloidogenic proteins and membranes using lipid

monolayers and bilayers. Advances in Colloid and Interface Science. 207, 81-92

http://www.alzheimers.org.uk/site/scripts/documents_info.php?documentID=418

http://community.frontiersin.org/people/GurjinderKaur/49146

http://www.frontiersin.org/Molecular_Neuroscience/10.3389/fnmol.2012.00079/abstract

Student: Laura Bruce Supervisor: Neil Kad Creating a nucleosome array for single molecule investigation Background: The development of single molecule techniques has allowed for in-depth investigation into proteins and DNA and how they interact with each other and other molecules that they may encounter within a cell. Of particular interest within the Kad lab is how DNA repair proteins locate damage in the genome. Using a single molecule technique known as DNA tightroping it is possible to directly image these interactions, these investigations have shown that proteins find their targets sites on DNA through a range to different mechanisms[1], however it has not yet been discovered what need there is for these multiple pathways. The eukaryotic genome is structured in a very organised way and there are many mechanisms in place to create a compact structure so that large amounts of DNA can be stored within the cell. The organisation of the DNA involves histone proteins that form a complex with DNA to create nucleosomes. These nucleosomes are arranged as beads on a string, as seen in figure 2, would act as a roadblock to the movement of proteins along the DNA Aim: To test the hypothesis that proteins use jumping as a means to negotiate physical boundaries created by the structural organisation of DNA and to understand the impact of roadblocks on the ability of proteins to find their target sites. To achieve this, arrays of nucleosomes on DNA tightropes must be created, and single molecule techniques used to image how proteins involved in DNA repair are impeded by these structures. Work carried out: Xenopus laevis histones H2a, H2b, H3 and H4 held in PET3a vectors were received, these were the original histones used by K. Luger et al[2]. These histones were transformed into BL21, TOP10 and XL10 GOLD competent cells and then the plasmid DNA isolated. These transformations were carried out in 3 separate competent cells as there was an issue with achieving the desired concentrations of plasmid DNA to perform sequencing. After isolating the DNA, it was digested with the restriction enzymes NdeI and BamHI. This was necessary to facilitate the subcloning of the histones into a vector that would incorporate a poly-histidine tag (histag). PET28b was

Figure 1. 147 bp of DNA is wrapped 1.65 times around the core histone complex to form a nucleosome[2]

Figure 2. (A) Chromatin isolated directly from an interphase nucleus appears in the electron microscope as a thread 30 nm thick. (B) This electron micrograph shows a length of chromatin that has been experimentally unpacked, or decondensed, after isolation to show the nucleosomes. (A, courtesy of Barbara Hamkalo; B, courtesy of Victoria Foe.) [3]

Figure 3. Plasmid map of PET3a showing the NdeI and BamHI restriction sites. [4]

http://www.ncbi.nlm.nih.gov/books/n/mboc4/A4754/def-item/A5350/






selected as the recipient vector because an in-frame histag sequence was accessible using the restriction enzymes BamHI and NdeI that were also present in PET3a. PET3a vectors containing H2a and H2b were selected and digested, the products were run on

an agarose gel to extract the inserts containing the H2a and H2b genes. PET28b was also digested and run on an agarose gel, the vector was gel extracted. If H2a and H2b inserts had been obtained the H2a and H2b would have been ligated into the PET28b and then transformed into competent cells. Results: Histone DNA concentration (ng/ul) H2a 124.5 H2b 1.5 H3 1.9 H4 2.5 Figure 5. Concentration of DNA after transformation into BL21 cells. Histone DNA concentration (ng/ul) H2a 22.5 H2b 23.4 H3 23 H4 34 Figure 6. Concentration of DNA after transformation into TOP10 cells. Histone DNA concentration (ng/ul) H2a 53.7 H2b 17 H3 22.6 H4 14.6 Figure 7. Concentration of DNA after transformation into XL10 GOLD cells.

Figure 4. Plasmid map of PET28b showing the NdeI and BamHI restriction sites and the in frame histag. [5]

Figure 8. Agarose gel. Lane 1: digested PET28a. Lane 2: digested PET3a containing H2a. Lane 3: digested PET3a containing H2b. Lanes 4, 5 and 6 are repeats of lanes 1, 2, and 3. The weights of DNA indicate the amount of DNA run on each lane. Figure 8 showed a large enough band of digested PET28a for gel extraction. No insert was seen here but the insert was not required and was expected to only be a few bases long. No band was seen in lane 2, even though an extremely high concentration of DNA was run on the gel, this indicated that the high concentration seen for H2a in BL21 vectors was most likely caused by a contaminant. Lane 3 showed a band, but no insert. The insert was the part of the vector containing the H2b so this could not be used. Techniques and skills acquired during studentship: -Making agar plates and growth media -Microbiology -Making and accurately measuring solutions -Isolating plasmid DNA -Gel extraction of DNA -Making agarose and acrylamide gels -Appreciation of single molecule approaches -Planning and executing experiments -Time management Value of Studentship: This studentship has given me confidence in the lab, it has also taught me skills that I hope to use if I undertake a Masters or PhD program. I have also learned a lot about reading and researching on an individual topic and have gained a detailed understanding of the molecular biology required in creating a nucleosome array. I have also undertaken a lot of reading on single molecule techniques and have a greater understanding of the research being undertaken at the moment in this field. I was interacting with the PhD students in the Kad lab on a daily

basis and experienced some of the research that is taking place in the lab at the moment, and gained an understanding of their projects. I also learned that in real-life research there can be a lot of setbacks and frustrations. Research is not predictable and takes much longer than is immediately apparent. This project came with a lot of challenges and it taught me how to appropriately deal with these setbacks and come up with a new plan. I gained a lot of independence over these 8 weeks and feel privileged to be given this opportunity. References:

[1] N. M. Kad and B. Van Houten, Mechanisms of DNA Repair, vol. 110. Elsevier, 2012.

[2] K. Luger, A. W. Mader, R. K. Richmond, D. F. Sargent, and T. J. Richmond, “Crystal structure of the nucleosome core particle at 2.8[thinsp]A resolution,” Nature, vol. 389, no. 6648, pp. 251–260, Sep. 1997.

[3] F. Malouin and L. E. Bryan, “Molecular Biology of the Cell,” no. 4th edition, 2002.

[4] “https://www.addgene.org/vector-database/2637/.”

[5] “http://www.helmholtz-muenchen.de/fileadmin/PEPF/pET_vectors/pET-28a-c_map.pdf,”

Identifying the intracellular survival strategies of chlamydiae Student: Laura Pokorny Supervisor: Dr Paul Pryor, Centre for Immunology and Infection, University of York Daily Supervision: Hayley Clissold Background Chlamydia trachomatis is an obligate, intracellular pathogen that can infect a broad number of organisms ranging from humans to free-living amoebae. C. trachomatis is a common cause of urethritis and cervicitis and long term effects include pelvic inflammatory disease (PID), ectopic pregnancy, tubal factor infertility and reactive arthritis[1]. It has a unique intracellular developmental cycle, whereby it alternates between two distinct morphological forms: the infectious and metabolically inactive elementary body (EB) which is taken up into a phagocytic vesicle, and the metabolically active and replicative reticulate body (RB), which replicates by binary fission inside the bacterium-containing vacuole termed the inclusion. These inclusions are non-fusogenic with the components of the lysosomal pathway[2] thus can establish an intracellular niche within the cytoplasm of the host cell, and quickly divert from the phagolysosomal maturation pathway. C. trachomatis expresses a type-III secretion system (T3SS) which enables delivery of the bacterial effector proteins directly into the host cytoplasm to modulate host cellular function[3]

, including inhibition of phagolysosomal destruction and modulation of intracellular trafficking. Thus, the T3SS is an appealing target for the identification of pathogen virulence factors. Aims and overview of the project The aim of the project was to identify chlamydial effector proteins secreted by the bacterium’s T3SS that are involved in disrupting intracellular trafficking pathways, using both a targeted approach and a random approach. The targeted approach uses the in silico prediction program, EffectiveT3 to predict proteins that are secreted by the C. trachomatis T3SS. These predicted proteins were then cloned into bacteria via a plasmid vector for amplification. The plasmid DNA is isolated and used to transform BHY10 yeast strains, which contains a copy of the vacuolar hydrolase carboxypeptidase-Y (CPY) fused to invertase (CPY-inv). The random approach uses a random genomic library of C. trachomatis genes to transform BHY10 yeast strains. If normal trafficking is perturbed, CPY-inv is missorted and thus secreted from the cell. An overlay solution will be laid over the colonies in order to test for CPY-inv secretion. A brown precipitate will be seen in the case of a positive result. Description of work A random genomic library extracted from C. trachomatis EBs was inserted into pVT100-U vectors prior to my arrival. The vectors were then used to transform Stellar competent bacterial cells and BHY10 yeast. Colony PCR of transformed Stellar Competent Cells: A colony PCR was performed to amplify plasmid DNA containing the gene of interest. 10 reactions for each gene of interest were performed (alongside a control containing 0.5µl stock undigested pVT100-U). These were then run on an agarose gel and imaged using the GeneSnap programme to check for inserts. MiniPrep: The plasmid from one colony that had been identified to contain the gene of interest was purified using the protocol provided by the QIAprep Spin Miniprep Kit[4]. Due to the low copy number of the plasmid, buffer concentrations were doubled. Transformations: 50µl Stellar Competent Cells were transformed with 5ng of DNA, 400µl of SOC medium added and plated on LB-amp plates. The yeast strain BHY10 was grown overnight in yeast extract-peptone-dextrose (YPD) broth at 30°C, 250rpm. Yeast were pelleted by centrifugation at 3.3 x g for 1 minute, mixed with 100µg of salmon sperm DNA (ssDNA) and 2.25µg purified plasmid DNA. 250µl of PLATE solution (10mM Tris, pH7.5, 1mM EDTA, 1M LiOAc, 50% (w/v) PEG4000) was added prior to overnight incubation at 30°C. Yeast were then pelleted by centrifugation at 3.3 x g for 1 minute, resuspended in water, plated onto SC-ura plates and incubated at 30°C to allow the transformed colonies to develop. Assay: Transformed BHY10 were streaked onto SC-ura plates alongside positive (BHY10 ΔVPS10 + pVT100-U) and negative (BHY10 + empty pVT100-U) yeast controls. An overlay solution containging 0.17M sucrose, 4mM sodium acetate buffer pH 5.5, 8µM N-ethylmaleimide, 1mg/ml horseradish peroxide (HRP), 160 units glucose oxidase and 10mg/ml o-dianiside was combined with agar (3% w/v) and poured over plates containing transformed yeast and controls. A brown precipitate is seen when the o-dianiside is oxidised. Results and Discussion In the whole library screen (the random approach), the yeast were grown covering the genome 38x. 18 brown colonies were identified after performing the assay, suggesting an intracellular trafficking defect. These colonies were restreaked onto new SC–ura plates, against positive and negative controls, in order to be reassayed the next day. In the reassay, 3 of the 18 colonies were identified to be convincing positives. These were named positive secreting colony (PSC) 6, PSC 8 and PSC 18. Through the targeted approach, two of the predicted effector proteins gave positive results. These were BOUR_00381 and BOUR_00916. These 5 colonies were retested on SC-ura plates with PSC 8, BOUR_00381 and BOUR_00916 still giving strong positive results after this second assay. A colony of PSC 8, BOUR_00381 and BOUR_00916 was picked and spread onto separate 5-FOA plates. The 5-FOA plates allow for selection against yeast carrying the plasmid. 5-FOA forms a product that is toxic to yeast containing the URA3 gene (which is encoded on the pVT100-U plasmid). However, as yeast naturally kick out plasmids, only those yeast that have got eliminated their plasmid will grow on the 5-FOA plate. A colony was picked from each 5-FOA plate and, after 2

days incubation at 30°C, spread onto yeast-extract-peptone (YEP) plates. These were then assayed to ensure that the result seen previously was plasmid dependent, and not a mutation in the yeast. This would be evident by the absence of a brown precipitate. However, in the assay on the YEP, all three showed the brown precipitate (fig. 1) suggesting that the positive results seen were due to a mutation and not the gene inserted into the plasmid. (a) (b)

Fig. 1. Assay of positive colonies on YEP after growth on 5-FOA. Positive colonies were spread onto YEP plates after growth on 5-FOA plates and assayed to screen for trafficking defects. All clockwise from top: (a) Positive control, BOUR_00916, BOUR_00381, negative control; (b) Positive control, PSC 8, negative control.

The whole library screen was repeated, with the genome being covered 36x this time, and 24 potential positively secreting colonies were identified and named PSC 19-42. Of these, 18 were shown to be still secreting in the second assay. After the third assay, three of these (PSC 20, 24 and 26) were shown to be convincing positives (figure 2). (a) (b)

Fig. 2. Third screen for PSCs on SC-ura. Colonies thought to be positive were spread again onto SC-ura and screened. All clockwise from top: (a) PSC29, PSC28, PSC27, PSC26 ; (b) PSC24, PSC23, PSC22, PSC21, PSC20, PSC19

Departure from the original protocol We aimed to isolate the plasmid DNA in the transformed yeast of any positive colonies and propagate it in bacteria. We would then isolate that plasmid DNA and transform yeast with it in order to reassay, as well as sequencing the plasmid to identify the gene of interest. This would be a way to check that the yeast has not mutated and to also double check that it is a plasmid dependant result we are seeing. Unfortunately, in the time I was in the lab we didn’t find any true positive colonies and so did not get onto this stage. Future directions The PSCs 20, 24 and 26 will be streaked from the 5-FOA onto the YEP once growth on the 5-FOA is seen. This will then be assayed in order to test that the positive results are plasmid dependent. Once any effector proteins have been identified, immunoprecipitation will be performed to identify binding partners of the effector protein and isothermal calorimetry can be used to characterise the strength of interaction. The identified chlamydial effector protein can then be crystallised so that the full structure can be analysed and, if possible, their biological activity determined. Value of the studentship Not only has the studentship been extremely enjoyable, it has also been instrumental in helping me decide what I want to do after my undergraduate degree. I now know I would like to apply for PhDs, and having the opportunity to talk to and work with the PhD students here has been incredibly helpful. Dr Paul Pryor and Hayley Clissold have been excellent teachers, teaching me not only important laboratory techniques but also how to methodically address unexpected issues that may arise – something that cannot be properly addressed during large, time-constrained undergraduate lab session. By the end of the 8 weeks I was truly invested into the project and will undoubtedly keep in touch to find out where it goes. My confidence in the laboratory has developed dramatically and I am now thoroughly excited and more prepared for undertaking my final year project. References 1. Paavonen J & Eggert-Kruse W. (1999) Chlamydia trachomatis: impact on human reproduction, Hum Reprod Update. Sep-Oct;5(5):433-47. 2. Fields KA, Hackstadt T. (2002) The chlamydial inclusion: escape from the endocytic pathway. Annu Rev Cell Dev Bio. United States. 18:221-45, 3. Betts-Hampikian HJ, Fields KA. (2010) The Chlamydial Type III Secretion Mechanism: Revealing Cracks in a Tough Nut. Frontiers in Microbiology.1:114. 4. QIAGEN (2012) QIAprep® Miniprep Handbook (PDF), p19, [Accessed: 28th July 2015]

Student : Leanne O’Sullivan, Biomedical Science Year 3, University College Cork

Supervisor : Dr. Paul Young, Dept. of Biochemistry, University College Cork

Construction of a novel bacteria-based DNA detection system as a simple,

low-cost alternative to current molecular diagnostics tools.

Background

Real time PCR and Quantitative PCR both currently play major roles in diagnostic labs in the detection and amplification

of target DNA sequences. These may be targets found in pathogens such as viruses or mutated genes such as in cancer

patients. The advantages of these methods is that they are quick, accurate and offer a detailed diagnosis unrivaled by

other tests. Molecular analysis makes it possible to distinguish viral and bacterial strains and also identify specific gene

mutations involved in disease, which then allows patients to receive more targeted treatments. However, these methods

of analysis are not universally available to patients. This is due to the costly nature of instruments and reagents, the need

for specialised training of staff and also the need for strict controls in procedure to prevent contamination between

samples. Where PCR diagnostics are not feasible, such as in under-resourced labs, temporary medical facilities and POC

tests in the GP office, an alternative is needed.

Aims

The aim of this project was to optimise and evaluate a novel diagnostic tool constructed by UCC iGEM Team 2014. The

system works on the principle that a DNA plasmid may only enter a competent bacterial cell and be transcribed if it is

circular. Hence by creating a linear detector plasmid that may only be circularised by binding to a specific nucleic acid

target, transformation with competent cells and transcription of a reporter gene acts as a simple readout. Binding of target

to detector is allowed by the presence of single stranded overhangs on the detector part complementary to the target. The

novel system constructed has been titled “Basehunter” by Cork iGEM team 2015. Over the summer, the aim of this project

was to optimise the construction protocol, assess selectivity of the system and construct multiple detectors to demonstrate

Basehunter’s customisability.

Methods

Results obtained by UCC iGEM 2014 suggest that construction of the detector plasmid by digestion with four enzymes

(NtbsI,NtBsQI, KpnI & HindIII) was not optimal due to presence of false positive colonies on negative control tests. It was

suspected and confirmed by sequencing of false positive colonies that this was due to undigested detector plasmid. To

overcome this, construction of the detector was attempted by PCR this summer.The detector was amplified using plasmid

pSB1C3 with cloned detector part containing the four restriction enzyme sites and the target sequence. Primers were

designed containing the target DNA sequence of the target size and annealed between nicking enzyme sites. A protocol

was designed involving amplification of the linearised (by KpnI) detector plasmid and subsequent digestion by the four

restriction enzymes. A decoy reaction was also carried out by reacting an excess of oligos complementary to the top

strand of the detector sequence with the digested plasmid to remove the top strand, yielding the single stranded

overhangs of the detector part. PCR cleanups were carried out to remove the decoy oligos and restriction enzymes and

produce the final activated detector.

Another aspect of the work carried out was the assessment of specificity of the detector system. Specificity is the ability of

the detector to select the correct target when non-target DNA is present, preventing false positives. The detector’s ability

to select the correct size target was initially tested. This was done by reacting a detector for a 30bp region of HPV L1

gene with targets of sizes ranging from 30bp to 9bp.

Also assessed was the detector ability to select the target in the presence of mutated targets. These mutations included

random sequences of correct target length, 3bp changes in correct sequence and insertions and deletions in correct

sequence. These mutated and varying length targets were synthesised commercially.

Finally to assess selectivity, the sry detector (targeting a region of the sry gene on the Y chromosome) was reacted with

genomic DNA samples to evaluate whether it could differentiate male (Y chromosome) and female (no Y chromosome)

genomic DNA. To carry this out, genomic DNA was obtained from mouse tails and digested with various restriction

enzymes to release fragment of correct size for detection. Another aspect of the work carried out was the design and

construction of detectors targeting various sequences. Constructed during the project were two HPV detectors (targeting

55bp and 30bp regions of L1 gene on Human Papillomavirus), two sry detectors (targeting 62bp and 32bp regions of sry

gene) and one mycobacterium detector (24bp region of ML0319 lipoprotein gene). These were designed using sequence

analysis software, Benchling and constructed by PCR method.

Results

It was deduced that construction of the detector was optimally done using PCR as background CFUs obtained was

reduced. A decoy reaction was included in the protocol and maximised CFU achieved by positive reactions.

The selectivity of the detector was assessed and gave mixed results. Reaction with targets of various size suggested that

the detector was able to select the correct size target significantly better than smaller targets and did not detect

sequences <50% target size. However it was less efficient in selecting correct target size when larger targets were

present. Sequences which had additional bases attached to target sequence were detected almost as frequently as target

of correct size. This suggests that background colonies may be obtained where a lot of DNA is present in sample but also

that detector may react with target even if not digested to correct length. Reaction of the detector with mutated targets

revealed that the detector was highly efficient in selecting correct target where a random sequence of correct length was

present. However, where minor base changes or deletions were present, a significant number of positive CFUs were

obtained, suggesting efficiency in detecting mutations was poor.

Experiments conducted using the sry detector and genomic DNA were inconclusive. Results obtained using the digested

detector suggest that the male sample returned the most positive CFUs. However when using the PCR constructed

detector, the female sample returned the most colonies. The detector size was then reduced from 62bp to 32bp in an

attempt to improve this result, however reaction of this detector with genomic DNA was not completed in time.

Results generated using various detectors were similar and comparable, suggesting all detectors worked equally well.

Design was simple and quick, highlighting a clear advantage of the Basehunter system.

Conclusion

Overall, results suggest that the Basehunter system shows promise as a viable diagnostic tool but further work is needed

for the optimisation of selectivity of the system. Construction has been optimised to maximise performance. Selectivity

assessment suggests that the detector may be used to identify presence or absence of a target sequence, as it

successfully selects target of correct length. However, the system is currently not capable of detecting minor mutations in

sequence and further work is needed to allow this function. Also, further work is needed to optimise ability to use detector

with genomic samples as this is ultimately where the detector will be of use. In the future, these faults may be corrected.

Also, the system may be improved further by the development of a software that could locate target sequences and

design detector part and primers for amplification automatically. In addition, protocol for detector reaction may be

simplified by automating reaction and transformation. Also, the readout system may be altered to improve specificity such

as by adding a reporter gene to detector part that may be expressed by detection of correct target sequence only.

Reflection

In carrying out the summer studentship, I gained valuable experience in the world of research.

It was a great opportunity to get insight especially into the development of diagnostic tools used

in my field, Biomedical science. I became highly interested in the standards and requirements

needed for the introduction of such a system into diagnostic use and would be interested in the

future in being a part of the research and development of diagnostic tools. I learned a lot about

recording and presenting results, especially while working with others in the lab.

Communication of ideas and findings was an important aspect of everyday work that I think I

improved upon. I also got the opportunity to travel to the iGEM Giant Jamboree to present

results with the team. I met many others enthusiastic about synthetic biology there and saw

limitless possibilities in that field.

References ■ UCC iGEM Team 2014, Wiki : http://2014.igem.org/Team:UCC_Ireland (2014).

■ Cork iGEM Team 2015,Wiki : http://2015.igem.org/Team:Cork_Ireland (2015)

■ Sails AD (2009). "Applications in Clinical Microbiology". Real-Time PCR: Current Technology and Applications.

Caister Academic Press. ISBN 978-1-904455-39-4.

■ Niemz, A., Ferguson, T. M. & Boyle, D. S. Point-of-care nucleic acid testing for infectious diseases. Trends

Biotechnol. 29, 240–250 (2011).

Biochemical Society Summer Vacation Studentship Report 2015

Student: Lee Ross Burdon

Supervisor: Dr Ritchie Williamson

Hyperthermia Induced Hyperphosphorylation of Tau in Alzheimer’s Disease

Alzheimer’s disease is a form of neurodegeneration that currently affects over 800,000 people in the UK and is the leading form of dementia. Two distinct features are extracellular senile neuritic plaques and intracellular neurofibrillary tangles (NFT). NFTs are composed of the protein tau which has been shown to be hyperphosphorylated causing a decrease in tau’s ability to stabilise microtubules. This leads to a loss of cytoskeleton integrity and affects the normal functioning of the cell. In addition, cognitive decline correlates with the number of NFTs and therefore identifying the kinases responsible for specific phosphorylation events would help researchers to develop methods to inhibit the formation of NFTs. It has been previously reported that hyperthermia can induce changes in the phosphorylation of tau similar to that found in Alzheimer's. The aims of the project were (I) to determine specific sites in tau that are phosphorylated in response to temperature change (a novel model of tau hyperphosphorylation) using phosphor-specific antibodies and phosphomapping , and establish which of those overlap with those found in tangle pathology. (II) To identify which kinases and phosphatases are responsible for the specific phosphorylation events in tau using the novel slices model.

In order to meet the objectives of the study, hippocampal brain slices were obtained using a McIlwain tissue chopper and stabilised at room temperature in oxygenated artificial cerebrospinal fluid (ACSF). After stabilisation, slices were incubated at two different temperatures (35°C or 37°C) in ACSF in order to determine changes in phosphorylation of tau dependent on temperature. Glucose use in the brain decreases as we age and is exacerbated in Alzheimer's and so the temperature-dependent changes in tau phosphorylation were also investigated using ACSF with different glucose concentrations (2 mM and 10 mM). The reversible nature of tau phosphorylation was also investigated by incubating slices at 35°C for 1 hr followed by incubation at 37°C for a further 1 hr and vice versa. After incubation, slices were collected and processed for downstream analysis of phosphorylation changes. Brain slices were homogenised in lysis buffer followed by centrifugation at 4°C for 20 minutes at 15,000 RPM. The supernatant was removed and protein concentration of lysate was determined using the Bradford assay. For analysis of tau protein phosphorylation changes, SDS-PAGE followed by immunoblotting was performed with a panel of total and phospho-specific antibodies (see Figure 1).

Tau phosphorylation appears to increase when slices are incubated at 35°C compared to 37°C. Total tau antibody detected a shift in the electrophoretic mobility of tau (indicative of increased phosphorylation) when slices were incubated at the lower temperature. This shift was reversed when slices were switched back to 37°C. In addition, tau phosphorylation was increased at the phospho 396 epitope (a site that displays increased phosphorylation in Alzheimer brain). This was more obvious for the slices incubated in 10 mM glucose containing ACSF, a change that was not reflected in total tubulin levels indicating a specific phosphorylation change. Further analysis revealed no change in glycogen synthase kinase 3 (GSK3) phosphorylation at its regulatory sites (Y216 and S9). The results indicate that hyperthermia leads to increased phosphorylation of tau but reverting back to normal body temperate (37°C) can reverse this phosphorylation providing the tissue has adequate glucose available.

Figure 1. Representative Western blots performed using various antibodies. Hippocampal slices were incubated in ACSF containing either 2 mM or 10 mM glucose at the indicated temperatures (°C) for 1 hour, or 1 hour followed by a different temperature for a further 1 hour.

We originally intended to identify which kinases and phosphatases are responsible for the specific phosphorylation events in tau. Unfortunately, there was insufficient time for this to be accomplished as the viability of the slices was initially difficult to maintain. The next step is to identify further phosphorylation changes in tau before trying to identify which kinases are responsible.

Value of the studentship

From the student:

The summer project has enabled me to develop confidence at using a variety of common laboratory techniques which will be invaluable in enabling me to perform well in my final year project, future research and job applications. It also made me consider my opinions on the ethics of animal research. I am still unable to condone any form of testing on animals that causes unwarranted suffering but I now accept that not all animal testing is beyond me. The mice were well looked after, with the ability to exercise on a wheel, and were euthanized quickly and with minimum suffering. I would be happy to continue performing this type of research on animals as the benefits of the research were to improve our understanding of human disease. As for my career aspirations, I now know that I definitely enjoy working within a laboratory but I am still unsure of the direction I will take. I would consider undertaking a PhD, although, I would prefer to gain some industry experience first and make that decision in the future.

I would like to say thank you to the Biomedical Society and Dr Ritchie Williamson for giving me this fantastic opportunity and also to everyone else that gave me help and advice throughout the project.

From the supervisor:

The benefits of this project were two-fold, it provided an excellent opportunity to encourage talented undergraduate students to pursue a career in basic research (especially in neurodegeneration), and to give real experience of research as opposed to the 'cook book projects' they undertake as part of their undergraduate program. It also validated our laboratories approach in trying to model complex neurodegenerative correlates without recourse to whole animal in vitro studies.

Background

Complement is a vital branch of the innate immune response and consists of numerous plasma proteins which have a

collaborative role in host-defence against foreign pathogens. The complement system can be activated by three activation

pathways: the classical, lectin and alternative pathways which congregate at C3, which is the central protein of the complement

system. Unlike the classical and lectin pathways, the alternative pathway is constitutively active; this “tick-over” mechanism

allows the system to be primed for full activation. The alternative pathway is initially activated via the spontaneous hydrolysis of

a thioester bond in C3; this initiates a cascade resulting in the formation of a C3 convertase called C3bBb. This convertase results

in a positive feedback amplification loop by cleaving more C3 into immunologically active C3b.

The effector functions of complement are extremely potent and have the potential to harm the host. Hence, the down-

regulation of these functions on host cells, whilst not detrimentally impacting their effect on foreign pathogens, is crucial. A

major complement regulatory protein is Factor H; it has a significant contribution to the protection of host cells. Factor H has

multiple roles; these include: binding C3b, C3bBb decay acceleration and cofactor activity for Factor I mediated C3b proteolytic

inactivation. C3b is degraded to iC3b and eventually to C3c and C3d. Three C3b binding sites on Factor H have currently been

identified and these are located on short complement regulator domains 1/4, 6/8 and 19/20 (1). As C3b and C3u are functionally

synonymous, structural studies carried out on C3b-FH complex formation are analogous to those with the C3u-FH complex

formation. Hydrolysis of the thioester domain in C3 produces C3u, whereas C3b is generated via C3 cleavage to remove C3a.

Recently, conflicting data has emerged which raises uncertainty of the previously assumed 1:1 binding valency between Factor H

and C3b; with studies suggesting a bivalent interaction with two C3b molecules binding at opposite ends of Factor H. (2;3)

Aims

Perform analytical ultracentrifugation studies in order to elucidate solution structures of the complex formed between Factor H

and C3u to determine whether this has a 1:1 or 2:1 binding stoichiometry under physiological conditions.

Experimental Procedures

Purification of Complement Factor H│ CFH was purified in micromolar amounts using five chromatographic stages via an in-

house protocol. CFH was purified from frozen human plasma stock; the plasma was thawed and centrifuged to remove

precipitated material. The supernatant was collected, filtered and then dialysed in running buffer. Subsequently, the

supernatant was passed through a gravity-driven non-immune immobilised IgG on Sepharose column. This was followed by a

Lysine-Sepharose column and then an immunoaffinity column using MRC-OX23 anti-FH monoclonal antibody; the CFH was

eluted from the column using 3M MgCl2 solution. Next, the CFH was repeatedly dialysed in HEPES buffer and then passed

through a GE HiTrap Protein G HP column to remove antibody contaminants. Finally, the CFH solution was concentrated in a

centrifuge using centrifugal filter units and gel filtrated using a GE Superose 6 column to remove any other contaminants.

Purification of C3│ Initially, EDTA was added to freshly collected venous blood in order to prevent clotting. The blood was then

spun down to separate the plasma from the other blood components. The plasma was centrifuged further and PEG was added

to promote precipitation; helping to increase the relative concentration of C3 in the plasma. Pefabloc® was then added to

prevent proteolytic degradation before purification and the plasma was manually filtered using a filter unit. The first

chromatographic stage involved using a GE Q-Sepharose anion exchange column run with an ÄKTA purification system, C3 was

eluted using a NaCl gradient. The second column used to purify C3 was a GE MonoQ anion exchange column; this removed

major contaminants in the plasma and C3 was again eluted with a NaCl gradient. The final purification step, which took place

after the C3 had been centrifugally concentrated, was gel filtration using a GE Superose 6 column. This step removed any

degradation products and other minor contaminants that may have been present. The purified C3 was then hydrolysed to C3u

by incubation with hydrazine. Finally a functional assay was carried out to confirm the C3 to C3u conversion using the

complement regulators CFH and CFI, which cause proteolytic degradation of C3u, but not C3.

Analytical Ultracentrifugation│ Analytical ultracentrifugation (AUC) is a versatile technique which allows quantitative analysis of

macromolecules in solution. Sedimentation data is logged by optical systems which observe samples during sedimentation

under a sufficient gravitational field. In recent experiments, AUC has shown the presence of the 2:1 complex; however, this

deduction was inconclusive as the concentrations of protein used were too low. After purification, various mixtures and

concentrations of CFH, C3, and C3u were sedimented to study the stoichiometry; the data was analysed using SEDFIT software.


Assay of cofactor activity for

Factor I-catalysed cleavage of

C3u. The inferred identities of

Coomassie-stained protein bands

on an SDS-PAGE gel are shown.

The functional assay was carried

out with excess FI and C3u in a 2:1

ratio with FH in micromolar

amounts.

Complement Factor H and C3u were used for two AUC analyses. The initial experiment was carried out using a 1:1 ratio of

C3u:FH at 6.2 µM and the second experiment at 9 µM. The AUC analyses were performed at 6oC, 20

oC and 30

oC and the data

collected were interpreted using a SEDFIT analysis. The first experiment resulted in three peaks which corresponded to FH, C3u

and a 1:1 complex. The binding complex peak was relatively low in comparison to peaks suggested by other studies; this factor

in addition to the lack of a 2:1 peak gave cause for further investigation in a second experiment at higher concentrations. Results

from the second analysis were very similar and peak integration allowed the Kd values to be calculated. The Kd values were 29.05

µM and 30.37 µM for the first experiment at 6oC and 20

oC respectively and 78.46 µM for the second experiment at 20

oC.

The overall results collected show a reproducible formation of a weak transient complex as indicated by the relatively high Kd

values. The lack of a 2:1 binding complex between FH and C3u suggests the non-existence of this form of complex, this may be

due to the complexes being mutually exclusive of one another which allows only a 1:1 complex to form.

Departures from Original Proposal & Future Directions

There were no deviations from the original proposal. The data will be used to supplement earlier studies of this complex with a

view to its eventual publication.

Value of Summer Studentship

This studentship has been instrumental in compounding my desire to pursue a research laboratory based PhD. Moreover, the

opportunity to develop my scientific writing, communication and presentation skills has substantially increased my confidence

levels, especially when interacting with colleagues of all ages in a work environment. My practical competence has improved to

a point where I actively search for occasions to try out new techniques and equipment. I have not only gained a deeper

understanding of life in the laboratory, but also how to deal with problems which arise from all possible aspects of a professional

setting. I have learnt to work collaboratively with other members of the group in addition to working independently without

needing assistance or reassurance. Accordingly, I am now looking forward to working on my final year project rather than being

subjected to my previous apprehension. I have truly appreciated being part of a project with significant scientific relevance and

also being provided with the opportunity to use and be trusted with niche technology which I haven’t experienced as part of my

degree course. Professor S. J. Perkins adds: The summer project by Louis Buckley progressed very well and we are more

confident of our understanding of the stoichiometry of the C3u-CFH complex. We hope to publish our conclusions in due course.

References

1. Jokiranta TS, Hellwage JF, Koistinen VF, Zipfel PF FAU, Meri S. Each of the three binding sites on complement factor H interacts with a distinct site on C3b.(0021-9258 (Print)).

2. Kajander T, Lehtinen MJ FAU - Hyvarinen S, Hyvarinen S FAU - Bhattacharjee A, Bhattacharjee AF, Leung E FAU - Isenman D, Isenman DE FAU - Meri S, et al. Dual interaction of factor H with C3d and glycosaminoglycans in host-non host discrimination by complement.(1091-6490 (Electronic)).

3. Wu J, Wu YQ FAU - Ricklin D, Ricklin DF, Janssen BJ FAU - Lambris J, Lambris JD FAU - Gros P, Gros P. Structure of complement fragment C3b-factor H and implications for host protection by complement regulators.(1529-2916 (Electronic)).

RNA processing in the Plasmodium falciparum apicoplast

Louis Wilson Department of Biochemistry, University of Cambridge Supervisors: Dr Ellen Nisbet, Prof. Chris Howe Introduction and Project Aims Apicomplexan parasites have become remarkable in recent years for the discovery of their algal ancestry. Most apicomplexans, including Plasmodium spp., possess a remnant chloroplast – the apicoplast – complete with its own genome and proteome (albeit small in size). Since antimalarial drugs such as doxycyline and clindamycin target apicoplast gene expression, it is in the interest of researchers to understand more clearly how apicoplastic genes are expressed – continued research may lead to the development of further drugs. In Plasmodium falciparum, it is currently understood that genes in the apicoplast are initially expressed as a small number of long, polycistronic transcripts, which, in order to be translated, are subsequently cleaved into individual open reading frames (ORFs) by RNA processing enzymes. It has also more recently come to light that the ribosomal protein-encoding gene rpl2 undergoes a G→A editing event towards the 3’ end (Nisbet et al 2015, submitted). The aim of this project was to observe snapshots of RNA intermediates at different stages of such processing, and determine the point at which RNAs are edited, as well as examining the general processing mechanism (an addition to the original proposal) using the procedure below. A bioinformatic analysis was also to be conducted in order to identify potential reasons for editing to be necessitated in vivo. Experimental methods Prior to the project, parasites were subjected to a treatment of rifampicin, an inhibitor of RNA polymerase, by Dr Nisbet for either 1, 2 or 6 hours before initiating the first step of RNA extraction. During the project, RNA extraction was completed; RNA was then circularised using an RNA ligase. First strand synthesis employed a primer complementary to a site near the 3’ end of rpl2, creating a cDNA concatemer (polymerisation continues several times around the circle). Reverse transcriptase was not added to a second set of similarly-treated RNA preparations, which were retained as negative controls. Outward-facing primers annealing to sequences at each end of rpl2 were then used to amplify the intervening sequence containing the regions adjacent to the gene by PCR (their termini ligated during the circularisation reaction). PCR using conventional inward-facing primers was also conducted as a control. PCR products were examined using gel electrophoresis, before cloning into a plasmid vector. Competent Escherichia coli cells were transformed with the plasmid by heat shock transformation, and were spread on ampicillin plates to permit blue-white selection of positive transformants. White colonies were checked for successful transformation by colony PCR (primers annealing to sequences either side of the insert) before culturing and mini-prepping. Purified plasmids were submitted to an internal facility for sequencing, and the sequences themselves analysed using NCBI’s BLAST algorithm.

Rpl2 protein sequence analysis was also conducted using BLAST, while structural analysis involved the use of the open-source molecular visualisation system PyMOL.

Results Obtaining the desired cDNAs with minimal contamination proved to be very difficult. This may have been due to RNA samples being of insufficient quality or quantity (rifampicin-treated cells intuitively contain less RNA than is typically encountered), but was at least partly caused by exogenous DNA contamination (or failure to fully digest DNA during the extraction procedure). However, one cDNA preparation appeared successful following diagnostic electrophoresis (see Figure 1), and provided the bulk of the data collected during the project. Unfortunately, due to unanticipated deterioration of the ampicillin stock, three quarters of the cells transformed with the PCR products originating from this cDNA had to be discarded due to bacterial contamination. Nevertheless, several colony PCRs were conducted to analyse the many remaining transformants. A large proportion of identified transformants proved to possess only a very small insert in the plasmid vector (a few tens of base pairs), which was demonstrated by the negligible difference in band size between these clones and blue colonies assayed as negative controls. Initially, efforts were primarily focused on purifying plasmids containing the larger inserts, which should yield the sequences of interest. A list of implied RNA species was then generated by analysing the derived sequences in silico (see Table 1). Most of the transcripts sequenced had already been edited by the time RNA was extracted; this suggests that the editing occurs quite early relative to mRNA cleavage events, although more data are required to confirm this conclusion owing to the surprising existence of unedited transcripts from the 6-hour treatment. In addition, the assay

Fig. 1. PCR amplification of cDNAs synthesised from

circularised rpl2 transcripts extracted after 1, 2 or 6 hour

treatment with rifampicin. Internal primers permitted

conventional amplification of the rpl2 locus, whereas

circular primers amplified the encompassing sequences.

100 kb DNA ladder was added to the first and fourteenth

wells.

lacks the capacity to detect editing in unprocessed transcripts, as they possess multiple phosphate residues at the 5’ end that prevent their circularisation. Examining the points at which the transcripts are cleaved provided some interesting observations. Several transcripts shared the same position of cleavage at one end (but not the other), advocating a degree of sequence-specificity in endonuclease activity. Many of the sites also corresponded to the precise 5’ or 3’ ends of ORFs. 3’ processing did not appear to be co-ordinated with 5’ processing. Contaminating sequences were also detected; while some of these were derived from other taxa, a large proportion of cloned contaminants were primer dimers of sorts. Many of these primer dimers contained small inserts of 3–15 bp length, and were often of indeterminable origin. However,

one such insert, of sequence ATGACCCCT, was consistently detected across several preparations, albeit sometimes truncated. Since the first four bases are identical to those following the forward primer in an edited transcript (the fourth being the edited base itself), and the next five bases are complementary to the site of editing in an unedited transcript, it remains to be seen whether this is a by-product of the editing mechanism itself, or simply an artefact of the proximity of reverse transcriptase priming. The G650A edit in rpl2 results in a codon change from glycine to glutamate at position 217 in the encoded protein, ribosomal protein L2 (Rpl2). By comparing the primary sequence of Rpl2 with those of orthologues in a wide range of taxa, it was found that the glycine edited in the P. falciparum apicoplast is highly conserved. Therefore, the edit does not correct an aberrant residue to one that is conserved, contrary to the previous hypothesis. Structural analysis of the Rpl2 orthologue in E. coli indicated that the comparable residue, Gly-232, was very close in space to a pair of residues, Asp-228 and His-229, that have been

reported to be of large importance to the peptidyl transferase activity of the ribosome (Diedrich et al., 2000). Future directions Clearly, more data are required to strengthen the findings of this project. Ideally, new cultures should be grown to provide fresh RNA samples, and systematic replacement of reagents might be warranted to eliminate DNA contamination. It might well also be prudent to repeat the experiment using different primers, especially given the proximity of current primers to the site of editing itself. An interesting subject for further investigation might be the enigmatic antisense mRNAs that appear to exist for every apicoplastic transcript. The processing of these transcripts could be analysed to determine whether they are processed and edited in the same fashion as conventional mRNAs. Value of studentship to student I have learnt a great deal from this studentship. This project has provided a unique opportunity to experience science in practice – to see how research is actually carried out in the laboratory. Owing to the difficulty in conducting the above experiments successfully, I spent a large amount of time working out why assays were not working – this, in itself, is a critical skill in research, and I am glad to have had to experience it. In addition to becoming more aware of the practical aspects of research, I have had the chance to practise many biochemical techniques (including some protein work on the side of the project) that will put me in good stead for the remaining years of my degree and, hopefully, a PhD. This studentship has strongly reinforced my aspiration to become an academic researcher. Value of studentship to laboratory We greatly enjoyed hosting Louis in the laboratory over the summer. He worked extremely hard and was able to produce some excellent data. These have confirmed our suspicion that RNA editing in the Plasmodium apicoplast occurs early, and is important. We hope to include his data in a future publication, and also in future grant applications for further analysis of apicoplast transcription. Dr Ellen Nisbet, 1 Oct 2015 References Diedrich, G., Spahn, C. M., Stelzl, U., Schäfer, M. A., Wooten, T., Bochkariov, D. E., … Nierhaus, K. H. (2000). The EMBO Journal, 19, 5241–50.

Table 1. The 5’ and 3’ ends of cloned apicoplast rpl2 mRNA molecules were mapped to positions in the genomic sequence. Due to primer locations, the assay lacks the power to detect transcripts cleaved between positions 2886 and 3453 (this region is contained by the rpl2 ORF).

Treatment 5’ end 3’ end Length Edited?

1 hr

2824* 3916* 1092 Yes

2392* 3738* 1346 Yes

2750* 4202* 1452 Yes

1909* 3961* 2052 Yes

2857 3846 989 Yes

1909 3846 1937 Yes

2773 3564 791 Yes

? 3739 N/A Yes

2 hr 2506 3876 1370 Yes

6 hr

2869† 4493† 1624 Yes

2756† 4026† 1270 No

2785† 4103† 1318 Yes

2873 4494 1621 Yes

2873 4494 1621 Yes

2756† 4026† 1270 Yes

2785† 4103† 1318 Yes

2756† 4026† 1270 No * ± 1; † ± 3

Interaction of Parkinsons’ and Lysosomal Related Genes

Background and Aims

Parkinson’s disease (PD) is primarily associated with movement disorders. However patients also report non-

motor symptoms including a range of visual defects such as dry eyes, double vision and hallucinations. The

Lrrk2-G2019S mutation is the most common form of genetic PD. Both fruit flies and mice are commonly used

to model PD. Mice models, however, fail to faithfully recapitulate phenotypes associated with visual

dysfunction, fly models conversely have been shown to produce reliable PD phenotypes associated with visual

system. Previous work has shown that expression of Lrrk2-G2019S in dopaminergic neurones results in an

initial gain in visual response in young flies followed by a decay consequently leading to compromised visual

function (1)

. The G2019S mutation has also been linked to disruptions to the endolysomal system (2-4)

. Evidence

suggests the function of Lrrk2 occurs in a Rab dependant manner; with both Rab7(2, 4)

and Rab7L1 (3, 5)

(drosophila orthologue Lightoid) implicated as potential interaction partners.

Using the visual system of model organism Drosophila melanogaster, we aimed to probe for genetic

interactions between Rab7 and Rab7L1 with Lrrk2-G2019S; to provide novel evidence for the function of Lrrk2-

G2019S and its’ role in the pathogenesis of PD.

Work Carried Out

Expression of Rab7 or Lightoid was upregulated by constitutive expression or knocked down by RNAi using the

GAL4/UAS system. Transgene expression was driven specifically in the dopaminergic neurones using Tyrosine

hydroxylase-Gal4 (TH-Gal4) and interactions were tested for with G2019S. Interactions were also tested for

between G2019S and wapr

(negative control) or α-Synuclein-30P (positive control). The visual response of the

transgenic flies was tested at 1 and 7 days using the SSVEP (steady state evoked visual potential) technique.

Whereby, flies were restrained in a Gilson pipette tip and exposed to full field illumination by a blue LED driven

by a flickering wave. The response of the visual network was then recorded by electrodes, one placed on the

eye and the other the mouth.

Results

Our results (Fig 1A, 1B) showed several things of note. Firstly, the results clearly show that the function of both

Rab7 and Lightoid in dopaminergic neurones is linked to the visual response; RNAi of either gene resulting in a

large gain in visual response. Moreover, Lightoid did not appear to interact with Lrrk2. The visual response of

Lightoid flies not differing significantly between flies expressing only the Lightoid transgenes or the transgenes

in the background Lrrk2-G2019S, when compared to the negative control flies. Conversely a clear interaction

was observed with Rab7. Flies constitutive expressing Rab7 in the G2019S background showed a significant

increase in visual response compared to those only expressing Rab7 alone. A similar but less pronounced result

was observed in the Rab7 RNAi flies. These phenotypic differences were visible in 1 day old flies but became

more pronounced by 7 days. By testing at both one and seven days we ensured small phenotypes, which did

not differ significantly from the controls at one day, yet increased overtime would not be dismissed, giving

more confidence in our conclusions. The α-Synuclein-30P mutant (positive control) has been linked to

inherited PD. Flies expressing α-Synuclein-30P showed a clear phenotype with a significant increase in

response when in the G2019S background, further reinforcing our conclusions. Surprisingly, our results did not

show a clear phenotype for G2019S alone. It is possible that this is a consequence of the genetic background

of the fly eyes studied; here flies with a wapr

eye were used, whereas the G2019S phenotype was reported in

flies with a w- eye. These results therefore support an interaction with Lrrk2 and Rab7 not Rab7L1, however

caution should be taken with the interpretation of data from recombinant animal studies and further work is

needed to validate this result.

Future directions

The G2019S mutation is characterised by increased kinase activity, to validate our results the transgenes used

here should be expressed with Lrrk2-G2019S and targeted kinase inhibition. Previous work showed the visual

Fig. 1. Rab7 but not Rab7L1 interacts with Lrrk2-G2019S. Visual response of flies measured using SSVEP. Wapr

(negative control) and α-Syn-30P (positive control). Error bars showing one standard error of the mean. At least n = 9

flies of each genotype were tested at 1 (Fig. 1A) and 7 (Fig. 1B) days of age.

1A 1B

response of Lrrk2-G2019S decayed over extended periods of time or if flies are repeatedly exposed to flashing

light and future work should look to see if this phenotype is replicated by the genotypes studied here.

Departures from the original proposal

It was originally intended that as a second objective the HRS and ALIX genes which function in lysosomal

trafficking by the ESCRT complex would be examined to provide evidence for a secreted role of Lrrk2. This was

abandoned and instead Lightoid was manipulated alongside Rab7 to test current hypotheses for the

interaction partners of Lrrk2. It was also intended to corroborate results from a physiological reporter with an

anatomical reporter however stock contamination of flies used for the later rendered this unfeasible in the

time frame.

From the Supervisor

Martin worked hard on this project, and collected an outstanding amount of good data. He learnt a great deal

about time management, record keeping and data analysis as well as a deep understanding of the roles of

Rabs and Lrrk2. He was able to test published hypotheses about the roles of Rab7 and Rab7L1 in the fly model

of Lrrk2-G2019S induced PD. He clearly showed that Rab7L1 interacts with Lrrk2-G2019S in a different way to

Rab7. We have already begun follow up experiments to confirm and extend his conclusions, as it is essential to

try and understand the interactions of Lrrk2-G2019S. We will study the effects of other Lrrk2 mutations

associated with PD on these two Rabs, the effects of synthetic, kinase dead, mutations, and of a protective

Lrrk2 variant. We will revisit the developmental phenotype using a cleaner GMR-GAL4 line.

The success of this project opens a new area of work for the lab. Once Martin’s conclusions have been

followed up in this way, we will be in a position to extend them to other Rabs with grant support.

From the Student

This grant allowed me the opportunity to undertake a summer studentship. Through which I have gained

valuable experience of working a laboratory and confirmed my interest in research and my decision to pursue

further study.

References 1. Afsari F, Christensen KV, Smith GP, Hentzer M, Nippe OM, Elliott CJ, et al. Abnormal visual gain control in a Parkinson's disease model. Human molecular genetics. 2014;23:4465-78. 2. Dodson MW, Zhang T, Jiang C, Chen S, Guo M. Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning. Human molecular genetics. 2012;21:1350-63. 3. MacLeod DA, Rhinn H, Kuwahara T, Zolin A, Di Paolo G, McCabe BD, et al. RAB7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson’s disease risk. Neuron. 2013;77:425-39.

4. Gómez-Suaga P, Rivero-Ríos P, Fdez E, Ramírez MB, Ferrer I, Aiastui A, et al. LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity. Human molecular genetics. 2014;23:6779-96.5. Beilina A, Rudenko IN, Kaganovich A, Civiero L, Chau H, Kalia SK, et al. Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease. Proceedings of the National Academy of Sciences. 2014;111:2626-31.

Biochemical Characterisation of Tsr3, a Novel Candidate Endoribonuclease in Yeast Ribosome Biogenesis, and its Putative Co-factor and Chaperone Tsr4

Student: Matthew Wilkinson, Supervisor: Dr Claudia Schneider, Newcastle University

Background Ribosome biogenesis is one of the most important tasks undertaken by any eukaryotic cell, requiring around 80% of a growing cell’s energy. It is also immensely complicated, taking place in both the nucleus and the cytoplasm, and involves over 300 assembly factors. In Saccharomyces cerevisiae (budding yeast), the 18S, 5.8S and 25S ribosomal RNAs (rRNAs) are transcribed in the nucleus as a single long precursor molecule (pre-rRNA) that is then cleaved into the mature rRNAs by multiple endo- and exonucleases. Nob1 is the endonuclease that cleaves the 20S pre-rRNA at site D to generate the mature 18S rRNA and it interacts with a binding partner, Pno1. Interestingly, abolishing Nob1 activity in vivo does not completely stop 18S rRNA formation, suggesting another endonuclease must be present that potentially works as a backup enzyme for Nob1. Tsr3 has been identified by bioinformatics (Burroughs and Aravind, 2014) as this potential backup site D endonuclease and it has been hypothesised to interact with a binding partner Tsr4, an essential protein in yeast. The role of Tsr3 is currently unknown and there are many suggestions as to its role. Ideas include that it directly modulates Nob1 activity, it directly cleaves the 20S pre-rRNA at site D or that it plays a more subtle structural role in the pre-ribosomal complex.

Aims The key aim of the project was to produce purified recombinant proteins of Tsr3, Tsr4, Nob1 and Pno1 that could be used in protein-protein interaction studies and to carry out pre-rRNA cleavage assays. Another aim was to apply site-directed mutagenesis to generate mutant constructs to probe the importance of residues in the hypothesised active site of Tsr3. In an addition to the original project, it was also aimed to co-express and purify another pre-rRNA endonuclease, Utp24, and its cofactor Utp23 in order to probe the interactions between the two through in vitro crosslinking. A very important aim of the studentship was also to give me an idea of what working in a busy research lab would be like in order to inform my career decisions and to help me develop important skills such as time management, decision making and key practical skills.

Methods The open reading frames for the Tsr3, Nob1, Pno1 and Tsr4 proteins were amplified from yeast genomic DNA and cloned into the pET100 vector to add an N terminal His tag to the expressed protein. Tsr3 and Nob1 were also sub-cloned into the pGEX vector to add an N terminal GST tag. Utp23 and Utp24 were subcloned into a single pGEX vector so that Utp23 had a GST tag and Utp24 had a His tag. Site-directed mutagenesis was carried out on the bacterial expression vector encoding His-tagged Tsr3 to create an Aspartate to Asparagine change at position 57 (D57N). It was attempted to mutate both GST- and His-tagged Tsr3 and Nob1 (D15N), but only the His-Tsr3 D57N mutant was successfully created within the given time frame. All vectors were then transformed into BL21 E.coli cells, grown up into large scale cultures and recombinant protein expression was induced with IPTG. Cultures were then lysed and protein purifications were performed using Nickel sepharose (for His-tagged proteins) or glutathione sepharose (for GST-tagged) proteins. Protein-protein interaction studies (pulldowns) were undertaken, where the GST-tagged proteins were immobilised on glutathione sepharose beads before being incubated with His-tagged proteins. The beads were washed to remove any unbound protein and the immobilised proteins were extracted, separated by SDS PAGE and analysed by Western blotting. Crosslinking of Utp23 and Utp24 was carried out by adding different concentrations of the chemical crosslinker BS3 to purified samples, followed by SDS PAGE and Western blot analysis.

Results All proteins were successfully expressed and purified including the mutant Tsr3 protein (data not shown). The D57N mutation in Tsr3 had previously been confirmed by sequencing. Pulldown studies showed that there is a multitude of interactions between all of the different recombinant proteins tested, which is summarised in figure 1. This can also be seen from the western blot that was performed to analyse the pulldown experiments, an example for this is shown in figure 2. Crosslinking was also successfully applied to Utp23 and Utp24 complexes (figure 3).

Discussion In this studentship, it has been shown that Tsr3 and Tsr4 do interact with each other as hypothesised, albeit only weakly, and that Tsr3 is likely to be involved in ribosome biosynthesis due to its strong association with Nob1. It was planned to carry out in vitro cleavage assays using both wild type and D57N Tsr3 on 20S pre-rRNA, however there was not enough time to accomplish this. However, by generating the purified proteins this will greatly help the Schneider lab to carry out these studies in the future. Further work to build on this studentship is currently being carried out in the Schneider lab through investigating the in vivo role of Tsr3 using S. cerevisiae. The next step in the crosslinking experiment is to optimise the process and then use mass spectrometry to identify the individual amino acids that are interacting with each other in order to describe the interaction surface between Utp23 and Utp24.

Value of the Studentship I found the studentship extremely enjoyable and it was valuable in giving me an idea of what doing a PhD would be like and how research works on a day-to-day basis. This has helped me to decide that I want to study for a PhD after I graduate. The lab experience and other valuable skills that I have gained from the studentship will be very useful not only to my final project but to my future career and studies. Furthermore, it was very motivating to hear from my supervisor that the data generated by myself has really kick-started a new research project in the laboratory.

References Burroughs AM and Aravind L (2014) Analysis of two domains with novel RNA- processing activities throws light on the complex evolution of ribosomal RNA biogenesis. Front. Genet. 5:424

Figure 3. As the ratio of crosslinker

to protein increases the weight of

the band increases to show

multimer formation

Figure 1. Each arrow shows a

protein-protein interaction as

seen in pulldown experiments.

Figure 2. Each dark signal

represents an interaction

between two proteins. GST was

used as a negative control.

Crosslinked

proteins

1

Biochemistry Society Summer Studentship Report 2015 Student: Michaela Dermendjieva Supervisor: Prof Robin Allshire Direct Supervisor: Dr Manu Shukla Affiliation: Institute of Cell Biology, University of Edinburgh

Evaluation of proximity tagging approaches for identification and analyses of CENP-A chromatin-associated factors in S. pombe

Introduction Centromeres are chromatin structures where kinetochores assemble during cell division. Kinetochores are large multiprotein complexes which facilitate the attachment of chromosomes to the microtubules and the proper segregation of sister chromatids. The sites of centromere formation are defined epigenetically by the presence of the specialized histone H3 variant CENP-A (centromere protein A). Some CENP-A-associated proteins have been identified so far [1] but further cataloguing of such factors is needed to elucidate the mechanisms of CENP-A assembly. Studying these proteins in the fission yeast S. pombe has been difficult due to technical limitations. To overcome these limitations, we evaluated two recently-described proximity tagging approaches. 1. BioID (proximity dependent biotin identification) is a method based on a mutated prokaryotic biotin ligase (BirA*) which is fused to a protein of interest and promiscuously biotinylates proteins in its vicinity [2]. The biotinylated product is then isolated by streptavidin-based biotin affinity capture. An additional benefit of this system is that transiently interacting proteins can be identified. 2. APEX is a system based on an engineered ascorbate peroxidase (APEX) which similarly to BirA* biotinylates neighbouring and interacting proteins when biotin-phenol is present in the reaction [3, 4].

Aim The aim of the project was to evaluate two proximity tagging approaches (APEX and BioID) in the fission yeast Schizosaccharomyces pombe for identifying CENP-A chomatin-associated proteins.

Departures from Original Proposal There were no major departures from the original proposal.

Description of work To target the engineered enzymes to the centromeres, we mainly used direct fusion constructs of BirA*/APEX with CENP-A. These constructs were introduced and expressed in S. pombe cells via plasmid transformation. In addition, this preliminary stage included performing colony PCR to determine transformation efficiency. An alternative indirect tagging system based on GFP-GBP interaction was used in the initial BioID experiments. To overcome the physical barrier of the yeast cell wall, we performed partial permeabilization of the cells by enzymatic digestion reaction. Then we isolated the insoluble chromatin fraction and performed in vitro biotinylation to facilitate the labelling of CENP-A-proximity proteins. To assess the final product, we performed western blotting and streptavidin-based affinity enrichment to detect the biotinylated proteins in the chromatin fraction. A series of experiments was aimed at optimization of the experimental conditions. Attempts to better suit the protein extraction method for both of our BioID and APEX experiments included performing co-IPs with streptavidin beads. Optimization of the biotinylation conditions for APEX included H2O2 titrations. To improve the development conditions we tested for a better enrichment reagent.

Results and Outcomes BioID experiments. We successfully managed to obtain a crude chromatin fraction of our strains and showed that the biotinylating proteins remain in the soluble fraction while the chromatin-associated biotinylated proteins remain in the crude chromatin fraction (Fig 1.).

2

APEX experiments. We could observe several unique bands of biotinylated proteins in one of our in vitro biotinylation experiments but due to much background noise no conclusions can be made about the success of our system (Fig. 2). However, overall increase in biotinylation could be observed in our co-IP experiment with APEX-CENP-A expressing strain (Fig. 3). In conclusion, some activity in the APEX system could be observed but further optimization of the development and extraction methods could increase the efficiency of this technique in the future.

Future Directions Further experiments can be performed with the APEX system to optimize the biotinylation conditions, for instance the timing over which the reaction should take place can be tested for. Additionally, a further improved version of APEX has been recently described which can be evaluated.

Value of the Studentship To the Student: This project has been an incredible insight for me into what it is like to be involved in research. It was an opportunity to not only learn new techniques and get hands-on experience, but also learn how to go about when trying to verify a system and what to take into account when designing an experiment. Adapting a method turned out to be a challenge but I learnt so much about the reasoning behind every step and the limitations of analytical tools. I made a lot of observations about the research profession which together with my own work made me very positive about pursuing a career in science. To the Lab: During her stay, the student managed to evaluate many optimization parameters towards setting up the methodology. These results will serve as valuable groundwork for further optimizations. Additionally, the studentship allowed a mentorship opportunity for a postdoctoral researcher in the lab, which is extremely important for their development as future mentors.

Control strain

APEX-CENP-A strains Figure 2. In vitro biotinylation experiment with APEX-CENP-A-expressing strains. Red arrows pointing towards unique bands.

- Bio

tin

, + h

em

in

+ B

ioti

n, -

he

min

+ B

ioti

n, +

he

min

Co

ntro

l Strain

AP

EX

strain

Figure 3. Co-IP protein extraction of biotinylated proteins after in vitro biotinylation of crude chromatin fraction. Silver staining method.

Figure 1. In vitro biotinylation performed on pombe strains expressing BirA*-GBP-NLS construct (indirect tagging) or BirA*-CENP-A construct (direct tagging). 1 – supernatant, 2 – crude chromatin fraction, 3 – crude chromatin fraction + biotin. Western blot with streptavidin-HRP.

BirA*

Endogenous

biotinylating

proteins 1 2 3

BirA*-GBP-NLS GFP-CENP-A

BirA*-GBP-NLS

1 2 3

APEX-CENP-A strains

3

References 1. Perpelescu, M. and Fukagawa, T. (2011) The ABCs of CENPs. Chromosoma, 120, 425-446 2. Roux, K.J., Kim, D.I., Raida, M. and Burke, B. (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. The Journal of Cell Biology, 196, 801- 810 3. Martell, J.D., et al.(2012) Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nature Biotechnology, 30, 1143-48 4. Rhee, H.W., Zou, P., Udeshi, N.D., Martell, J.D., Mootha, V.K., Carr, S.A. and Ting, A.Y. (2013) Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science, 339, 1328-1331

Mónica Esteban Summer Studentship Report

Student: Mónica Esteban Garcia Summer 2015

Supervisor: Dr Akane Kawamura CRL-Department of Chemistry, University of Oxford

Oxidative Stress and Epigenetics: Study of the inhibition of KDM4A by H2O2

Aims of the project:

Epigenetic regulation is the mechanism by which cell differentiation and specific cell expression is

possible. Its deregulation has been linked to many disorders, such as different types of cancer,

inflammation-related diseases or aging(1). A wide array of enzymatic families is responsible for

genome’s integrity and accessibility to transcriptional machinery, by inducing modifications on

histones such as demethylation(2). JmjC-KDMs belong to a family of Fe(II)/ 2-oxoglutarate (2OG)

dependent oxygenase, that require oxygen to carry out their function. KDMs are histone

demethylases that target specific lysine residues on histones, presenting different states of

methylation. KDM4 can demethylate tri-methylated lysine residues of H3K9me3 and H3K36me3. It

can also use H3K9me2 and H3K36me2 as a secondary substrate, so it can also result in formation of

H3K9me1(3). H3K9 methylation is often associated to silenced parts of chromatin, but has also been

found in transcriptionally active genes, and vice versa applies to H3K36, so the final effect of KDM’s

action seems to fall into a more complex set of interactions(4).

Detrimental epigenetic modifications have been associated with oxidative stress, resulting in

aberrant patterns of gene expression. Oxidative stress is the result of many cellular metabolic

processes, such as imbalance of cellular redox haemostasis due to excessive production of reactive

oxygen species (ROS)(5). Hydrogen peroxide is one of the naturally produced ROS in the human

body, and is neutralized to a certain extent by antioxidant. However, an imbalance in the

compensation, whether due to endogenous or exogenous causes, can result in DNA, lipids and

protein damage(6). The aim of this project is to determine whether KDM4 is sensitive to hydrogen

peroxide, and whether the epigenetic consequences of increased amounts of ROS pass through

inhibition of these histone-demethylases. Exposure to hydrogen peroxide (H2O2) and tert-

butylhydroperoxide (tBuOOH) of KDM4 are to be performed in vitro on isolated KDM enzyme, and in

cells on overexpressing KDMs.

Departures from original proposal:

Due to initial variability in the assay data obtained (difficulties in obtaining consistent results

initially), and the expansion of studies on isolated KDM enzyme, the cellular part of the project was

not possible to accomplish within the 10 week project. Instead, in vitro experiments were not limited

to the determination of IC50, but extended to cleavage assays of KDM4 by H2O2.

Description of work and result assessment

(1) In vitro IC50 determination of H2O2 for isolated KDM4A. Recombinant KDM4A was exposed to

different concentrations of H2O2 and its enzymatic activity was determined using MALDI-TOF mass

spectrometry (MS) where the reaction was monitored by the removal of methylation on histones

(Figure 1). Assays were carried out at optimal cofactor concentrations, based on previous work

performed by the group, and H2O2 was added only before the enzyme, so as it would not react with


the cofactors (Fe(II), 2-oxoglutarate (2OG)) beforehand. All materials used in this study were

provided by the laboratory.

Figure 2. Sensitivity of recombinant KDM4A

to H2O2. The inhibition assays were carried

out on 96 well plates, 40µL per reaction.

Each reaction contained 100µM ascorbate,

100µM 2OG, 10µM Fe(II), 100µM H31-

15K9me3 peptide, and 1 µM KDM4A with

different concentrations of H2O2. All

reactions were run in triplicates at 37°C and

quenched with 0.1% Formic Acid. H2O2

(17.5M) was diluted in water to give the

desired concentrations.

The designed assay produced IC50 values (µM) that were reproducible (IC50 = 23.2 µM and 18.23µM

for reactions stopped after 5 min and 40 min (at linear range of the reaction) respectively) (Figure 2),

demonstrating that the KDM4A is highly sensitive to H2O2.

(2) Mechanism of KDM4A inactivation by H2O2

To determine the exact mechanisms of the inhibition, further assays were undertaken with variation

in ascorbate and iron concentrations to study the direct effect of peroxide on the enzyme using SDS-

PAGE .

KDM4A protein was mixed with components in various combinations of KDM4A, Fe(II) and ascrobate

concentrations in 10 µL final volume and incubated for 90 min at 37°C. Reactions were quenched

using an equal volume of 2 x sodium dodecyl sulfate (SDS) loading buffer (50 mM Tris-HCl pH 6.8,

SDS (2% w/v), glycerol (12 %), β-mercaptoethanol (5%), Bromophenol blue (0.2% w/v)), then

analysed via SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) (Figure 3).

Resulting gels were imaged and revealed that surprisingly, H2O2 did not seem to induce structural

changes on KDM4A, whose original weight is 45kDa, as demonstrated by the lack of KDM4A

fragmentation. However, intriguingly, its incubation with highly concentrated Fe (II) and Ascorbate

(with or without H2O2) led to increased fragmentation and aggregation, which is translated by lighter

bands of 30 kDa and heavier bands of 130 kDa respectively.

Figure 1: MALDI-TOF MS spectrum showing

demethylation of 15mer H3K9me3 (mass

1603.53 Da) by KDM4A to produce

H3K9me2 peptide product (mass 1589.51

Da). The percentage of dimethylated

peptide relative to the substrate peptide

was calculated to find the extent of

demethylation.

m/z

-14Da


Figure 3. Cleavage assay of KDM4 in presence of ascorbate. 15 µM protein was loaded. Concentrations listed in mM unit. * denotes assays where the reagent was added first.

The gel shows degradation and aggregation bands (lighter and heavier than 45 kDa respectively), that increase in intensity between lanes 8 to 12, corresponding to the lanes where highly concentrated ascorbate was added.

Future directions of the project

This project was framed within the larger project of studying the interactions of H2O2 and the 2OG-

oxygenases family. Studies so far have revealed different sensitivities against H2O2 for different

enzymes. The assays carried out in this project showed that KDM4A is sensitive to H₂O₂, following

the lines drawn by the HIF-hydroxylases(7). However, the inhibition assays revealed IC50 values

between 18.23 and 23.20 µM, with KDM4A proving to be very sensitive to H₂O₂. This low value is

very different to the IC50 for PHD2 of 782 µM (unpublished data, H.R. William), showing the potential

for differential sensitivity of 2OG oxygenases. A low tolerance of KDMs towards ROS could have

important consequences at an epigenetic level, inducing diseases or accelerating cellular apoptosis.

The cleavage assays had the initial objective of studying the possible degradation of KDM4A by H₂O₂.

However, they did not reveal any structural damage induced by ROS, its inhibitory mechanism

remains to be studied. It did show that ascorbate, which is used in the assays to maintain Fe(II),

might be a double-edged sword, as it induced structural degradation and aggregation of the protein.

In the future, it would be interesting to continue studying the mechanisms by which H₂O₂ inhibits

KDM4, expanding the study to other KDMs and eventually to other members of other 2OG

oxygenase family. Furthermore, the role of ascorbate and whether it truly acts as a protector or if it

can also be damaging is also of interest for further study.

Value of the studentship

From the student: This summer studentship has provided me with the opportunity of truly

discovering a laboratory environment. Coming from a medical background, this experience has given

me the chance of working on a wide array of skills that I consider very important for my future

professional life. Having my own project assigned and feeling responsible for it has increased my

autonomy and my curiosity for the laboratory world. The atmosphere in the lab was very enjoyable,

and everyone was very welcoming and patient. The projects being followed in the group have

triggered in me a new found interest for epigenetics that I will like to follow in my future career. I

Lane2 3 4 5 6 7 8 9 10 11 12 13 14 [H2O2] - - - - -20 -20 20* - 20 - 20 [Fe(II)] -0.020.2 - - -0.20.2* 0.20.02 0.020.2 0.2 [Asc]- - -0.2 2 - 2 2 2 2 2 0.2 0.2

5540

70100

362515

kDa

KDM4A45kDa


would like to thank the Biochemical Society for this incredible opportunity, as well as everyone in

the lab for making my summer such an interesting and lively one. I would like to thank Dr Akane

Kawamura and Rebecca Hancock for their patience and countless help.

From the supervisor: Monica has demonstrated a very strong work ethic, dedication to the project

and the ability to apply critical thinking. Even though it was her first time working in a laboratory,

she was highly competent and picked up techniques very quickly. The activity assay she was using

showed variability due to the inherent instability of the protein, but she persisted and worked

through the problems to successfully obtain robust dataset for this report. During the 8 weeks,

Monica learnt a number of techniques, including enzyme assays, mass spectrometry, peptide

purification and SDS-PAGE. She produced two well-written reports (above, and a more detailed

report for the lab), and presented at the lab meeting (to approx 15 people) at the end of her project.

She was confident and delivered a professional presentation, demonstrating her clear understanding

of the subject area, her project, and discussed the significance of her results. She answered the

questions well which led to a fruitful discussion about future work / directions for this project. She

has made important contributions towards this new project and achieved a great deal in a short time

period. It was an absolute delight to have worked with Monica and she is more than welcome to

come back in the future. I’m sure she will go on to achieve many things in her future studies and

career.

References

1. Mikhed Y, Daiber A, Steven S. Mitochondrial Oxidative Stress, Mitochondrial DNA Damage and Their Role in Age-Related Vascular Dysfunction. Int J Mol Sci [Internet]. 2015 Jan [cited 2015 Jul 31];16(7):15918–53.

2. Klose RJ, Kallin EM, Zhang Y. JmjC-domain-containing proteins and histone demethylation. Nat Rev Genet [Internet]. 2006 Sep [cited 2014 Dec 8];7(9):715–27.

3. Hancock RL, Dunne K, Walport LJ, Flashman E, Kawamura A. Epigenetic regulation by histone demethylases in hypoxia. Epigenomics [Internet]. Future Medicine Ltd London, UK; 2015 Apr 2 [cited 2015 Apr 7];1–21.

4. Klose RJ, Zhang Y. Regulation of histone methylation by demethylimination and demethylation. Nat Rev Mol Cell Biol [Internet]. Nature Publishing Group; 2007 Apr [cited 2015 Jun 16];8(4):307–18.

5. Kowluru RA, Mishra M. Oxidative Stress, Mitochondrial Damage and Diabetic Retinopathy. Biochim Biophys Acta [Internet]. 2015 Aug 3 [cited 2015 Aug 10]

6. Rhee SG, Chang T-S, Jeong W, Kang D. Methods for detection and measurement of hydrogen peroxide inside and outside of cells. Mol Cells [Internet]. 2010 Jun [cited 2015 Jul 10];29(6):539–49.

7. Masson N, Singleton RS, Sekirnik R, Trudgian DC, Ambrose LJ, Miranda MX, et al. The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity. EMBO Rep [Internet]. 2012 Mar [cited 2015 Jul 6];13(3):251–7.

Targeting the NAD+

Synthetase from Mycobacterium Tuberculosis using fragment-based approaches

Student:Nikhil Sathyan*

Supervisor:Prof.Sir.Tom Blundella

Day-to-Day Supervisors:Dr.Michal Blaszczyka and Dr.Vitor Mendes

a

* Dept of Life sciences,UM-DAE Centre for basic sciences,Mumbai-400098,India

a Dept of Biochemistry,University of Cambridge,Cambridge-CB2 1GA,United Kingdom

BACKGROUND AND AIMS

Fragment-based drug discovery (FBDD) has emerged as a successful method to design inhibitors for biomacromolecules of therapeutic interest. Herein, we

describe the fragment-based approach of targeting the NAD+ synthetase from Mycobacterium, an enzyme necessary for both the de-novo pathway and

salvage pathway of NAD+ biosynthesis(Fig.1.). It makes use of a screening pipeline of biophysical and biochemical techniques .A NadE-C176A expression

construct with an inactive glutaminase domain and an active NAD+ synthetase domain, was used for fragment screening. The top fragment hits from thermal

shift assay were validated biophysically by ligand based NMR techniques and by a biochemical assay.

Fig.1. De-novo and salvage pathway of NAD+ biosynthesis.

Source: NAD+ auxotrophy is bacteriocidal for the tubercle

bacilli.Catherine Vilcheze.2010.Molecular biology

2. Experimental

2.1. Cloning, test trials, Protein Expression and Purification

Prior to the start of the project, the NadE gene was amplified by PCR dire

ctly from M. tuberculosis genomic DNA and cloned between BamHI and

HinDIII restriction sites into an in-house modified pET28 vector containing an Nterminal SUMO tag with an internal hexahistidine tag.

The C176 mutation was introduced by site directed mutagenesis.

Testing for expression of NadE was carried out in four different strains

:BL21,BL21 Star, Origami and Tuner under different conditions of

expression temperature and IPTG concentration using an in-house protocol (M.Hyvonen et al, 2013, http://camelot.bioc.cam.ac.uk/~marko/

methods/expression_test_2013.pdf). The transformed cells were flash

frozen in LB with 50% glycerol using liquid nitrogen and stored at -80 C for subsequent purification.

For protein expression, LB media was inoculated with scrapings from the 80 C stock and incubated overnight (37 C). This culture was then used to

inoculate 2xTY media for expansion at 37 C until cultures reached an OD

600 of 0.4-0.5. These were then induced with IPTG (400 mM)

.Temperature was lowered to 16 C and incubated overnight. Post expression overnight , the cells were harvested by centrifugation. Cell

pellets were re-suspended in lysis buffer supplemented with EDTA-free

complete protease inhibitor cocktail (Roche). The cells were lysed by sonication. Debris was removed by and the supernatant was passed

through a 5 ml HiTrap IMAC Fast Flow column charged with Ni. After

washing with 50 ml of HisTrap A buffer, tagged NADE was eluted with a 0-100% gradient of HisTrap B buffer. The eluate was incubated with Ulp1

protease in a dialysis bag and dialysed overnight with gel-filtration buffer

(20 mM TrisHCl pH 8.0, 100 mM NaCl, 15% glycerol). NADE was

separated from the cleaved SUMO tag by size-exclusion chromatography

(Superdex 200) and concentrated(4500 g at 4 C) using 10 kDa Amicon Ultra concentrators.

2.2.Thermal shift assay:Fluorescence based thermal shift assays were performed using Bio-Rad CFX Connect real-time PCR machine to

identify hits. Each 25 µl reaction mixture in 96-well plate format

contained 5 µM NADE, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2.5×Sypro Orange dye, 5 mM test fragment and 5% DMSO (fragment

were prepared in DMSO). The temperature of the sample was raised in 0.5°C increments from 25°C to 90°C and the fluorescence measured at

each step, with excitation/emission wavelength’s of 490/575 nm.

2.3.Ligand based NMR(STD and CPMG) :Ligand based 1H-NMR

experiments were performed at 278 K on a Bruker AvanceIII AV600

equipped with a 5 mm QCI cryo probe. Samples (550 µl) containing 3

mM fragment and 1% (v/v) DMSO-d6 in the presence and absence of 10

µM NADE was prepared in NMR buffer (50 mM Tris-HCl (pH 8.0), 50

mM NaCl) and loaded into 3 mm NMR tubes.. The resulting spectra were

analysed with Bruker TopSpin software.

2.3 .NADE Biochemical assay

A biochemical assay of the synthetase domain of NadE was optimized on

a BMG Pherastar plate reader based on the previously published work of LaRondeLeBlanc et al. (3). The coupled assay consisted of two steps

: the synthetase reaction where NaAD is converted to NAD+ by NadE

and the dehydrogenase reaction where alcohol dehydrogenase converts produced NAD+into NADH, the presence of which can be detected by

measuring absorbance of the reaction mixture at 340 nm. Each 100ul of

the inhibition reaction consisted of 1mM ATP, 4mM Mg2+, 1mM NaAD , 1mM DTT and 2.67 uM of NadE. For IC50 calculations, the inhibition

of NadE due to binding of the ligand was measured at various

concentrations of the compounds.

NadE-SUMO

(90 kDa)

http://camelot.bioc.cam.ac.uk/~marko/

3. Results and Discussion

The expression trials showed that at 37 C a large amount of the

recombinant NadE form inclusion bodies compared to 17 C samples. Based on the visual analysis of fractions on a 12 % SDS- PAGE gel

(Fig.3.), Tuner(DE3) is the best system for obtaining pure NAD+

synthetase at a growth temperature of 17 C and IPTG 400uM. Expression of N-terminal SUMO-tagged NadE-C176A in Tuner (DE3)

gives a higher yield (6 mg of protein per litre of cell culture) than that

originally reported by Bellinzoni et al. who used a thioredoxin-NadE fusion protein in E. coli Origami(DE3) and reported a yield of 4 mg of

protein per litre of cell culture. This is may be due to the fact that Tuner

cells produces a concentration-dependent, homogeneous level of induction and thus helps in increasing protein solubility.

Bl21(DE3) TUNER BL21(STAR) ORIGAMI(DE3)

M 400 800 400 800 400 800 400 800

1 2 3 4 5 6 7 8 9

Fig.3. Expression test trials of NadE at 17 C . Soluble fractions

were visualized on Coomassie stained 12% SDS–PAGE;

recombinant enzyme is indicated by the arrow. Lanes: 1, molecular

weight markers (Bio-Rad); 400 and 800 indicates the amount of

IPTG(uM)

A thermal-shift screen was carried out using a library of 1152 fragment

molecules. After analyzing the ∆Tm data any fragment displaying

thermal shift (∆Tm) of more than 3 C was classified as a hit. In total, 47

fragment hits were identified. All the hits identified from the screen

showed negative thermal shifts (Fig..4.).Out of these, 12 fragments

showed a negative shift of value more than 6 C. These large negative

thermal shifts probably have resulted from the octameric nature of

NadE, and binding of these ligands may have disrupted/destabilized the

quaternary structure. The top six hits were screened by thermal shift

assay with varying concentration of ligands and increase in the shift in

melting temperature with respect to ligand concentration was

confirmed for all(Fig.5.).

Fig..4.No of hits corresponding to the thermal shift observed.

Thermal Shift values shown are absolute values. All the hits caused

a negative thermal shift indicating a destabilization of the enzyme.

Fig.5..Thermal shift experiments with varying concentration of

ligands. Each data point is an average of three independent

experiments in which every condition was reproduced thrice. Error

bars shows the standard deviation.

NadE-SUMO

(90 KDa) NadE-

SUMO

90 KDA

NadE-SUMO

(90 KDa)

Fig.6.Ligand based NMR for fragments. (A) STD for NMR 352

bound to NadE. (B) CPMG for NMR 352(red) and NMR 352

bound to NadE (blue line).The flat blue line for CPMG indicates

that the signal obtained when NMR 352 is bound to NadE is

negligible in comparison to NMR 352 alone in the solution.

The top six hits were further validated by ligand based NMR

techniques namely STD (saturation transfer difference) and CPMG(Carr-Purcell-Meiboom-Gill). In STD, if the fragment binds to

the target protein, the buildup of NOE that is transferred to the ligand

results in enhanced signal corresponding to the resonances of that ligand in the STD spectrum. Here a representative STD NMR spectra

for ligand no.352 is shown (Fig.6.A). In CPMG, when a fragment binds

to the protein, resonances due to the fragment protons broaden and a decrease in signal is expected. For ligand no.352 it can be seen that the

signal is almost a flat line indicating a very strong binding(Fig..6.B)

The biochemical assay also agreed with this result as NMR 352 gave the strongest inhibition (Table.1).

Fig.7. Inhibition of catalytic activity of NadE by fragments:

representative examples of dose-response curves for NMR 352 and

NMR 658.

Thermal shift and Ligand based NMR characterize the protein ligand

interactions on the basis of their physical binding and does not give an exact idea about the inhibitor potency of the ligand on the enzymatic

activity of the enzyme. Hence the fragment hits was validated by a

biochemical assay. Dose-response curves were produced to determine half maximal inhibitory concentrations (IC50s) of the top 5 fragment

hits.(fig.7,Table.1)

Table.1. IC50 values of ligands determined by biochemical assay.

Thermal shift values are given for ligand concentration at 5mM

and NadE at 5uM.

NMR 352 seems to be allosterically inhibiting NadE since the

concentration of the substrate (1mM) is considerably high compared to

the IC50 value (675uM). This needs to be verified through kinetic experiments.

Summary and Future directions

In summary, this project has helped in discovering inhibitors of Mtb

NadE. Also the optimised biochemical assay can be used for screening the library for any ligands causing positive thermal shifts which may

not have been detected with thermal shift assay. This would both detect

hit fragments as well as allowing fragments to be ranked according to inhibitor potency.

However the affinity of the fragments and the how and where it binds

needs to be determined by techniques like ITC and X-ray crystallography as both these factors are very important for enhancing

the potency of the hit fragment .

Value of the studentship to the student:

Before this summer studentship I didn’t have the confidence to enter

into a PhD program, but after the research experience , exposure and the brain storming sessions with my team in Prof.Tom Blundell’s lab, I

can’t wait to get into a research program of my choice .This studentship

also enabled me to make full use of the intellectual community at Cambridge by participating in various seminars and lectures organised

in different institutes and departments of Cambridge university. All this

was possible with the generous support from the Biochemical Society. I hope many other organisations follow the example of the Biochemical

society and open their doors to students of all nationalities.

-Nikhil Sathyan

Value of the studentship to the lab:

Nikhil joined the research team funded by the Gates Foundation to work on Fragment-Based Drug Discovery targeting Mycobacterium

tuberculosis. We selected the synthesis of NaD as an important enzyme

pathway, focusing on the enzyme NaD synthetase. He developed an expression construct of NaDE-C176A to provide an inactive

glutaminase domain and an active NaD synthetase domain. He then

developed fragment-based screening using several biophysical assays

including thermal shift and ligand-based NMR. Most significantly he

developed a biochemical assay for the system.

Nikhil is a very thoughtful and bright scientist, much more relaxed and

confident than many other Indian visitors much senior to him who have

joined our research team. He very quickly became a full member of the group focusing on the multi-disciplinary approach in fragment based

drug discovery, learning techniques quickly and working closely with

his day-to-day supervisor Dr. Michal Blaszczyk. Michal and I feel that his contributions were significant and he will be included on a paper

that we eventually publish on this target. He has been of real value to

the progress of the research team.

I had long conversations not only about the biochemistry but also about

aspects of Indian life and indeed about philosophy. Nikhil is very independent in his thinking and has some very carefully thought-out

ideas about his research career and about life in general. This was one

of the best summer internships we have had and we were delighted that the Biochemical Society could provide support to make his visit

possible. -Prof.Sir.Tom.Blundell

Ligand IC50 (uM) ∆Tm (C)

288 1000 -7

352 675 -18

527 2168 -7.5

658 980 -7

744 822 -11.25

Oliver Prosser- Biochemical Society Studentship Report

The content of my summer project departed significantly from the original proposal. The original aim

of the project was to purify and crystallise Ebola virus nucleoprotein (EBV NP), in order to gain

structural data on EBV NP using X-ray crystallography. However, prior to beginning work on the

project the structure of EBV NP core domain was solved by another group (Roa et al. 2015). Wanting

to perform a project using similar techniques, we switched the focus of the project to solving the

crystal structure of another haemorrhagic fever virus nucleoprotein, namely Lujo virus (LUJV), an

arenavirus found on the African continent.

The laboratory work associated with this project involved the expression and purification of the LUJV

NP, using Rosetta 2 cells that I transformed with a Pet 28C vector containing a sumo-His tagged LUJV

NP. Within the early weeks of the project I managed to express LUJV NP using auto induction and

purified a small amount of the protein by using affinity chromatography using Ni-NTA resin. This

involved lysing the bacteria by freeze thawing and sonication, binding the lysate to a small amount

of nickel resin in a falcon tube and then washing with of a succession of buffers containing an

increasing concentration of imidazole, preforming all wash steps using centrifugation. The protein

was then incubated overnight with sumoprotease to remove the sumo-His tag.

LUJV NP appears to be very temperature sensitive, making purification of the protein more difficult

as all buffers used needed to be at 4˚C during the purification. Because the buffers contained Tris,

which varies pH with respect to temperature, all buffers also had to be at the operating pH of 7.4

when at 4˚C.

I was able to increase the amount of protein being produced by making a glycerol stock of my

bacteria and using this to inoculate the cultures, in addition to increasing the volume of my preps

from an initial 1 litre volume to an eventual 6 litres of culture. Due the increase in volume I moved

from using a centrifuge based purification method to performing the purification using a glass

column.

X-ray crystallography requires very pure protein, so I performed size exclusion chromatography to

remove the sumoprotease and any other contaminants that remained after the nickel resin

purification. This also required concentration of the protein prep down to a volume of 5 ml. I first

attempted this using a concentrator column and centrifugation, but found that this resulted in the

loss of a large amount of protein. For later preps I concentrated using polyethylene glycol, which

proved much more successful.

As described above, the use of size exclusion chromatography resulted in the loss of a significant

amount of LUJV NP from the protein preparation. I then attempted both cationic and anionic

exchange as alternative methods for removing contaminants, both however were unsuccessful.

Another problem identified at this stage was that the Rosetta 2 cells appeared to be expressing LUJV

NP poorly. After performing a western blot I was surprised to discover that the contaminants

present in my elutions contained epitopes present in LUJV NP, suggesting that the bacteria were

producing truncated forms of the protein in addition to the full-length version. It also appeared that

small amount of LUJV NP still retained the sumo-His tag after incubation with sumoprotease,

perhaps suggesting that a small amount of the protein was misfolding.

I transformed a new set of Rosetta 2 cells and performed another western blot to check expression.

Whilst none of the new cells were producing the truncated variants of LUJV NP, expression of the

protein varied dramatically from colony to colony, although all colonies were expressing the protein

to some extent. I then used a culture that appeared to express well on the western blot and created

a new glycerol stock. Despite using this new glycerol stock and increasing the volume of culture to 10

litres I was still unable to produce enough clean protein to carry out crystallography, as a large

amount of the protein I produced was being lost during size exclusion chromatography.

For future work I would suggest increasing the amount of protein produced by the bacteria in order

to still have enough left for crystallography after size exclusion. This could involve using a different

form of induction such as IPTG. Also, growing up an overnight starter culture beforehand to

inoculate the cultures may have an impact on expression. Increasing the volume of culture appeared

to have little effect on the amount of protein purified and similarly increasing the amount of nickel

resin did not appear to increase yield.

Although I didn’t manage to obtain any crystallography data for LUJV NP, my studentship has

benefited the lab by helping to optimise LUJV NP purification. For example, we now know that

polyethylene glycol can be used to successfully concentrate the protein preparation. I have also

found that individual colonies express LUJV NP at very different levels despite being transformed

with identical plasmids and being grown on the same plate. My work has also identified that

sumoprotease cleavage of the sumo-His tag is partially incomplete, likely due to mis-folding of the

protein, and that the proportion of LUJV NP that mis-folds varies between preps.

Being awarded this grant and undertaking this project has enabled me to gain experience of working

in a world class research laboratory, allowing me the opportunity to improving my practical skills,

communication in a group environment, abilities in troubleshooting problems, connections with

professionals within the field and giving me an improved sense of what it takes to have a career in

research science. I now feel I am better prepared to move to the next step when I begin a master’s

degree this September and will carry the skills learnt during this studentship hopefully to a PhD and

a career in academic research.

Student: Ryan Squire

Characterisation of FeFe-hydrogenase maturation enzyme HydE

Supervisor – Professor Peter Roach, University of Southampton

Background:

FeFe Hydrogenases undergo a catalytic reaction for the reversible formation of Hydrogen, where the widespread

applications of Hydrogen production from a renewable source range from transportation, coolants or use in the

chemical industry as a reagent.

The Maturation of the FeFe Hydrogenase involves the formation of the cluster HydA [1], which is thought to be

assembled by three corresponding enzymes: HydE, HydF and HydG. Much work has already been done on HydF and

HydG, with some links to their roles in the cluster formation, with HydF acting as a scaffold for the cluster to assemble

and HydG is involved in CO and CN bound ligands to the cluster. However little work has been done on the role of

HydE, where it is hypothesised that it is involved of the addition of the Dithiolate Bridge to the cluster.

[1] H-Cluster HydA, where [4Fe4S] is a Cubane structure with Iron and Sulphur.

My task for this 8 week project was to express the HydE protein in transformed E.coli, where we could then go forward

to isolate/ purify the protein, where we could carry out quantification, characterisation and potentially crystallographic

studies.

New techniques learnt on this placement included:

Agarose Gel electrophoresis SDS-PAGE Bradford assay analysis

Streptavidin column purification Gel extraction procedure DNA purification

Nucleic Acid Quantification Small scale culture growth Large scale culture growth (5L)

Plasmid Transformation Using the Glove box Liquid Nitrogen Flash freezing

Autoclave operation Centrifuge operation MSC operation

Enzymatic double digest Ligations Agar plate preparation

Results and placement progression

Weeks 1-2: transformation of the HydE Thit1265 and Thit1675 gene containing vectors into E.coli, with subsequent

double digest studies and DNA purification to purify the isolated DNA for later use.

Week 3: Small scale expression studies of HydE in 100 mL cultures, by incubating at 37 oc until an OD600 of 0.6, then

inducing with 5 mL 20% w/v Arabinose and left to grow for 6 hours. Analysis of the cell pellet after cell lysis on the

SDS-PAGE, showed presence of HydE protein being expressed [2]:

[2] [3]

Weeks 4-5: Large scale expression in a 5L culture gave a cell pellet of 14.6 g, this was flash frozen and thawed out in

the anaerobic glove box, where the following purification was performed.

The HydE being expressed has a Strep-tag on the N-terminus, which has high affinity for a Strep-Tactin® binding

column. This meant that the sample could be washed through the column, with only the desired protein being bound

to the column, which was eluted after. This was found to be in high purity, with only one band being seen on the SDS-

PAGE [3].

Characterisation showed that the protein was in low concentration and was inactive after a couple of days (tested by a

SAM cleavage assay), this lead to a repeat expression, where the culture was induced at 27 oc as opposed to 37

oc,

and additional Fe, S and L-Cysteine were supplemented for the growth. However this provided only 13.7 g of cell

pellet, and too small a concentration of Protein to work with. Progress was also halted by inefficient column

regeneration, meaning some of the initial protein was eluted and not binding to the Strep-Tactin® column.

Weeks 6-7: A new direction for this project was to attempt the co-expression of HydF and HydE proteins in the hope

that when expressed together, they would be less toxic and damaging to the cell growth culture. A vector with the

HydF gene was cut with Xho1/Nde1 enzymes where the vector fragment was isolated and the HydE 1265 and 1675

were also digested, with the smaller inserts being isolated. There were problems with the following ligation reaction, as

insufficient concentration of the DNA fragments were observed, with concentrations between 2-10 ng.uL-1

being

achieved. This was repeated several times with variable conditions on the digestion, different quantities and gel

extraction procedures. Unfortunately this lead to several unsuccessful ligation reactions between the vector and the

insert, where no growth was seen on plates with chloramphenicol and streptomycin (present in the bacteria strain and

the plasmid, therefore only resistant against both with a complete plasmid). It was concluded that the ligation was the

problem due to a control HydF plasmid growing, ruling out errors in the transformation or the plate’s antibiotics. No

further progress has been made on this area of the project.

Week 8: New competent cells were prepared with HydE plasmid, tested on small scale growth with induction with

arabinose at OD600, for 4 hours, found to produce HydE.

This lead to a 5L expression being performed. This unfortunately was unsuccessful due to growth beginning to occur,

then no further growth being observed.

Future directions for the project:

Isolating a larger quantity of the HydE protein will be attempted either by the co-expression with HydF, or by

optimising the growth conditions or the bacteria strain to achieve a higher yield of protein compared to what was

achieved before. Then following successful purification at a reasonable yield, to attempt various assays to find out the

mass of the protein, cleavage assays to measure its activity and potentially to crystallise the protein, with the hope that

the thiol containing substrate will be located in the active site.

Student statement:

This placement has been valuable in the sense that it has opened up my knowledge to the field of Biochemistry,

exposing me to techniques and theory that I haven’t experienced before. I now feel more confident in my ability to

work in a professional laboratory and have grasped a deeper concept of what it would be like to undertake a PhD,

which this has inspired me to pursue.

The project has also shown me the true value of what it is to do research, where a lot of the time things can, and will

go wrong, where you will have to try another approach and think logically to move forward, all of which makes it feel

more deserved when the results finally go your way.

I would like to thank Professor Peter Roach for allowing me into his lab and to use his resources and time, I would like

to thank both Pedro Dinis and Beata Wieckowski for spending a lot of their time in the lab teaching and guiding me

through this placement. Finally I would like to thank the Biochemical society for giving me funding to do this

placement.

Biochemical Society: Studentship report

Circadian rhythms possibly evolved through photo sensing, with the intention of minimizing UV-light

induced damage during DNA transcription. Alternative splicing (AS) is a powerful mechanism capable

of applying the information within one genetic sequence into the expression of thousands of different

proteins. These two powerful mechanisms are found universally in higher eukaryotes, emphasizing

their importance through evolution and contrarily also in instantaneous response to external stimuli.

Circadian genes function using the identical principal of negative feedback loops in clock gene

expression, driving oscillations in the levels of clock proteins with a period of approximately 24 hours.

This lab analyses the additional mechanism to expressional control of AS. The mechanism of AS

allows plants to control slightly deviant functions of AS circadian clock protein and thereby respond to

temperature shifts. (James et al., 2012) Here, we focus specifically on the light induced AS of A.

thaliana splicing factor Polypyrimidine Tract Binding protein-1 (PTB1) and briefly on LHY: a

transcriptional factor of the circadian clock.

Acquired methods and work contributed during placement

The placement consisted of assisting many aspects of the laboratory process leading up to qPCR

quantification of the plant tissue. I have successfully attempted plant growth in hydrophonics. Next,

many transgenic 3 lines (=transgenic F2) needed selection for fitness, hence the quicker sand method,

as described in Davis et al., 2009, was used. The laborious process of seed sterilization was thus not

needed, considering the likelihood of CO2 intake by micro-organisms facilitating their growth is

depressed in a sand medium. Subsequently, T3 seed growth fitness selection was carried out using

antibiotic hygromycin and analysis of successful germination. Additionally, the routine techniques of

seed sterilization and seed harvesting were also taught to me. Finally, I thoroughly learned the steps

from mRNA extraction to cDNA synthesis for quantification and the technique of qPCR.

Results

The work contributed to cDNA synthesis and qPCR were mostly related to the analysis of light-

sensitive AS of splicing factor polypyrimidine tract binding protein-1 (PTB1) and LHY: a transcriptional

factor of the circadian clock. Figure 1a-b indicates there is light mediated AS of the PTB1 splice

variants SPI and SPII. There is a difference of statistical significance between the two splice variants.

SPI expression increases compared to the total PTB1 expression with decrease in light intensity from

150 to 75uE. Conversely, SPI expression decreases compared to total PTB1 when light intensity is

increased 150 to 300 uE. Hence, the reverse can be said for SPII. See Figure 1c. Figure 2 indicates

there is no statistical proof to light mediated AS of the LHY splice variants 5’UTR FS and LHY I1R.

1A

1B

Figure 1: a. PTB1 SPI expression increase after decrease in radiation from 150 to 75uE. SPI expression

decreases upon increase of light intensity from 150 to 300uE. b. PTB1 SPII mRNA expression increase when

light intensity is increased from 150 to 300 uE and has weak trend when light intensity decreases form 150 to

75uE. c. Splice ratio: SPI/(SPI+SPII). The total amount of PTB1 SPI increase slightly, in comparison to total PTB1,

when light intensity decreases from 150 to 75uE. The total amount of PTB1 SPI decreases, in comparison to total

PTB1, when light intensity is increased from 150 to 300uE. Difference of statistical significance between the 2

groups (the 150-75 and 150-300 groups (ZT78, 81 and 84h) as determined by one-way ANOVA: F(5,12) = 5.079,

p=0.0099.

Figure 2: There was no statistically significant difference between the 2 groups (the 150-75 and 150-300 groups

(ZT78, 81 and 84h) as determined by one-way ANOVA: F(5,12) = 0.8759, p=0.5255

Discussion and future directions

PTB1 is a splicing factor which influences AS, by association of the polypyrimidine tract to a

pyrimidine-rich sequence within pre-mRNA. (Wachter et al., 2012) PTB exploits various mechanisms

for AS, it competing with U2 auxiliary factor 65 in binding to the pre-mRNA (Saulière et al., 2006), the

U2 auxiliary factor being a non-snRNP protein is required for the binding of U2 snRNP to the pre-

mRNA branch site. PTB1 also allows for looping of RNA regions (Spellman and Smith, 2006) and its

interference with splicing factor interactions is required for exon or intron definition (Izquierdo et al.,

2005; Sharma et al., 2005).

The results describe how alternative splicing can be regulated via a gene which is itself regulated by

alternative splicing, in this case the trigger for the splicing of the splicing factor PTB1 is a variation in

light intensity. The data also indicates there is no significant evidence LHY is alternatively spliced by a

change in light intensity. Further research which could lead from this is investigation into the splicing

factors which influence splicing of PTB1 and determining whether they too are light or temperature

mediated. This could possibly enlighten whether the regulation of eventual downstream genes are

modulated by primarily one external factor or a combinatory effect of different circadian oscillations

within the environment.

1C

2

Sarah Neely Biochemical Society Summer Studentship Report Form:

Project Aims: The primary aim of my project was to construct Multiple Antigen-Presenting System (MAPS)

conjugates from Shigella proteins to allow us to determine their potential as a vaccine against

Shigellosis in the mouse lung infection model. This research aimed to contribute to our

understanding of how, in the future, we may answer the increasing antibiotic resistance and lack of

vaccine that is currently available against Shigella. I specifically investigated serotype 5a O-antigen

and three highly conserved proteins of Shigella, MxiH, IpaD and IpaB in making the MAPS to be

tested. To produce the full MAPS constructs there were 2 main project aims: preparation of Shigella

lipopolysaccharide O-antigen and its labelling with biotin, and production of Shigella antigens as

fusion proteins with a biotin-binding moiety, rhizavidin. Upon successfully obtaining both of these

products I then aimed to assemble MAPS conjugates by a mixture of these 2, for later purification

and quality assessment.

Work done during the studentship: To investigate the viability of Shigella proteins as conjugates for MAPS production I first

concentrated on the design and production of the fusion protein constructs. To do so the mxiH,

ipaD129-322, ipaD149-304 and ipaB were firstly isolated and inserted in the pET21B plasmid, and

fused to the rhizavidin gene. Whereas ipaB was cloned as a full length fusion protein and co-

expressed to maintain solubility, two different lengths of IpaD fragments were used to establish the

most useful for later neutralising antibody generation. To then establish the base constructs these

PCR products were then ligated into the pACYC plasmid. After which I carried out expression trials of

fusion constructs in E.coli and scale-up purifications of those that were successfully expressed and

stable. Finally to confirm their abundance and purity both SDS-PAGE and Coomassie staining were

used.

At the same time, I also worked on preparation of Shigella lipopolysaccharide O-antigen and its

labelling with biotin. To do so in the time frame of the project I planned to extract Shigella flexerni

serotype 5a, detoxifiy it via the removal of its Lipid A portion, and biotinylated this product using

amine-biotin and CDAP. To plan this part of the project, with adaptations to the time frame, and

make contact with relevant persons who had previously described use of these techniques took

significantly longer than planned. Therefore, although we managed to produce a viable plan for the

desired technique, including details such as concentrations and volumes of reagents needed, we did

not have time to carry out this portion of the procedure.

Results of my research: One of the most notable findings of the research was the fact that within the 6 week timeframe all 4

of the planned fusion constructs were successfully produced, with their sequences and therefore

identities confirmed. Although they have not yet been fused with the Shigella lipopolysaccharide O-

antigen, the fusion complexes produced do provide the first step in the application of MAPS

technology to Shigella.

Departures from the original plan: I experienced somewhat unexpected results occurring when testing the solubility of the fusion

constructs that I had produced. Despite using different methods to test the solubility of the proteins

and repeating these protocols to optimise the procedures, I still found that none of the fusion

constructs produced appeared to be suitably soluble. Importantly this highlights a fundamental need

for further research and development of this technique before MAPS technology is a viable

approach to Shigella vaccine development.

Future direction of the research: As discussed, the apparent insolubility of the fusion constructs must first and foremost be

investigated fully. If found to be accurate, the insoluble nature of the fusion constructs produced

highlights a need for further research, to investigate the cause of this insolubility and from this

investigate the possibility of alternative routes to produce the constructs. Additionally however, the

sequence verified constructs themselves produced and the planned optimisation of the LPS

extraction method do provide a firm foundation as to the possibility and potential of MAPS

technology being applied to Shigella in the future.

Professional and personal development during the project: I cannot fully express how lucky I feel to have had this opportunity. Not only have I really been able

to develop my confidence around the lab in techniques both familiar and new to me, but I have

gained such an invaluable insight into scientific research. An environment of asking questions, seeing

exciting possible links in the data to be explored and coming up with novel hypothesis is one which I

now fully realised that I want my future career to be in, and am determined to strive towards.

Student: Simona Andreea Vatavu Supervisors: Professor Sherif El-Khamisy and Dr Swagat Ray Exploiting DNA damage/repair to improve the clinical outcome of prostate cancer Background Several anti-cancer therapies are based on the accumulation of DNA strand breaks that lead to genome instability [1]. . One method by which DNA strand breaks can arise is through the abortive activity of DNA topoisomerases that usually remove torsional stress through generating and resealing DNA strand breaks [2] . The cleavage complex that arises as the enzymes form a covalent phosphotyrosyl bond to the DNA can become trapped and form protein-linked DNA breaks upon collision with RNA polymerases or replication forks [3] . Tyrosyl-DNA phosphodiesterase-1 (TDP1) is responsible for hydrolyzing the 3’-phosphotyrosyl bond covalently linking topoisomerase I to DNA breaks, thus aiding DNA strand repair [4] . Its depletion leads to the accumulation of TOP1-linked DNA breaks, and cells are sensitized to camptothecin (CPT), a TOP1 poison that stabilizes cleavage complexes and induces cytotoxicity [5, 6] . Tyrosyl-DNA phosphodiesterase-2 (TDP2) possesses robust 5’ tyrosyl DNA phosphodiesterase activity to resolve TOP2-linked DNA breaks [7] , and its depletion sensitizes cells to etoposide, a TOP2 poison [1, 8]. .TDP2 also possesses weak 3’ tyrosyl DNA phosphodiesterase activity that can repair TOP1-induced DNA breaks when TDP1 is absent [1] . In hormone-driven cancers such as prostate cancer, exposure to androgens induces the co-recruitment of topoisomerase II beta (TOP2B) and the androgen receptor to the promoter regions of androgen-driven genes(10). TOP2B is needed for their efficient transcriptional activation, as it mediates DNA double strand breaks to relieve torsional stress, though its abortive activity can lead to the generation of DNA double strand breaks(10). TOP1 is also involved in transcription, and the prevention of R-loop formation that can otherwise lead to DSBs(11). It has previously been shown that genetic deletion of TDP1 and TDP2 results in human, murine and avian DT40 cells becoming hypersensitive to transcription-associated topoisomerase-linked DNA breaks[1, 9] . As prostate cancer is initially hormone-responsive, and as topoisomerases are important in the transcription of androgen-induced genes[ 9] , it is predicted that depletion of TDP1 and TDP2 may result in sensitizing hormone responsive prostate cancer cells to androgen through the accumulation of androgen-driven topoisomerase-linked DNA breaks, thus triggering cytotoxicity. Aims To investigate whether depletion of TDP1 and TDP2 increases the number of androgen-induced cytotoxic DNA double strand breaks in hormone-responsive prostate cancer cells.

1. To grow androgen-responsive LNCaP prostate cancer cells in culture 2. To knockdown TDP1 and TDP2 separately and together through siRNA transfection 3. To validate knockdown by Western Blotting and TDP activity assays 4. To compare the number of double strand breaks in TDP proficient and deficient LNCaP

cells before and after androgen treatment, with or without CPT/etoposide by comparing the number of γH2AX foci using immunofluorescence.

Experimental design and methods Transient transfection with siRNA and Western blotting to validate TDP1 and TDP2 knockdown LNCaP cells (maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and L-glutamine at 37°C in 5% CO2) were plated into 6-well plates at a density of 0.8x105 cells/well, with the aid of a haemocytometer. DharmaFECT siRNA transfection (with scrambled RNA, siTDP1 and siTDP2 in the corresponding wells) was carried out when cells were 20-40% confluent, with a final siRNA concentration of 25nM. (The transfection protocol was repeated for siTDP2 after 24 hours since the initial single hit was not successful) Cells were incubated for a further 48 hours, after which they were washed twice and harvested in ice-cold PBS using a cell scraper. The cells were subsequently centrifuged at 4,500 rpm for 5 minutes at 4°C, the pellet was resuspended in 30ul SDS, and the DNA sheared using a 21G syringe. This was followed by incubation at 95°C for 5 min in a heating block, and centrifugation at room temperature, with the supernatant retained. The samples were loaded into a 10% resolving gel with a 4% stacking gel on top, run for 10 minutes at 120mV to allow the samples move into the resolving gel, after which they were run at 200mV for 45 minutes. The proteins were then transferred from the gel onto a nitrocellulose membrane at mixed MW for 7 minutes, which was then incubated with 5% milk powder and PBST for one hour in order to block non-specific binding. A rabbit anti-TDP1 and anti-TDP2 antibody were used at 1:1000 in milk solution for 4°C overnight incubation with the corresponding membrane. This was followed by 3 washes with PBST buffer, and incubation of the membranes with anti-rabbit secondary antibody conjugated with HRP at 1:4000 for one hour before visualization by the ECL technique. While the TDP1 knockdown was validated, the siTDP2 has been unsuccessful at knocking down TDP2 even after a double hit.

Figure 1: Western blot showing that only TDP1 knockdown has been successful Transfection with siTDP1, DHT and CPT treatments, and γH2AX immunostaining assays

LNCaP cells were grown on glass coverslips in 24-well plates, at 0.3x105 cells/well and with 1ml full medium at 37C. DharmaFECT transfection with 25nM siTDP1 (and 25nM scrambled RNA as a control) was conducted after a 24 hour incubation when 20-40% confluence was achieved. A GFP plasmid was also co-trasnfected at this time at 250ng/well in order to verify the proportion of cells that have taken up the siRNA. The full media was replaced for hormone starvation with 5% charcoal-stripped FBS media 24 hours post transfection. The media was again replaced two times at 24-hour intervals, the second time immediately prior to dihydrotestosterone (DHT) induction at 100nM. CPT was directly added to the wells at 10uM for one hour, after a one-hour incubation with DHT. The immunostaining protocol was carried out at room temperature. Coverslips were washed 3x with ice-cold PBS and cells were fixed with 10% paraformaldehyde for 10 minutes. They were again washed 3x with ice-cold PBS and subsequently permeabilised with 0.25% Triton X-100 for 2 minutes, followed by another 3x PBS wash and blocking of non-specific binding through a one hour incubation with 3% bovine serum albumin (BSA) in PBS. Cells were then incubated for one hour with mouse anti- γH2AX monoclonal antibody diluted 1:1000 in 3% BSA, washed with ice-cold PBS, followed by a one-hour incubation with Alexa Fluor 555 goat anti-mouse secondary antibody and DAPI stain at 1:1000. Cells were washed with ice-cold PBS and the coverslips were mounted to glass slides with Immu-Mout (Thermo Scientific). 100 cells were scored at random from each coverslip, to obtain the average number of DNA double-strand breaks per LNCaP cell by counting the number of gamma-H2AX foci using an epi-fluorescence microscope.

Figure 3: Experimental set-up

Figure 4: immunofluorescence staining of transfected LNCaP cells , DAPI (blue), GFP (green), γH2AX foci (red).

Results

Figure 4: Graph showing the average number of γH2AX foci/LNCaP cell ± s.e.m. of four biological replicates T-tests: sample P-value

scr vs scr, DHT 0.018766975

scr, DHT vs siTDP1, DHT 0.107800908

scr, DHT + CPT vs siTDP1, DHT + CPT 0.001250194

scr, CPT vs scr, DHT + CPT 0.112594448

siTDP1, CPT vs siTDP1, DHT + CPT 0.001930557

siTDP1 vs siTDP1, DHT 0.04102077

siTDP1, DHT vs siTDP1, DHT + CPT 0.000247387

Figure 5: statistical analyses conducted using a student’s t-test (two-tailed distribution, two sample unequal variance) Induction of TDP1-depleted cells with DHT resulted in the accumulation of significantly more DNA double strand breaks compared to non-DHT induced TDP1-depleted cells (P=0.04), supporting the hypothesis that DHT can sensitize androgen-responsive LNCaP cells to androgen via the accumulation of more DNA double strand breaks when TDP1 is depleted. Likewise, the same comparison in which both samples were additionally subjected to the TOP1 poison CPT showed a statistically significant result (P<0.01),. Statistically significant results were also obtained in the case of scrambled-RNA treated cells incubated with DHT compared to scrambled-RNA treated cells with no DHT induction, (P=0.02) and knocking down TDP1 led to a statistically significant increase in foci compared to the scrambled control when incubated with DHT and CPT (P<0.01). Adding CPT to TDP1 knockdown cells under DHT induction also significantly increased the number of foci observed compared to TDP1 knockdown cells with DHT (P<0.01). Future directions

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Since the siTDP2 had not been effective in knocking down TDP2, another siTDP2 must be ordered and tested to achieve TDP2 knockdown, after which the number of γH2AX foci must be compared in cells depleted of TDP1 and TDP2 separately and together under DHT induction. Due to time constraints, the TDP knockdown was not further verified by TDP activity assays, though cells were co-transfected with a GFP plasmid and it was found that siRNA had been taken up by 78% of cells. To further reduce TDP protein levels, genetic deletion could be carried out. Value of the studentship This project has provided me with training in a number of techniques including cell culture, transfection, western blotting, and DNA break measurements using immunofluorescence. It has also given me the opportunity to develop my skills in planning experiments and troubleshooting, as well as other transferable skills including project management skills, communication skills, independence and also learning to record and present data effectively. Weekly lab meetings helped me to learn more about each lab member’s research and more about the field of DNA repair as a whole, and I was also given the opportunity to learn other techniques including Northern blotting and immunoprecipitations. This studentship has been exceptionally valuable in strengthening my desire to pursue a PhD and to work in scientific research. Supervisor’s comments Dr Swagat Ray (day-to-day supervisor): Simona’s time in the lab has been very well utilized both in terms of generating interesting data for her project and learning varied experimental techniques while shadowing other senior members in the laboratory. She has shown a keen interest in how things work in a laboratory set up, including day to day running of routine experiments like Western Blotting, protein immune-precipitation, RT-PCR and other biochemical techniques. Her analytical skills are showcased in how she approached each data set and tried to explain the experimental findings in a logical fashion. I hope that this experience would give her enough encouragement to pursue a career in research. Sherif El-Khamisy (PI): It was a pleasure to see Simona flourishing in the lab. She has been very committed, driven and able to swiftly grasp both the intellectual and technical aspects of the project. Simona generated high quality data in such a short time in the lab and she continued analyzing and writing her report and a literature review for the most part of the summer holidays. It was a pleasure supervising Simona and I look forward to witnessing her continued successes. References

1. Zeng, Z. et al. (2012). TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1. Nucleic Acid Research. 40(17) pp. 8371-830

2. Pommier, Y., et al., (2010). DNA Topoisomerases and Their Poisoning by Anticancer and Antibacterial Drugs. Chemistry & Biology 17, pp. 421-433

3. Nitiss, J. (2009). Targeting DNA topoisomerase II in cancer chemotherapy. Nature Reviews Cancer 9, pp. 338-350

4. Zhou, T., et al. (2009). Tyrosyl-DNA phosphodiesterase and the repair of 3′-phosphoglycolate-terminated DNA double-strand breaks. DNA Repair 8, pp. 901-911

5. Alagoz, M. et al. (2013). TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy. Nucleic Acids Research. 42(5) pp. 3089-3103

6. Solier, S. et al. (2013). Transcription Poisoning by Topoisomerase I Is Controlled by Gene Length, Splice Sites, and miR-142-3p. Cancer Research 73, pp. 4830-4839

7. Ledesma, F. C., El-Khamisy, S. F. et al. (2009). A human 5’-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage. Nature. 461, pp. 674-678

8. Zeng, Z. et al. (2011). TDP2/TTRAP Is the Major 5′-Tyrosyl DNA Phosphodiesterase Activity in Vertebrate Cells and Is Critical for Cellular Resistance to Topoisomerase II-induced DNA Damage. Journal of Biological Chemistry. 286(1), pp. 403-409

9. Gomez-Herreros, F. et al. (2014). TDP2 protects transcription from abortive topoisomerase activity and is required for normal neuronal function. Nature Genetics. 46, pp. 516-521

10. Haffner, M., et al. (2011). Transcription-induced DNA double strand breaks: both oncogenic force and potential therapeutic target? Clinical Cancer Research. 17, pp. 3858-3864

11. Ashour, M., et al. (2015). Topoisomerase-mediated chromosomal break repair: an emerging player in many games. Nature Reviews Cancer. 15, pp. 137-151

Stephen Power

Membership Number: 1068995

Investigation of the Relationship between Insulin

Resistance and Fyn during the development of

Alzheimer’s disease

Student Report 2015

Background

The effects of Alzheimer’s disease (AD) are well

documented but there is much to learn regarding

disease etiology. Research in laboratories,

including the UCC neurodegeneration lab, has

shown that neurons become resistant to insulin in

AD at early stages of the disease [1]. This insulin

resistance is characterised by abnormally high

levels of insulin receptor substrate-1

phosphorylated at serine 616 (IRS-1 [pS616

]).

Insulin resistance occurs at very early stages of

AD, is found in neurons with the defining tangle

pathology of AD and can be caused by the build-up

of amyloid beta peptide which is believed to be the

primary initiator of AD. There is a lot to be learned

about the cell and molecular mechanisms that cause

and result from the development of neuronal

insulin resistance in AD. Preliminary data from the

O’Neill lab at UCC indicates that neuronal insulin

resistance in AD may link to defective activation of

the Src kinase Fyn which has also been strongly

linked to the development and pathogenesis of AD,

but mostly in animal and cell models of AD.

Exploring the functional link between Fyn and

insulin resistance heightens the capacity to

understand early signalling causes of AD, to best

develop better AD diagnostic and therapeutic

systems

Aims and Objectives

Most of the work on Fyn in AD emanates from

preclinical AD animal models and cell culture

work. However, very little is known about Fyn

integrity in AD brain, with just two published

papers existing, nor has the relationship between

Fyn and insulin resistance been explored The aim

of this project was to use cell and molecular

approaches to investigate the relationship between

neuronal insulin resistance and Fyn in the AD brain

compared to normal aged brain.

Description of Work

Human brain tissue was obtained in an established

collaboration with the Netherlands Brain Bank.

Temporal cortex lysates and formalin-fixed paraffin-

embedded sections of the hippocampus and temporal

cortex from AD (n=7) and control cases (n=7) were

used in this research.

Initially, the study focused on western

immunoblotting analysis of Fyn and IRS-1 [pS616

]

levels in total homogenate, membrane (100,000 x g

pellet) and cytosolic fractions (100,000 x g

supernatant) lysates from the control and AD cases

previously prepared in the lab as described [1]. These

nitrocellulose blots were re-probed with β-actin to

verify the quality of protein loading. Control and AD

membrane preparations were also analysed for total

human tau (HT7) and pathological paired helical

filament (PHF-1) tau

This research also utilised double immuno-

fluorescence microscopy to determine the

relationship between Fyn and IRS-1 [pS616

] in

control and AD hippocampal and temporal cortex

brain sections. The Leica DMI3000b was used to

observe immunofluorescence alongside the Leica

Application Suite to capture images. Adobe

Photoshop allowed the green and red channels of the

image to be layered to allow visualisation of co-

localisation.

Results

Comparative Western immunoblot analysis revealed

that IRS-1 [pS616

] levels increased in AD compared

to matched control cases in the homogenate and

membrane (Fig 1) fractions as previously described

in the O’Neill lab [1].

Supervisor: Dr Cora O’Neill

Student: Stephen Power

Stephen Power


Figure 1: Levels of IRS-1 [pS616] in membrane lysates from

Alzheimer’s disease (AD) n=7) and control(C) brain

(n=7) showing increased IRS-1 [pS616] in AD cases

compared with controls

Levels of Fyn appear unchanged when comparing

control and AD samples for both homogenate (not

shown) and membrane fractions (Fig. 2) This

result is interesting, as further analysis in the lab

indicated levels of cytosolic Fyn decreased in AD

cases compared with controls (Fig. 3).Together this

indicates that the majority of Fyn in AD cases

localises to membrane fractions whereas the

cytosolic component of Fyn diminishes in the

disease, which could be associated with increased

activation of Fyn in Alzheimer’s disease.

Figure 2: Levels of Fyn in membrane lysates in

Alzheimer’s disease (AD) (n=7) and control (C brain

(n=7)

Figure 3: Reductions in cytosolic Fyn in AD (n = 7) compared

to control (n =7) temporal cortex brain samples

Western blots of membrane fractions probing with

PHF-1 primary antibodies revealed that PHF-1 was

entirely absent from control brain, while high

amounts of PHF-1 were visible within the AD brain

lysates (not shown). HT7 primary antibodies,

which detect total human tau, showed that 6

isoforms of human tau were visible in both the

control and AD brain (Fig. 4). However, the AD

tau was increased and showed a higher molecular

weight than the control tau due to the characteristic

hyperphosphorylation of tau as part of AD

pathogenesis. The presence of high molecular

weight tau immunoreactive banding was noted in

AD which is likely to be higher molecular weight

tau aggregates.

Figure 4: Levels of total human tau in membrane lysates

in Alzheimer’s disease (n=7) and control brain (n=7)

Together this immunoblot analysis showed

increased insulin resistance in AD, typified by

increased levels of IRS-1 (pS616) associated with

reduced levels of cytosolic Fyn although overall

levels of Fyn or Fyn levels in membranes did not

change in AD. This changed cytosolic/membrane

partitioning of Fyn in AD cases tentatively

indicates insulin resistance in AD may be linked

with increased Fyn activation.

Immunofluorescence microscopy revealed an

increase in the levels of IRS-1 [pS616

] as previously

shown in the lab [1], but also an increase in Fyn

expression in AD neurons compared with control

neurons (Fig 5). Also evident was the strong co-

localisation of Fyn and IRS-1 [pS616

] in AD

neurons. This result does not quite agree with the

results of the western blot which suggested that Fyn

levels were not increased in AD but rather altered

in their membrane to cytosolic expression. This

may be explained by the use of different antibodies

for immunoblotting and immunofluorescence

where the antibody used for immunofluorescence

may detect both active and total Fyn. Future studies

are investigating this further. Together the results

reveal that alterations in Fyn in AD are typified by

increased cytosolic to membrane migration of this

Src kinase that is found concurrent with, and can

co-localise with, increased neuronal resistance.

This gives a preliminary indication that increased

activation of neuronal Fyn and insulin resistance

may be mechanistically linked in Alzheimer’s

disease.

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Stephen Power


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IRS-1 [pS616

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Value of the Studentship

My experience in the lab has been truly invaluable.

The Biochemical Society summer studentship has

provided me with the unique opportunity to hone

and expand upon my laboratory practical skills and

my theoretical knowledge of Alzheimer’s disease

and its mechanisms of pathogenesis. This research

experience has allowed me to gain confidence

working in a research environment, and working

here in the BioSciences Institute has provided me

with the opportunity to attend several seminars

based in the field of biochemistry. I have really

enjoyed my hands-on experience researching AD

under the guidance of Dr Cora O’Neill and my time

spent here has further cemented my ambitions to

pursue a PhD following the completion of my

degree course. I am very thankful to the

Biochemical Society for providing me with this

opportunity.

Benefit to the Laboratory

Stephen was an excellent summer student,

extremely motivated and enthusiastic and a great

addition to the lab. His work over the 8 weeks was

valuable in many ways and gave us significant

preliminary data indicating that insulin resistance in

AD neurons may be linked to changes in Fyn

activation that have been described in the disease.

Stephen’s data will form part of future studies in

our lab in this area. We are very grateful to the

Biochemical Society for providing the funding to

support Stephen.

Bibliography

[1] Moloney, “Defects in IGF-1 receptor, insulin receptor and IRS-1/2 in AD indicate possible

resistance to IGF-1 and insulin signalling,” Neurobiology of Aging, vol. 31, pp. 224-243, 2010.

Alzheimer's Association, “2014 Alzheimer's Disease Facts and Figures,” Chicago, 2015.

Figure 5: Immunofluorescent microscopy of the

CA1 region of control and AD

hippocampus. Co-localisation of Fyn

and IRS-1 [pS616] within neurons is

evident

Can N-Srcs help re-program fibroblast cells into neurons? Victoria Scott- Summer placement in Evans’ lab, University of York 2015 Aim: The conversion of fibroblasts to neurons (induced neurons, iNs) has the potential to treat neurodegenerative diseases, and thus research in this field is very important. Recent work in the Evans’ lab has found that transfecting fibroblast cells with N2 Src, a tyrosine kinase, can differentiate them to have a neuronal phenotype, becoming iNs. In addition researchers have found that several transcription factors, including Ascl1, are important for the differentiation of iNs, (1,2). So the aim of this project was to determine whether the transfection of Cos 7 cells with N Srcs along with Ascl1 can increase the complexity of iN morphology. In addition, I aimed to confirm the neuronal status of N-Src-iNs by western blotting. Work carried out: Cell culture and transfections Initially I spent much of my time working out the optimal conditions for transfection efficiency and cell health. When I had found the best conditions, I transfected Cos7 cells with N1, N2 and C Srcs, alone and with Ascl1, as I wanted to see whether Ascl1 would increase the neuronal phenotype. I repeated this experiment so I had at least 50 cells for each condition to analyse. I then spent a few days learning how to use the fluorescent microscope and taking pictures of my cells I had stained with antibodies. Western blotting I then lysed cells transfected with the same conditions and used western blotting to analyse whether there was a change in Src and Ascl1 protein when transfected singly and together. I also used a pY146 antibody as this sight is auto phosphorylated when Src is active. Cloning Alongside this experiment I used two different methods to attempt to clone Ascl1 into a bacterial vector, but unfortunately this was unsuccessful. North of England Cell Biology meeting I attended the Biochemical Society sponsored NECB meeting at the end of my placement and had my first experience of a scientific conference, I was inspired by the research of the young scientists who presented their work. Results:

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Figure 1: Transfection with Ascl1 alone and in combination with Src kinases changes the morphology of Cos7 cells.

A: Fluorescence microscopy images taken with a 40X lens. Images are representative of the total sample from two Cos7 transfections of at least 50 cells from 20 fields of view. Scale bars represent 20 microns. B: The mean number of neurites per cell at each of the transfection conditions. Error bars represent 2X standard error of the mean. C: The percentage of cells with neurites.

D: The mean area of the cell body (μM2). Error bars represent 2X standard error of the mean.

Transfection conditions: 1= Ascl1 + Empty GFP. 2= Empty GFP+ Empty Flag. 3= N1 Src+ Empty GFP. 4= N1 Src+ Ascl1. 5= N2 Src+ Empty GFP. 6= N2 Src+ Ascl1. 7= C Src+ Empty GFP. 8= C Src+ Ascl1.

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There was a significant difference between the mean number of neurites per cell in the different transfection

conditions, (Kruskal wallis: 2 =34.49, df=7, p=0.00), however pairwise comparisons showed the only significant

differences to be between all of the transfection conditions and the control, except C Src+ Ascl1. This suggests that the addition of Src kinases and Ascl1 changes the morphology of Cos7 cells into a neuronal phenotype but they are equally effective in doing this. It also suggests that the co-transfection of Srcs and Ascl1 is not significantly improving the complexity of neuronal morphology compared to Ascl1 alone. There was also a significant difference between the

mean cell body area of cells transfected in the different conditions, (Kruskal wallis: 2 =24.07, df=7, p=0.001), but

pairwise comparisons showed this to only be between the cells transfected with Ascl1+ Empty flag and Empty flag+ empty GFP, thus suggesting that Ascl1 alone can reduce the size of the cell body. Previous work on this project in the Evans lab has found N Srcs to reduce cell body area, so perhaps more repeats are necessary. The western blots suggest that when Cos7 cells are co transfected with Ascl1 and C or N1 Src, the amount of Src kinase produced is increased, this could explain the neuronal phenotype. However C Src protein has been detected to be the highest concentration in the cell lysate despite the cells transfected with this kinase having the least neuronal phenotype. pY146 is auto phosphorylated when Src is active, however I believe the results are inconclusive and such they need to be repeated. Ascl1 was detected in all of the cell lysates when co transfected with Src, however it was not detected when alone, this suggests an interaction between the expression of Ascl1 and Src kinases. However it is possible that there is less protein in the lysate for Ascl1 alone, as shown by the weaker actin signal. Departures from the original proposal and future directions for the project: In addition to the original aim, I attempted to clone Ascl1 into a bacterial vector in order to complete a kinase assay. However this was unsuccessful by using both restriction enzymes and PCR. This part of my project will be continued by my Supervisor, who will hopefully be more successful! Also due to time restrictions, I did not blot for neuron specific proteins and thus I was unable to conclude whether the cells I have analysed are Cos7 cells with neurite growth or whether they have differentiated into neurons so I think the future direction of this project would be to distinguish between these. In addition, it would be interesting to discover whether different Srcs co-transfected in Cos7 cells could increase the complexity of neuronal morphology, and whether they are involved in the neuronal differentiation at different time points. Value of the studentship to me and Career aspirations: I have fully enjoyed my time in the lab and I feel I have learnt not only multiple important biological techniques but I have gained a real understanding of the day to day running of the lab and what a career in research is like. This placement has allowed me to put my knowledge into practice and has immensely improved my practical skills and so I am now ready to begin my final year project. I am also enthused to start a career in science so at the end of my degree I will be applying for positions in this field. I would like to thank Gareth, Sarah, Laura and Inés for all their help and support during my project. I am also very grateful to the Biochemical Society for this scholarship. Value of the studentship to my supervisor: It was a real pleasure having Victoria in the lab for her 6 week studentship. She achieved a lot in a short time using a wide range of techniques and was pretty independent towards the end. She has been able to validate the preliminary experiments of a previous project student, which has provided us with sufficient evidence to take the project further in the future. Although Victoria's attempts to prepare some new bacterial expression plasmids did not come to fruition, she has made reagents that we can use to finish the cloning. The whole experience has been highly beneficial to the lab and to Victoria's aspirations to pursue a research career. References

1. Vierbuchen T et al (2010) Direct conversion of fibroblasts to functional neurons by defined factors, Nature. 463(7284):1035-41

2. Wapinski OL et al (2013) Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons, Cell. 155(3):621-35

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Actin 42 kDa

Src-pY146 60 kDa

Figure 2: Cell lysate western blotting

Cells were lysed after transfection with the above plasmids for 24 hours. They were then blotted with various antibodies before being exposed on X ray film for approximately 15 minutes. Actin acts as a loading control. Ascl1 was detected at a very low level in the C+Ascl1 GFP lysate, but it is difficult to see on this image.

Biochemical Society Studentship Report 2015

Vitalii Mudryi

Supervisor: Dr. Shozeb Haider, University College of London School of Pharmacy

Investigating structure – phenotype relationship in Apparent Mineralocorticoid Excess Disease

Introduction

Apparent Mineralocorticoid Excess (AME) is a rare autosomal recessive genetic disease caused by mutations in the

11β-hydroxysteroid dehydrogenase type 2 gene (11BHSD2) leading to a deficiency in the enzyme. The 11BHSD2

enzyme belongs to the short-chain dehydrogenase/reductase family (SDR) and is a 405 amino acid protein of 42kDa

that catalyses oxidation of cortisol to inactive metabolite cortisone in minaralocorticoid target tissue, mostly in distal

nephrones. This protects the mineralocorticoid receptor (MR), which has equal affinities for both cortisol and

aldosterone, from excessive stimulation by cortisol which circulates in the body at 100-1000 times higher concentration

than aldosterone. The mechanism allows aldosterone to bind to the MR receptor with consequent downstream effects on

transcription. Activation of MR receptors leads to the expression of proteins regulating ionic and water transport,

mainly epithelium sodium channel or EnaC, Na+/K+ pump. In case of deficiency of enzyme 11BHSD2 MR receptors

are highly activated that increases renal sodium reabsorbtion and potassium extraction. Low sodium level means blood

volume expansion and increase in pressure. This resemble an increase of blood pressure caused by aldosterone excess,

this is why disease was called AME. [1-3]

Aims

Aim1.Construction of the next generation of structural model of enzyme 11BHSD2 with coenzyme NAD+ and cortisol

as ligand

Significant structural conservation is observed in the SDR family of proteins. This allowed us to construct a reliable

homology models. Firstly we have to choose our templates. We took to account features such as maximum sequence

overlap, resolution of the template structure, presence of coenzyme and ligands, possibility of a dimer formation, lack of

missing regions etc. In order to define maximum overlap we used BLASTp tool to search the PDB database. Our

template sequence was extracted from UniProt database (DHI2_HUMAN (P80365)). From the BLASTp result the top

three templates that were selected were two isoforms from 17β-hydroxysteroid dehydrogenase type 2 (17BHSD2) and

11β-hydroxysteroid dehydrogenase type 1 (11BHSD1). Due to the lack of structural resolution for the N- and C-

terminal regions in the templates, the model had to be constructed within 81- 370 residues. The best query cover was

89% for 17β-hydroxysteroid dehydrogenase type 2, with 29% identity E-value 3e-15 (pdb id 1IOL). For 11β-

hydroxysteroid dehydrogenase type 1 the cover was 61%, identity 23% ,E-value 1e-11 (pdb id 3PDJ). Besides, 17β-

hydroxysteroid dehydrogenase type 1 had NAD+ as coenzyme bound in the structure, which would be transferred from

the template to the model. The models was constructed using Modeller software (Mod9.14) [4]. Both monomer (active)

and dimeric (inactive) forms of the enzymes were constructed. The three templates that were chosen were PDB id

1IOL, 1JTV and 1FDV. We chose the multiple template modelling method to fill missing regions in 1JTV, while 1FDV

structure was used as the template for the dimeric form of the 17BHSD2 enzyme. Multiple alignment of 1IOL, 1JTV,

1FDV amino acid sequences was performed using ClustalX software [5] and then refined manually to avoid gaps in

secondary structure regions. After model construction, ligands were docked in the structure. Due to structural similarity

between cortisol and estradiol, we assumed that cortisol should bind at the same site as estradiol does. Thus the receptor

binding site was defined around estradiol. To prove that cortisol has the best affinity to 11BHSD2, we docked several

other ligands (cortisone, progesterone, estrogene, testosterone), and the binding energy for cortisol was the strongest at

-23,44 kcal/mol. For this task we used ICM Molsoft Pro software [6].

Aim2.Optimisation of the model via Molecular dynamics simulations

We performed Molecular Dynamics (MD) refinement of our monomer and dimer models. For MD simulations we used

AMBER software [7], employing the ff14SB force field. Parameters for the ligands were created using antechamber

tools. The system was solved with 8 Å water TIP3PBOX and neutralized with 5 chlorine atoms. Firstly the

minimisation was performed, than NVT equilibration for 5ns with heating to 300K, finally MD Production NPT for 100

ns.

Figure1: 3D structures of HSD11B2 in monomer and dimer forms after Molecular dynamics simulations

Figure2: a)the model of 11HSD2 before molecular dynamics refinement b)the model of 11HSD2 after molecular

dynamics refinement. (The lowest normalized residue energy is shown by blue color, the highest one by red.)

c)superimposed a - black, b – red with the RMSD value of 1.739 Å)

Aim3.Map known mutations and predict their effect on the structure.

Searching through databases and reading papers we created a list of mutations. The mutations were mapped on created

before structures. This allowed to see the influence of each mutation on protein features such as stability, dimer

formation, ligand binding.

Figure3: Missense mutation R186C causes disruption in R186 – E190 amino acids electrostatic interaction, which is

important for dimer formation. This could be the cause of AME type1, more severe type.

The value of the studentship to the student

The studentship has enabled me to experience what carrying out research in a leading institution. It has opened for me

perspectives in structural bioinformatics. It has allowed me to use most up-to-date and popular bioinformatic tools and

what's more important how to plan an in silico experiment by myself. I am in debt to Biochemical Society for providing

me this opportunity, which has strengthened my resolve to pursue becoming a scientist as a career.

The value of the studentship to the lab

The studentship allowed the Haider lab to generate valuable structural data. The models created by Vitalii were then

used to map all known mutations of AME and provide a structural rationale for the defective enzyme. This finding was

exemplary as it helps clinicians in quickly predicting the severity of the disease by assessing these mutations. This work

is a part of a study that is being submitted to the PNAS journal.

References:

1)Blood Press.2014;23(3):189-92

2)Mol. Cell. Endocrinol 2014;384:71-82

3)Physiol Genomic 2010;42(3):319-330

4)J. Mol. Biol. 1993;234: 779-815

5)Bioinformatics 2007;23: 2947-2948

6)Proteins 1997;1:215-220

7)J. Computat. Chem. 2005;26:1668-1688

Biochemical Society Summer Vacation Studentship 2015

| 1

High-throughput screening to identify novel inhibitors of

human α-methylacyl-CoA racemase 1A (AMACR; P504S)

Student: Yoana Petrova. Supervisor: Dr. Matthew Lloyd.

Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, U.K.

Introduction Pristanic acid [2R,S,6R,10R-(2,6,10,14-tetramethyl)pentadecanoic acid] is a 2-methyl saturated fatty acid derived from phytanic acid [3R,S,7R,11R-(3,7,11,15-tetramethyl)hexadecanoic acid] by α-oxidation in the body. Phytanic acid is obtained from the diet and is particularly abundant in foods such as red meat and dairy products.

1 The α-oxidation of phytanic acid produces a mixture of

2R- and 2S-methyl pristanic acid epimers.2 However, the acyl-CoA

oxidases responsible for the further metabolism of pristanic acid by β-oxidation have the absolute requirement for the 2S-methyl acyl-CoA esters.

3 The enzyme involved in the conversion of the 2R to the

2S isomer is α-methylacyl-CoA racemase (AMACR).4

AMACR has also the enzyme responsible for the activation of Ibuprofen in the pathway, converting the R-Ibuprofen to the pharmacologically active S-Ibuprofen.

5 The reactions catalysed by AMACR proceed via

an enolate intermediate.6

AMACR levels are increased (up to 9 fold) in all types of prostate cancers and phytanic acid has been shown to have a role in regulating levels.

7,8 Studies have shown a correlation between the

consumption of products high in phytanic acid (red meat and dairy products) and advanced prostate cancer.

9,10 It was demonstrated

that reducing AMACR levels using siRNA reduced the proliferation of prostate cancer cells, making AMACR an attractive target.

7

There are two main problems hindering the discovery of inhibitors against AMACR: the lack of a convenient assay to measure the activity of the enzyme and the fact that the CoA moiety is essential for binding of the inhibitor. Many CoA analogues were synthesised and showed inhibition of AMACR, however these are not drug-like molecules due being zwitterionic and having high MWs. The first problem was resolved when a colorimetric assay was developed by Dr. Maksims Yevglevskis (unpublished work), who is part of Dr. Lloyd’s research group. The colorimetric assay uses the reaction, in which a colourless acyl-CoA substrate is converted to a yellow product by AMACR (Scheme 1). The formation of the yellow product allows the activity of the enzyme to be assayed in 96-well plates using a spectrophotometer. The second problem concerning the lack of drug-like inhibitors for AMACR is addressed in the aims of this study.

Aims and objectives The aim of the project is to discover a novel class of small molecule compounds, which inhibit the activity of AMACR and exhibit drug-like properties. The objectives were to: 1) Optimise the colorimetric assay conditions for high throughput screening of compounds against AMACR; 2) Complete high throughput screening of 7680 compounds and identify potential inhibitors (‘hits’); 3) Further characterise the ‘hits’ with respect to their inhibitor properties. Materials and Methods All materials and reagents are obtained from the Sigma-Aldrich Chemical Co. or Fisher Scientific Ltd unless otherwise stated. The compound libraries were obtained from MRC Technology. AMACR expression, extraction and purification Competent E. coli Rosetta2 (DE3) cells (Novagen) were prepared and transformed with plasmid encoding for human His-tag AMACR enzyme

6 using the CaCl2-heat shock method. Recombinant cells

expressing the enzyme were lysed with the One Shot cell disruption system. The AMACR enzyme was purified by metal chelate chromatography. SDS-PAGE was performed to confirm the presence of the enzyme in fractions. Visking tubing dialysis was used to buffer exchange the enzyme, and the concentration of the enzyme was determined by measuring the absorbance at 280 nm using a Helios Omega spectrophotometer .

11

High throughput screening assay conditions The assay was performed in 96-well half-area plates. The activity of the enzyme was assayed by measuring the absorbance at two wavelengths (354nm and 390nm) using a BMG LabTech FluoStar Omega spectrophotometer. Measurements were taken every minute for 8 minutes. The total assay volume used was 100μL. The library compounds were used at 30μM in the assay, giving a final DMSO concentration in the assay of 3% (v/v). The library compounds were incubated with the AMACR enzyme for 10 minutes before addition of the substrate. The substrate concentration in the assay was 18μM, which is equal to the Km value (unpublished work). The enzyme was used at 0.086mg.mL

-1 in

the assay, which is within the concentration range previously used in Dr. Lloyd’s group. The positive control had 3% (v/v) DMSO to replace the library compound and the negative control had phosphate buffer to replace the enzyme and 3% (v/v) DMSO to replace the inhibitor. IC50 determination The IC50 determination was performed on inhibitors identified from the screening under the same assay conditions, with a substrate concentration of 40μM in the final assay. The top concentration of the selected inhibitors in the assay was 30μM. A 3-fold dilution series was used with 8 concentrations of inhibitor in total. All dilutions of the inhibitors were performed in DMSO in order to avoid precipitation of the inhibitors.

Scheme 1 The scheme shows the novel E1cB reaction catalysed by AMACR. The colourless acyl-CoA substrate is converted to a yellow 2,4-dinitrophenolate product and a colourless acyl-CoA product in one-step irreversible reaction.

2 |

Reversibility experiments The assay conditions were consistent with those used in the IC50

determination. The inhibitors were incubated with the concentrated enzyme for 10 min. The inhibitor concentration in the inhibitor-enzyme mixture was 30μM. Before the addition of the substrate, the inhibitor-enzyme mixture was diluted with phosphate buffer to give 0.086mg.mL

-1 enzyme and 0.66μM of

inhibitor in the final assay. Rates were determined as above Results and Discussion High throughput screening Possible inhibitors (‘hits’) were identified by comparison of the absorbance trace to positive and negative controls, and were taken forwards for IC50 determination. Approximately 70 ‘hits’ were identified during the screening, giving a ‘hit’ rate of 0.9%, which is consistent with the hit rate of <1% expected from a diverse and unbiased library.

12

IC50 determination The results from the IC50 determination are displayed in Figure 1. The top concentration of inhibitors used in the IC50 determination was 30μM due to the availability of limited amounts of compound. This is approximately 3 times lower than the top concentration of 100μM used previously in the IC50 determination of various CoA analogues, known competitive inhibitors of the AMACR. The IC50 values reported for some rationally designed 2-methylacyl-CoA analogues, including R- and S-Ibuprofenoyl-CoA, ranged between 0.5μM and 20μM.

13, 14 The inhibitors reported in this study have IC50

values in that range, suggesting that they have similar potency to the known inhibitors of the enzyme.

Reversibility experiments The reversibility experiments were performed in order to define the mechanism of inhibition (Figure 2). The experiment required incubation of the enzyme with inhibitor at a concentration 10 times the IC50.

12 However, due to insufficient amount of compound, 30μM

of inhibitor was used, approximately 2-3x IC50. It can be seen that the progress curve for the diluted inhibitors (0.66μM) is parallel to the one for the positive control, suggesting a reversible mode of inhibition because the activity of the enzyme is fully restored upon diluting the inhibitor to 0.04-0.07x IC50. The reversibility experiments also suggest that the compounds identified are enzyme inhibitors as opposing to non-specific denaturing agents. Future Directions The future directions of the project will involve performance of the IC50 determination and reversibility experiments to all of the ‘hits’ identified from the screening. Furthermore, comparisons

between the chemical structures of the inhibitors by computational chemistry methods will allow the identification of a common pharmacophore and the synthesis of analogues with improved activity. The development of a fluorescent assay is another future goal, as it will allow the measurement of the activity of AMACR in cell cultures and the evaluation of the inhibitors in vivo. Deviations from original project The original proposal for the project aimed for developing a convenient assay for measuring the activity of the AMACR. The reaction shown in Scheme 1 had been characterised by Dr. Lloyd’s research group at the time the project proposal was drafted. Therefore, the determination of the kinetic parameters of known inhibitors and the use of the assay to determine the inhibitors’ potency were part of the original project proposal. However, by the time I started the project, that part of the work in the proposal had been completed and the project I embarked on was a continuation of the original one.

Value of the studentship to the student The 8-week placement funded by the Biochemical Society allowed me to gain a valuable experience in variety of laboratory techniques such as: preparation of competent cells; protein expression using recombinant DNA technology; protein purification using His-tag; SDS-PAGE analysis. I was actively involved in the optimisation of the assay conditions for high throughput screening and in the process of screening. The placement gave me the opportunity to talk to other members of the team and to learn what the life of a researcher entails. As a consequence of the 8 weeks in the lab, I will definitely be considering a PhD after graduation. References 1) Lloyd, et al., Prog. Lipid Res., 2013, 52, 220-230; 2) Ackman, et al., Lipids, 1967, 2, 357-362; 3) Battaile, et al., Lipid Metab., 1998, 1390, 333-338; 4) Schmitz, et al., Eur. J. Biohem., 1995, 231, 815-822; 5) Woodman, et al., Chem. Commun., 2011, 47, 7332-7334; 6) Darley, et al., Org. Biomol. Chem., 2009, 7, 543-552; 7) Zha, et al., Cancer Res., 2002, 62, 2220-2226; 8) Mobley, et al., Cancer Epidemiol. Biomarkers Prev., 2003, 12, 775-783; 9) Wright, et al., Prostate, 2011, 71, 498-506;

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Figure 2 Reversibility experiments curves for four selected inhibitors (drugs 1-4). The positive control is shown in red and the negative control in yellow. The black line represents the drugs at 30μM and the green line the diluted drug at 0.66μM.

Drug1 Drug2

Drug3 Drug4

Figure 1 IC50 curves for four selected inhibitors (drugs 1-4). The IC50 values are : 16.2μM (Drug 1), 15.2μM (Drug 2) 9.3μM (Drug 3) and 12.8μM (Drug 4).

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10) Wright, et al., Int. J. Cancer, 2012, 131, 1396-406; 11) Yevglevskis, et al., Chem. Commun., 2014, 50, 14164-14166; 12) Copeland, Evaluation of enzyme inhibitors in drug discovery A guide for medicinal chemists and pharmacologists, John Wiley&Sons, 2005; 13) Carnell, et al., J. Med. Chem., 2007, 50, 2700-2707; 14) Carnell, et al., ChemMedChem., 2013, 8, 1643-1647.

Biochemical Society Summer Vacation Studentship Report Supervisors: Dr. Rachel Williams & Dr. John Taylor Student : Ngai Tsz Wai

Characterisation of the effect of IL-24 on human gingival keratinocytes and fibroblasts in vitro

Aim:

Our recent discoveries show a synergistic effect of IL-1 and leptin on the secretion of interleukin-24 (IL-24) in primary gingival fibroblasts. IL-24 is a cytokine of the IL-10 family with known immunomodulatory effects, such as, promoting inflammatory responses in epithelial cells. However, there is no information about similar effects in gingival keratinocytes and gingival fibroblasts. This project aimed to investigate the expression of IL-24 heterodimeric receptors consisting of either subunits IL-20R1/IL -20R2 or IL22R1/IL-20R2 on human gingival fibroblasts (HGF) or primary gingival keratinocytes and to find out the effects of IL-24 on these cells. Methods: Within the first two weeks, I looked at the expression of IL-24 receptors in primary keratinocytes. First, I observed the culturing of primary keratinocyte samples, extracted and analysed their RNA. Then, I converted RNA into cDNA through reverse transcription and amplified DNA segments through polymerase chain reaction. After that, I ran 3.5% Agarose Gels using those DNA segments along with molecular size markers at 70 Volts for 2 hours to look at the expression of IL-24 receptors. I also performed a Human IL-8 ELISA Assay to assess the effects of IL-24 on IL-8 secretion in HGF using HGF from three patients. In the following three weeks, I looked at the expression of IL-24 receptors in HGF using the same methods mentioned previously and performed a Human CXCL-12 ELISA Assay using the same set of HGF donors in the Human IL-8 ELISA Assay. In the remaining three weeks, I further ran two gels using DNA segments of three other HGF donors. I also acquired invaluable tissue culture techniques of cells feeding and splitting as well as the actual culturing of primary keratinocytes from donors’ gums. Result: Expression of IL-24 receptors in primary keratinocytes IL-20R2, IL-20R1 and IL-22R1 subunits that make up IL-24 receptors are expressed in primary keratinocytes as reflected by visible bands in the gels (not shown) at 75bp for IL-20R2, 125bp for IL-20R1 a d 150bp for IL-22R1. Effects of IL-24 on IL-8 secretion in HGF HGF donors secrete IL-8 at varying concentrations under different sample conditions as shown in Fig 1. HGF cultured from patient A secretes low IL-8 concentration with a mean of 7pg/ml under both unstimulated and IL-24 stimulated conditions whereas HGF cultured from patient B and patient C donors secrete even lower IL-8 concentration with a mean of 0pg/ml. In contrast, HGF from all patients stimulated with IL-1 secrete high IL-8 concentrations. HGF cultured from patient A secretes the highest concentration with a mean of 2098pg/ml, HGF cultured from patient B secretes the second highest with a mean of 1437.374pg/ml that is slightly higher than the secretion of the HGF cultured from patient C with a mean of 1294.699pg/ml. Similar results are obtained when both IL-24 and IL-1 were used to stimulate HGFs from all patients. HGF from patient A secretes the highest concentration with a mean of 2098pg/ml, HGF cultured

Fig 1. Human IL-8 ELISA Assay (HGF donors: patient A, B and C)

from patient C secretes the second highest concentration with a mean of 1608.71pg/ml that is greater than T22/11 2’s secretion with a mean of 979.0262pg/ml. Expression of IL-24 receptors in HGF Visible bands in gels (not change) are seen in all three HGF donors at 75bp for IL-20R2, 125bp for IL-20R1 and 150bp for IL-22R1 confirming that IL-20R2, IL-20R1 and IL-22R1 are expressed in HGF. Effects of IL-24 on CXCL-12 secretion in HGF HGF donors secrete CXCL-12 at varying concentrations under different sample conditions as shown in Fig 2. HGF cultured from patient B secretes the highest with a mean of 69.295pg/ml, HGF cultured from patient A secretes the second highest concentration with a mean of 16.3785pg/ml and HGF cultured from patient C secretes the lowest with a mean of 2.712pg/ml. When stimulated with IL-1, CXCL-12 is also secreted yet with a much lower concentration compared to that of IL-24 stimulated. HGF cultured from patient B secretes the lowest with a mean of 2.712pg/ml and HGF cultured from patient A secretes the second lowest concentration with a mean of 5.9525pg/ml. Both HGFs cultured from patient A and B secrete CXCL-12 under stimulation of both IL-24 and IL-1 with a mean of 16.575pg/ml and 38.17pg/ml respectively. Exceptionally for HGF cultured from patient C, a much higher CXCL-12 concentration is obtained instead with a mean of 6.25pg/ml and no CXCL-12 is secreted when stimulated under both IL-24 and IL-1.

Conclusion: The RT-PCR results confirm the expression of IL-24 receptors in both primary keratinocytes and HGF. Overall the ELISA data suggests IL-24 stimulated HGFs do not secrete IL-8 but secrete CXCL-12 whereas stimulation with IL-1 and both IL-1 and IL-24 results in the secretion of both IL-8 and CXCL-12 with varying concentrations. This suggests a synergistic effect of IL-1 and IL-24 on the secretion of both IL-8 and CXCL-12. Future directions: Further research into effects of IL-24 on human periodontal epithelia can be done using other cytokine or chemokine assays. Departures from the original proposal: There is no departure from the original proposal. Value of studentship: Throughout the studentship, I have learnt and practiced wide range of techniques from accurate and precise pipetting, culturing, feeding and splitting cells to running ELISA assays and agarose gels. This opportunity has also answered my doubts regarding my ability to take up a PhD after my undergraduate studies. I would also like to express my gratitude to my supervisor, John Taylor for his support and my postdoc, Rachel Williams for her patience and teachings throughout these eight weeks.

Fig 2. Human CXCL-12 ELISA Assay (HGF donors: patient A, B and C)

Role of cdc42 targets in Cancer cells interaction with endothelial cells

Student: Yunhui Zhuang Supervisor: Professor Anne Ridley and Dr Camilla Cerutti, King’s College London, UK

Background and objectives

Metastasis is the major cause of death in cancers. During cancer metastasis, cancer cells from the

primary tumour site invade their surrounding tissues and migrate through lymphatic or blood vessels

to enter the circulations. They can then cross through the endothelium of blood vessels and migrate

into a new tumour site where they survive and proliferate. This involves cell adhesion and

interaction of cancer cells with the endothelium in the blood vessels to allow migration and

development of secondary tumours (Reymond et al., 2013).

Rho-GTPases have been extensively studied for their effects on cell adhesion and migration through

remodelling of the actin and microtubules cytoskeletons. Cdc42 is one of the Rho GTPases that has

been found to mediate transendothelial migration of cancer cells through regulating β1-integrin at

transcriptional level (Reymond et al., 2012). IQGAP1 and N-WASP are both effectors of Cdc42

(Fukata & Kaibuchi, 2001), but their effects and roles in transendothelial migration are not yet fully

understand.

Brain metastasis from prostate cancer is infrequent, but have very poor prognosis (Tremont-Lukats

et al., 2003). Here, the aim was to study the effect of IQGAP1 and N-WASP on β1-integrin level, and

how they affected prostate cancer cells interaction with brain endothelial cells.

Methods and Materials

The prostate cancer cell line DU145 was primary used to study the effect of IQGAP1, N-WASP and

β1-integrin on cancer cells interaction with hCMEC/D3 brain endothelial cells. DU145 cells were

cultivated in RPMI-1640 medium (Gibco®) with L-glutamine, 10% foetal bovine serum (FBS), 100U/ml

penicillin and 50µg/ml streptomycin. The hCMEC/D3 brain endothelial cell line (Weksler et al., 2005)

was grown in EBM2 medium (Lonza®) on flasks coated with 5% college from calf skin (Sigma-Aldrich)

DU145 cells were transfected with siRNAs targeting IQGAP1 (GE-Dharmacon), N-WASP (Sigma-

Aldrich) and β1-integrin (Thermo Fisher Scientific) or control siRNA in 6-well plates using

oligofectamine™ (Invitrogen). After 72 h, cells were lysed in sample buffer and cell lysates stored at -

20°C.

Cell lysate were loaded and separated on a 6% SDS polyacrylamide gel, then transferred to

nitrocellulose membrane. Membranes were blocked with 5% skimmed milk powder, then incubated

overnight with mouse anti-IQGAP1 (Invitrogen), rabbit anti-N-WASP (Cell Signalling Technology®)

and mouse anti-β1-integrin (R&Dsystem®) antibodies, as well as mouse anti-GAPDH (Merck Millipore)

antibody (house-keeping gene as a loading control) for 1h. This was followed by washing with Tris

buffered saline with Tween 20 (TBS-T) and incubation with HRP-conjugated secondary antibodies

(GE Healthcare). Blots were developed by enhanced chemiluminescence (ECL) and exposed to the

film. Results on the film are then analysed in ImageJ software (NIH), where bands are quantified and

data are calculated against GAPDH and compared with the control.

For adhesion assays, siRNA-transfected DU145 cells were detached 72h after transfection using non-

enzymatic cell dissociation buffer (Sigma-Aldrich) and labelled with cell tracker™ green CMFDA (Life

Technologies). For live adhesion assays, images were taken after adding DU145 cells to confluent

hCMEC/D3 cells in μ-slide Ibidi 8 well plates for 15 min at 37°C, followed by two washes with PBS.

For each condition, 10 fields of view were acquired with a confocal microscope (Zeiss LSM510) and

the number of adherent DU145 cells were counted using ImageJ software. For fixed adhesion assays,

4% paraformaldehyde was added after incubating DU145 cells with D3 cells for 15 min in 24-well

plates. Cells were then washed with PBS and stored at 4°C for imaging on Nikon epifluorescence

microscope the next day. For each condition, cells were plated in duplicate wells and 2 field of views

were taken per well.

Intercalation assays were performed by adding CMFDA-labelled DU145 cells to confluent hCMEC/D3

cells in 24 well plates in a time-lapse microscope (Nikon), where plates were kept at 37°C and 5%

CO2. Images were captured every 5 min for 10 h using MicroManager software. For each condition

were plated in duplicate wells and 2 field of views were taken per well. Images were analysed using

ImageJ software and DU145 cells were counted as intercalated when they were flattened and

became part of the monolayer with hCMEC/D3 cells.

Results

Transfection of DU145 cells with siRNAs successfully reduced IQGAP1, N-WASP and β1-integrin

protein expression, in comparison to the control siRNA (Fig. 1). Two different siRNAs targeting each

gene were tested. β1-integrin antibody detected two bands by western blotting, which are most

likely be the glycosylated form and unglycosylated (lower bands) form. It was consistently observed

that knockdown of IQGAP1 and N-WASP reduced the level of β1-integrin, affecting the lower band

(unglycosylated) the most.

IQGAP1-1 siRNA reduced IQGAP1 expression by approximately 60%, and IQGAP1-3 siRNA has an

effect of 50% reduction. Both N-WASP2 and N-WASP3 siRNAs have reduced N-WASP expression

level by 40%. Both β1-integrin1 and β1-integrin2 siRNAs have reduced β1-integrin level by 90%.

Figure 1. Two representative blots were shown here. On the right, DU145 cells were transfected for 72hours

and total expression of IQGAP, N-WASP and β1-integrins were analysed in western blot. DU145 cells were

transfected with single siRNA oligos, as well as double and quadruple knock down with same total

concentration of siRNA oligos. Mock: reagents without siRNA; I+I: IQGAP1-1 and IQGAP1-3 siRNAs; N+N: N-

WASP2 and N-WASP3 siRNAs; I+I+N+N: IQGAP1-1, IQGAP1-3, N-WASP2 and N-WASP3 siRNAs

Figure 2. Western blot results of DU145 cells transfected with two siRNAs targeting IQGAP1, N-WASP, β1

integrin or control siRNA (control) after 72 h. Mock: cells treated with transfection reagent alone; Cells:

untransfected cells. Bars indicate mean ± SEM relative to control siRNA, n=3.

Experiments were performed to study the effect of IQGAP1, N-WASP and β1-integrin depletion on

cancer cell interactions with brain endothelial cells. Adhesion assays were performed, with one live,

and two fixed assays with DU145 cells on confluent HMEC/D3 cells. β1-integrin significantly reduced

adhesion of DU145 cells onto D3 brain endothelial cells by approximately 50% (Fig. 3). However,

IQGAP1 and N-WASP did not alter adhesion compared to the control siRNA-transfected cells.

Figure 3. Individual results of each adhesion assay. Exp1 was done with live cells in μ-slide Ibidi 8 well plates,

whereas Exp2 and 3 were fixed adhesion assay on 24-well plates with two duplications per plate.

When looked at individual results for each adhesion assay (fig.4), N-WASP3 seems to have an effect

on adhesion ability of DU145 cells on D3 cells in fixed adhesion assay of Exp2 and 3. However, this

observation is not consistent with Exp1 when images were taken live.

Summary of DU145 ADHESION TO hCMEC/D3 cells

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In parallel to adhesion assays, transfected DU145 cells were used to analyse intercalation with

hCMEC/D3 cells. Cells were imaged by time-lapse microscopy. Over 6 hour period, there was no

clear difference in intercalation of DU145 cells depleted of NWASP, IQGAP-1 or β1-integrin (Fig. 5) in

the two assays.

Figure 4. Adhesion of DU145 cells to

hCMEC/D3 cells. DU145 cells were

transfected with the indicated siRNAs.

After 72 h, they were labelled with

CDFMA before imaging. Results show

mean +/- SEM relative to control cells,

n=3.

Figure 5. Summary of duplicate results of intercalation assays of DU145 cells on hCMEC/D3 cells. In each assays,

eight conditions were duplicated in each plates and two field of views were imaged over 10 hours with 5

minutes interval.

Discussion

Western blotting analysis showed that all IQGAP1, N-WASP and β1-integrin siRNAs were able to

reduce total protein expression levels. It was interesting to observe that IQGAP1 and N-WASP

depletion reduced β1-integrin protein levels, and especially, the unglycosylated form. This may

suggest that both IQGAP1 and N-WASP are involved in internal cell trafficking and processing of β1-

integrins via the same or a different pathway. Since IQGAP1 is known to contribute to exocytosis in

cells (Ory & Gasman, 2011), it could affect endoplasmic reticulum (ER)-Golgi trafficking of β1-

integrins. N-WASP is an actin nucleating protein which may affect vesicular delivery of β1-integrin

between the ER and Golgi. However, both IQGAP1 and N-WASP are effectors of Cdc42 (Fukata &

Kaibuchi, 2001), and Cdc42 has been found to regulate β1-integrin transcription (Reymond et al.,

2012), and thus they could also act on transcription level. Further work could be done, such as qPCR,

to determine whether IQGAP and N-WASP affect β1-integrin mRNA levels.

Integrins are cell-matrix adhesion receptors that regulate cell adhesion and transduction of signalling

pathways from the extracellular matrix to the cell (Miao et. al, 2002). From the results summary of

the adhesion assays, it is apparent that β1-integrin knock down significantly reduced DU145 cell

adhesion to hCMEC/D3 cells. This indicates that DU145 cells may use integrins to adhere to

hCMEC/D3 brain endothelial cells. N-WASP seems to reduce DU145 adhesion to hCMEC/D3 cells,

which could be due to N-WASP reducing β1-integrin levels (as suggested in western blots). However,

this needs to be repeated to determine if the effect of N-WASP on adhesion is significant.

Interestingly, β1-integrin, IQGAP1 and N-WASP knock down seem to have no significant effect on

DU145 cell intercalation between hCMEC/D3 brain endothelial cells. This suggests that DU145 cells

may utilise use a mechanism independent of β1-integrin to intercalate between hCMEC/D3 brain

endothelial cells. This is in contrast to previous observations with β1-integrin-depleted PC3 cells,

which had strongly impaired intercalation between human umbilical cord endothelial cells (Reymond

et al., 2012). This may due to different properties between human umbilical cord endothelial cells

and hCMEC/D3 brain endothelial cells. Brain endothelial cells have specialised tight and adherent

junctions that forms the blood-brain barrier (BBB). This endothelial barrier can regulates its

transcellular permeability by responding to different signalling molecules, which might be β1-

integrin independent. On the other hand, DU145 cells may differ to PC3 cells by using different

mechanism that is β1-integrin independent to intercalate into endothelial cells. Further study can be

done to test these hypotheses by performing adhesion and intercalation assays with PC3 cells on

hCMEC/D3 cells, and DU145 cells on human umbilical cord endothelial cells.

Value of Studentship

This summer vacation research project funded by Biochemical Society has enabled me to work at

cutting-edge research. I have learnt several techniques in cell and molecular biology which I could

not possibly learn in my undergraduate degree. This has developed my confidence in research and

confirmed that academic research is the career path I wish to pursue after graduating.

References

Fukata, M., and Kaibuchi, K. (2001) Rho-family GTPases in cadherin-mediated cell-cell adhesion. Nat.

Rev. Mol. Cell Biol. 2(12) 887-897

Miao, H. Li, S. Hu, Y., Yuan, S. Zhao, Y., Chen, B.P.C, Puzon-Mclaughlin, W. Tarui, T. Shyy, J. Takada, Y., Usami, S., Chien, S. (2002) Differential regulation of Rho GTPases by β1 and β3 integrins: the role of an extracellular domain of integrin in intracellular signalling. J. Cell Sci. 115(10) 2199-2206

Ory, S., Gasman, S. (2011) Rho GTPases and exocytosis: what are the molecular links? Seminars in

Cell and Developmental Biology. 22(1) 27-32

Reymond, N., Im, J.H., Garg, R., Vega, F.M., Borda d’Agua, B., Riou, P., Cox, S., Valderrama, F., Muschel, R.J., Ridley, A.J. (2012) Cdc42 promotes transendothelial migration of cancer cells through β1 integrin. J. Cell Biol. 199, 653-668.

Reymond, N,, Borda d'Água, B,, Ridley, A,J. (2013) Crossing the endothelial barrier during metastasis. Nat. Rev. Cancer 13, 858-870.

Tremont-Lukats, T.W., Bobustuc, G. Lagos, G.K. Lolas, K. Kyritsis, A., Puduvalli, V.K. (2003) Brain

metastasis from prostate carcinoma. Cancer. 98(2) 363-368

Weksler B.B, E.A. Subileau, N. Perriere, P. Charneau, K. Holloway, M. Leveque, H. Tricoire-Leignel, A. Nicotra, S. Bourdoulous, P. Turowski, D.K. Male, F. Roux, J. Greenwood, I.A. Romero, P.O. Couraud (2005) Blood–brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J., 19, pp. 1872–1874

Isolation of secondary metabolites from Pseudomonas mesoacidophila

Student: Zainab Khan Supervisor: Dr Joel Loveridge

Abstract: Secondary metabolites were isolated from a

Pseudomonas mesoacidophila fermentation broth grown in

two different types of media. The extracted metabolites were

purified by prep-TLC followed by prep-HPLC and studied

by UV, IR, MS, LA-ICP-MS and high resolution NMR

analysis. In addition, flash chromatography was also used to

purify two of the cell extracts from the PF broths; however

due to time restraints the structures of the isolated

metabolites were not elucidated.

Introduction: Antibiotic resistance has been on the rise for many

years and many important antibiotics have become inactive. In

recent years, creating analogues of existing antibiotics by chemical

modification has been the preferred method of drug development;

however, this approach has resulted in fewer viable antibiotics.1

More recently, due to advances in technology, natural products are

once again becoming an area of interest in drug discovery.2 This

involves screening soil for bacteria which have evolved to produce a

diverse array of potent antimicrobial metabolites to kill their

competitors, such as simple phenylpyrrole derivatives like

pyrrolnitrin [Prn, 3-chloro-4-(2’-nitro-3’-chlorophenyl)-pyrrole, Fig

1]. Prn is a broad-spectrum antifungal compound that is active

against a wide range of fungi, yeast and Gram-positive bacteria.3 It is

an active component of PYRO-ACE and is one of the secondary

products of tryptophan metabolism.4

Figure 1: Structure of pyrrolnitrin

Aim: Initially, the aim of my project was to understand the

biosynthesis of the sulfated glycopeptide bulgecin in Pseudomonas

mesoacidophila.5 However, upon commencing my summer

placement the genome sequence of Ps. mesoacidophila became

available in our laboratory, revealing a Prn biosynthetic gene cluster.

This cluster seemed very interesting as previously Prn hasn’t been

found in or isolated from this bacterial species. Thus from there

onwards the aim of my project became the isolation, purification and

characterisation of Prn, and other organic-soluble secondary

metabolites from the culture broths of Ps. mesoacidophila.

Materials and methods

Growth media

PS (Pseudomonas seed) medium: 1.5% w/v Nutrient Broth, 1%

glucose, pH 7.0

PF (Pseudomonas fermentation) medium: 3% w/v glycerol, 1.5%

Nutrient Broth, 0.1% tryptophan, 0.1% glucose, pH 7.0 PF (Pseudomonas fermentation) with KBr: 3% w/v glycerol, 1.5%

Nutrient Broth, 0.1% tryptophan, 0.1% glucose, 0.1% KBr, pH 7.0 BSM-G (Basal salts minimal growth media with glycerol): 0.324%

w/v K2HPO4, 0.1% NaH2PO4.H2O, 0.2% NH4Cl, 0.02%

MgSO4.7H2O, 0.05% tryptone, 0.05% yeast extract, 0.4% glycerol,

0.06% v/v trace elements solution, pH 7.0. All media for bacterial growth were autoclaved at 121 oC for 15

minutes prior to use.

Extraction and purification of metabolites

A single colony of Ps. mesoacidophila was transferred to 100 mL PS

medium and grown at 28 oC, 150 rpm for 24 hrs. This seed culture

was then sub-cultured (1:50 dilution) into three 1 L portions of PF or

BSM-G medium, and grown at 28 oC, 150 rpm for 5 days. The

fermented broth was then pelleted at 6000 rpm at 4 oC for 20

minutes and the supernatant was separated from the bacterial cell

pellet. The pellet was suspended in acetone (200 mL), cells were

lysed by sonication, and the suspension filtered to remove any cell

debris. The acetone extract was concentrated under pressure; the oily

matter obtained was further extracted with DCM and concentrated

under pressure to yield a crude cell extract. The supernatant was extracted with ethyl acetate and concentrated

under pressure to yield a crude supernatant extract. The crude cell

and supernatant extracts were then tested for antimicrobial activity

against E. coli JM109 and XL1-Blue by the disc diffusion assay

method. Plates were incubated at 37 oC and diameters of inhibition

zones were measured in mm. For purification of the secondary

metabolites, preparative TLC followed by prep-HPLC of the crude

cell extracts were performed. For prep-TLC, chloroform, ethyl

acetate and formic acid (5:4:1 v/v) was used as a solvent system, and

for prep-HPLC, the isolates of prep-TLC were eluted with a 0-100%

gradient of acetonitrile in water using a reverse phase C18 column.

For one of the PF broths supplemented with KBr, flash

chromatography was employed, the crude extract for this was eluted

with a 0-100% gradient of acetonitrile in water using a reverse phase

C18 column.

Findings:

- All of the crude cell and supernatant extracts prepared were

compared against a standard of pure Prn using thin-layer

chromatography (TLC; Rf 0.91); the results showed that none of the

crude extracts contained Prn. - In addition, LA-ICP-MS was also carried out on the standard of

pure Prn (Fig 2). However, in general, ICP is a poor method of

detection for chlorometabolites as Cl has a high ionisation energy;

this can be seen from the small peaks observed in the pure Prn

sample even though the spot was concentrated. Negative results for

chlorometabolites are therefore not very informative.

- Moreover, the standard of pure Prn had a UV absorption band at

248 nm in chloroform.

Figure 2: LA-ICP-MS of pure Prn

- The crude cell and supernatant extracts of the first PF broth

prepared were tested for antimicrobial activity against E. coli JM109

and XL1-Blue using the disc diffusion assay method. The results

showed no clear inhibition zones for the crude supernatant extract;

however clear inhibition zones were observed for the crude cell

extract for both strains. As the concentration of the crude cell extract

was increased, the size of the clear inhibition zones increased (Fig

3). Subsequent work therefore focussed on the crude cell extract.

- The crude cell extract obtained from the first PF broth (PF1) was

purified by prep-TLC and all the different components were

separated. The spot at Rf 0.78 yielded an off-white solid (10 mg) and

this was further purified by prep-HPLC to yield an off-white solid

(Pm1; 8.6 mg).

- The mass of Pm1 was determined by electrospray mass

spectrometry to be 564 (Fig 4). The isotope ratio pointed towards a

compound containing two bromine atoms. LA-ICP-MS on a TLC

plate spotted with Pm1 confirmed the presence of bromine (Fig 5).

This finding was very interesting as terrestrial bacteria such as Ps.

mesoacidophila usually produce chlorinated metabolites, whereas

marine bacteria produce brominated compounds. Terrestrial bacteria

are usually able to synthesise the corresponding bromo-analogues

only when the culture medium is supplemented with bromide instead

of chloride.6

Figure 3: Disc diffusion assay of 5, 10 and 20 μL of PF crude cell

extract against E. coli XL1-Blue

Figure 4: ESI-MS of Pm1

Figure 5: LA-ICP-MS of Pm1

- The 1H NMR spectrum of Pm1 (Fig 6) showed the presence of four

aromatic proton environments and a broad singlet peak at 4.66 ppm

which was due to protons exchangeable with the solvent (most likely

an amino group). The other peaks at around 2 ppm were due to

impurities such as water and acetonitrile. The 13C NMR (Fig 7)

showed the presence of 14 carbon environments. Moreover, the

results of the COSY, HSQC and HMBC were very hard to interpret,

due to the similarity between the chemical shifts for both 1H and 13C.

Figure 6: 1H NMR of Pm1 in CDCl3

Figure 7: 13C NMR of Pm1 in CDCl3

- For the BSM-G crude cell extract, purification was unsuccessful

and no metabolites were isolated.

- Another two PF crude cell extracts (PF2 and PF3) were also

prepared to isolate the spot at Rf 0.78; however this spot was not

present in the new crude cell extracts and the results of Pm1 were not repeatable.

- A further two PF crude cell extracts were prepared but this time

one of the PF broths was supplemented with KBr to promote

production of bromo-metabolites, and both of these crude extracts

were purified by flash chromatography (Fig 8). Analysis of the

resulting fractions was not completed and TLC of both crude extracts did not show the presence of Pm1.

Figure 8: Flash chromatogram of PF crude cell extract

supplemented with KBr

- A disc diffusion assay was also carried out on pure Prn against E.

coli JM109 and XL1-Blue and no antimicrobial activity was observed.

- Furthermore, when the TLC plates of the crude cell and supernatant

extracts of both PF and BSM-G broths were visualised under UV,

blue/green fluorescent spots were observed; these are probably due

to siderophores (iron-sequestering compounds).7,8

- The fluorescence of the PF and BSM-G crude cell extracts was

therefore measured using a fluorimeter with excitation at 365 nm.

The fluorescence spectra confirmed that two different coloured

fluorescences were observed for PF with emission peaks at 440 and

465 nm. Whereas, only one coloured fluorescence was observed for

BSM-G crude cell extract with an emission peak at 445 nm (Fig 9).

Figure 9: Fluorescence spectra of BSM-G and PF crude cell extracts

Future directions in which the project can be taken: Crude cell extracts should be purified by flash chromatography and

the bioactivity of the resulting fractions should be tested against

fungi and both gram positive and negative bacteria. The genome of

Ps. mesoacidophila contains a number of putative biosynthetic genes

and gene clusters, including for non-ribosomal peptides, polyketides,

terpenes, butryolactones, lassopeptides, and bacteriocins, so a variety

of compounds should be produced. Once the antimicrobial activity is

known, the structure of purified isolates can be elucidated by NMR

and MS. Alternatively, bioautography could be performed on the

crude extracts by using developed TLC plates sprayed with a

suspension of fungal spores in broth, as a quick way of determining

which components of the extract show antifungal activity. In

addition, reverse transcriptase PCR could be performed on Ps.

mesoacidophila culture to determine whether the Prn gene cluster is

expressed under the experimental conditions used. Also, agar-based

growth media can be used to see if it has different effects to broths.

The value of the studentship to the student:

The summer studentship has been a very enjoyable experience and I

have relished every moment of it. It has significantly increased my

confidence in a research environment and I now feel more competent

at carrying out practical work. During my studentship, I was closely

supervised and taught by PhD student Gosia Kahl and my supervisor

Dr Joel Loveridge which was a great privilege, as I have now

acquired so many new skills and techniques beyond my chemistry

undergraduate studies. Overall, the studentship has been an excellent

experience and it has given me a valuable insight into what a career

in research entails on a daily basis. It has taught me that research is

all to do with patience, dedication and perseverance. Undertaking

this project has reassured me that a PhD in medicinal chemistry is

the correct path for me.

The value of the studentship to the lab (supervisor’s comment):

Zainab has done some excellent work in my laboratory this summer.

She worked hard, quickly learned the techniques required, and

managed her time well. At the end of her project, Zainab gave a very

good presentation to the Biological Chemistry group, and was able

to answer some quite challenging questions from the audience. She

will be co-author on at least one publication as a result of her work.

Furthermore, Zainab worked closely with my PhD student Gosia

Kahl, and I believe that both benefitted from this. I would strongly

recommend the Summer Vacation Scholarships to any

undergraduate student with an interest in research, and to any

academic with an interest in attracting excellent students to their

laboratory.

References

1. Coates, A. R. M. & Hu, Y. Novel approaches to developing new

antibiotics for bacterial infections. Br. J. Pharmacol. 152, 1147–54

(2007). 2. Ling, L. L. et al. A new antibiotic kills pathogens without

detectable resistance. Nature 517, 455–459 (2015).

3. Ligon, J. M. et al. Natural products with antifungal activity from Pseudomonas biocontrol bacteria. Pest Manag. Sci. 56, 688–695

(2000).

4. Van Pée, K. H. & Ligon, J. M. Biosynthesis of pyrrolnitrin and other phenylpyrrole derivatives by bacteria. Nat. Prod. Rep. 17,

157–164 (2000).

5. Imada, A., Kintaka, K., Nakao, M. & Shinagawa, S. Bulgecin, a bacterial metabolite which in concert with .BETA.-lactam

antibiotics causes bulge formation. J. Antibiot. (Tokyo). 35, 1400–

1403 (1982). 6. Van Pée, K. H. Biosynthesis of halogenated metabolites by

bacteria. Annu. Rev. Microbiol. 50, 375–99 (1996).

7. Cody, Y. S. & Gross, D. C. Characterization of Pyoverdin(pss), the Fluorescent Siderophore Produced by Pseudomonas syringae pv.

syringae. Appl. Environ. Microbiol. 53, 928–34 (1987).

8. Bultreys, A., Gheysen, I., Maraite, H. & de Hoffmann, E. Characterization of fluorescent and nonfluorescent peptide

siderophores produced by Pseudomonas syringae strains and their

potential use in strain identification. Appl. Environ. Microbiol. 67, 1718–27 (2001).

summer vacation studentship reports 2015

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