BMB & Pharm Research Conference 2012 ___________________________________________________________________

RETREAT MISSION STATEMENT

The Verna and Marrs McLean Department of Biochemistry and Molecular Biology

and The Department of Pharmacology

We take pride in the enormous breadth of biomedical research carried out within our two departments. This broad scope provides unique opportunities for cross-field education and intra- and inter-departmental collaboration. It is in the spirit of this cooperative venture that we hold the annual retreat, the goals of which are threefold: 1. To inform the departments as a whole about the state of research in the specific fields represented

within the departments. 2. To provide a congenial atmosphere where department members can present their research in a

manner conducive to collaboration. 3. To allow scientists at all levels the opportunity to hone their professional skills in an informal setting.

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BMB-PHARM RESEARCH CONFERENCE

OCTOBER 9 & 10, 2014

SCHEDULE OF EVENTS

Thursday, October 9 8:00 am – 8:45 am Continental Breakfast Salon A & B

8:00 am – 8:45 am Registration East and West Mainsail Lobby

8:50 am Opening Remarks East and West Mainsail

9:00 am – 10:00 am SESSION 1 East and West Mainsail

10:00 am – 10:15 am Departmental Photo

10:15 am – 10:30 am Break

10:30 am – 11:00 am Poster Ranking Salon C & D

11:00 am – 12:00 pm SESSION 2 East and West Mainsail

12:00 pm – 1:00 pm Lunch Salon A & B

1:00 pm – 2:00 pm SESSION 3 East and West Mainsail

2:00 pm – 3:30 pm POSTER SESSION 1 (Odd) Salon C & D

3:30 pm – 5:30 pm Free time

5:30 pm – 7:00 pm Cocktails & Costumes Poolside

7:00 pm – 8:30 pm Dinner Salon A & B

8:30 pm – 11:00 pm Dancing Salon C & D

Friday, October 10 8:30 am – 9:30 am Breakfast Buffet Salon A & B

9:30 am – 10:30 am SESSION 4 East and West Mainsail

10:30 am – 12:00 pm POSTER SESSION 2 (Even) Salon C & D

12:00 pm – 1:00 pm Lunch Salon A & B

1:00 pm – 2:00 pm SESSION 5 East and West Mainsail

2:00 pm – 2:15 pm Break

2:15 pm – 3:15 pm SESSION 6 East and West Mainsail

3:15 pm Awards and Announcements

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BMB-PHARM RESEARCH CONFERENCE

OCTOBER 9 & 10, 2014 THURSDAY, OCTOBER 10

8:00 am – 8:45 am CONTINENTAL BREAKFAST Salon A & B

8:00 am – 8:45 am Registration East & West Mainsail Lobby

8:50 am Opening Remarks: John Wilson East & West Mainsail

SESSION 1 Session Moderator: Ming Zhou [F]

9:00 am – 9:15 am Olivier Lichtarge [F] Evolution and Disease: The Calculus of Life

9:20 am – 9:35 am Josephine Ferreon [F] Allostery in 'Molecular Hub' Intrinsically Disordered

Proteins

9:40 am – 9:55 am Damian Young [F] Synthesis and Screening of Novel Fragments Derived

from Diversity–oriented Synthesis

10:00 am – 10:15 am Departmental Photo

10:15 am – 10:30 am Break

10:30 am – 11:00 am Poster Ranking Salon C & D

SESSION 2 Session Moderator: Carolyn Adamski [GS] (Palzkill Lab)

11:00 am – 11:15 am Michael Chen [GS] (Barth Lab) Trapping G Protein-coupled Receptors in

Active Signaling States Through Protein Design

11:20 am – 11:35 am Michele Darrow [GS] (Chiu Lab) Elucidating the Interactions of Mutant

Huntingtin with the TRiC-like CCT5 Complex

11:40 am – 11:55 am Melena Agosto [PD] (Wensel Lab) Oligomeric State of Purified TRPM1, a

Protein Essential for Dim Light Vision

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12:00 pm – 1:00 pm Lunch Salon A & B

SESSION 3 Session Moderator: Tim Farinholt [GS] (Kuspa Lab)

1:00 pm – 1:15 pm Zao Li [GS] (Z Zhou Lab) How Are Necrotic Cells Recognized by

Engulfing Cells in Caenorhabditis Elegans?

1:20 pm – 1:35 pm Milenka Arevalo-Soliz [PD] (Zechiedrich Lab) Mini Vectors Deliv er

Split Genes with No Cell Toxicity

1:40 pm – 1:55 pm Jessica Moore [GS] (Hastings/Rosenberg Lab) Reactive Oxygen

Species Are Required for Environmental Stress-induced Mutagenesis

In Starving Escherichia Coli Cells

2:00 pm – 3:30 pm POSTER SESSION 1 (Odd) Salon C & D

3:30 pm – 5:30 pm Free time

5:30 pm – 7:00 pm Cocktails & Costumes Poolside

7:00 pm – 8:30 pm Dinner Salon A & B

8:30 pm – 11:00 pm Dancing Salon C & D

FRIDAY, OCTOBER 11

8:30 am – 9:30 am Breakfast Buffet Salon A & B

SESSION 4 Session Moderator: Yun-Min Sung [GS] (Wensel Lab)

9:30 am – 9:45 am Rose Mikulski [PD] (Palzkill Lab) Sequence Determinants of Class

C Beta-lactamase Substrate Specificity at High Resolution

9:50 am – 10:05 am James Arnold [GS] (Sreekumar Lab) Epithelial-Mesenchymal Tran-

sition Metabolomic Signature (EMS) Significantly Improves Breast

Cancer Outcome Prediction

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10:10 am – 10:25 am Xiqian Jiang [GS] (J Wang Lab) Towards Quantitative Imaging

of Intracellular Glutathione Dynamics

10:30 am – 12:00 pm POSTER SESSION 2 (Even) Salon C & D

12:00 pm – 1:00 pm Lunch Salon A & B

SESSION 5 Session Moderator: Gilbert Huang [GS] (Kim Lab)

1:00 pm – 1:15 pm Sophie Xu [GS] (Schiff Lab) Clonal Evoluation of the HER2

L7558 Mutation Leads to Aqcquired HER-targeted Therapy Re-

sistance that Can be Reveresed by the Irreversible HER 1/2

Inhibitor AFATINB

1:20 pm – 1:35 pm Tiffany Hsu [GS] (Westbrook Lab) Oncogenic MYC Induces a

Dependency on the Spliceosome in Human Breast Cancer

1:40 pm – 1:55 pm Fangrui Wu [PD] (Song Lab) Cyclopropylamine Containing In-

hibitors of LSD1 A Potential Therapeutics for MLL-Rearranged

Leukemia

2:00 pm – 2:15 pm Break

SESSION 6 Session Moderator: Kim Tolias [F]

2:15 pm – 2:15 pm Jason Chen [F] The Role of Calcium-dependent Interaction Be-

tween Recoverin and Rhodopsin Kinase in Retinal Rods

2:20 pm – 2:50 pm Sunny Songyang [F] Telomere Signaling Networks in Aging and

Cancer

2:55 pm – 3:10 pm Alan Ferreon [F] Single-Molecule Conformational Maps of Intrin-

sically Disordered Proteins

3:15 pm AWARDS CEREMONY: Ted Wensel and Timothy Palzkill

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THE JOSHI

MEMORIAL PRIZE Dr. Vadusev C. Joshi was born in Gujarat, India. He

received his B.S. degree in pharmacy from Madras

Medical College in 1959, ranking among the top 5%

of students in the state. After earning his M.S.

degree in pharmacy from Andra Pradesh University in

1961, Dr. Joshi began his Ph.D. studies in the

Department of Biochemistry at the Indian Institute of

Science in Bangalore. Under the tutelage of Dr.

T. Ramasarma, he studied ubiquinone and related

compounds, earning his doctorate in biochemistry in

1964. While a postdoctoral fellow at the Indian

Institute of Science, Dr. Joshi was awarded a Ford

Foundation Research Fellowship. In 1966, he was

awarded the prestigious Fulbright Fellowship, which

allowed him to come to the United States to further his education at Duke University in the

Departments of Pediatrics and Biochemistry. There, he continued his studies of ubiquinone

and eventually became interested in fatty acid synthesis. In 1972, Dr. Joshi accepted a

position as an assistant professor in the Department of Biochemistry at Baylor College of

Medicine under the leadership of Dr. Salih J. Wakil. While at BCM, he continued his

investigations of fatty acid synthesis. He was promoted to associate professor in 1977 and

was honored with a prestigious Research Career Development Award from the National

Institutes of Health the same year. During his tenure at BCM, Dr. Joshi published numerous

papers and was an active member of the Admissions Committee. In 1980, Dr. Joshi spent a

year in the laboratory of Dr. Hargovind Khorana in the Department of Chemistry at the

Massachusetts Institute of Technology. After returning to BCM in late 1981, he continued his

research in fatty acid synthesis and gene regulation until his untimely death in 1982. Dr.

Joshi not only was a committed and hardworking scientist; he was an active member of the

Indian community and a strong patron of the arts. Most importantly, Dr. Joshi was a devoted

husband to his wife Yogini and a doting father to his three daughters, Harshini, Yamini, and

Meena.

As a tribute to Dr. Joshi for his dedication to excellence in education and research, his family,

friends, and colleagues established a memorial fund in his honor in 1986. The Joshi

Memorial Prize is granted to students and postdoctoral trainees within the Department of

Biochemistry in recognition of their outstanding oral and poster presentations at the annual

research conference.

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JUDGING CRITERIA Oral Presentations

I PRESENTATION (30%)

Organization of talk, clarity of presentation

Quality of slides, legibility & clarity of labeling, color & contrast

Engaging, entertaining, style

Time management

II RESEARCH (40%)

Significance for biomedical or basic science

Quality of data, difficulty of experiments

Interpretations justified, conclusions sound

Innovative approach or techniques

Clear acknowledgment of work by others

III INTERACTION (30%)

Did the talk stimulate questions

Ability to respond to questions

Display knowledge outside prepared presentation

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JUDGING CRITERIA Poster Presentations

I POSTER (30%)

Organization of poster, clear flow, aesthetics

Abstract, hypothesis or question, conclusions, acknowledgments

Legible figures, clearly & concisely labeled

II RESEARCH (40%)

Significance for biomedical or basic science

Quality of data, difficulty of experiments

Interpretations justified, conclusions sound

Innovate approach or techniques

How much work is done by the presenter

III INTERACTION (30%)

Was the poster presented clearly

Ability to respond to questions

Knowledge outside prepared presentation

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POSTER RANKING CRITERIA

I POSTER (40%)

Organization of poster, clear flow, aesthetics

Abstract, hypothesis or questions, conclusions, acknowledgments

Legible figures, clearly & concisely labeled

II RESEARCH (60%)

Significance for biomedical or basic science

Quality of data, difficulty of experiments

Interpretations justified, conclusions sound

Innovative approach or techniques

How much work is done by the presenter

9

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Table of Contents Name Type Page Carolyn Adamski Poster #1 11 Melina A. Agosto Talk 12 Ivan Anastassov Poster #2 13 Lirio M. Arevalo-Soliz Talk 14 James Arnold Talk 15 Yonghong Bai Poster #3 16 Salil Kumar Bhowmik Poster #4 17 Alex J. Brewer Poster #5 18 James Campbell Poster #6 19 Gong Chen Poster #7 20 Bo Chen Poster #8 21 Kuang-Yui Chen Talk 22 Ching-Kang Jason Chen Talk 23 Jinxuan Cheng Poster #9 24 Audrey Christiansen Poster #10 25 Audrey Christiansen Poster #11 26 Zachary Conley Poster #12 27 Michele C. Darrow Talk 28 Rocio Dominguez-Vidana Poster #13 29 Timothy Dosey Poster #14 30 Timothy Farinholt Poster #15 31 Xiang Feng Poster #16 32 Allan Chris Ferreon Talk 33 Josephine Ferreon Talk 34 Monica Galaz-Montoya Poster #17 35 Franklin Gu Poster #18 36 Feng He Poster #19 37 Tyler Hilton Poster #20 38 Matthew Holt Poster #21 39 Benjamin Hornstein Poster #22 40 Teng-Kuei Hsu Poster #23 41 Tiffany Hsu Talk 42 Liya Hu Poster #24 43 Gilbert Huang Poster #25 44 Amy Hurwitz Poster #26 45 Xiqian Jiang Talk 46 Hye Jin Kang Poster #27 47

Name Type Page Akash Kaushik Poster #28 48 Beom-Jun Kim Poster #29 49 Brian Kirk Poster #30 50 Zao Li Talk 51 Olivier Lichtarge Talk 52 Boxue Ma Poster #31 53 Shrenik Mehta Poster #32 54 Rose Mikulski Talk 55 Phillip Minnick Poster #33 56 Sharmistha Mitra Poster #34 57 Sayantan Mitra Poster #35 58 Jessica Moore Talk 59 Ilya Novikov Poster #36 60 Maha Patel Poster #37 61 Liying Qin Poster #38 62 Sam Regenbogen Poster #39 63 Zhenning Ren Poster #40 64 Michael Robichaux Poster #41 65 Soung-Hun Roh Poster #42 66 Zhou Songyang Talk 67 Vitali Stanevich Poster #43 68 Vlatko Stojanoski Poster #44 69 Zhizeng Sun Poster #45 70 Tingting Sun Poster #46 71 Yun-Min Sung Poster #47 72 María Elisa Terrón-Díaz Poster #48 73 Lin Tian Poster #49 74 Mengyu Wang Poster #50 75 Sara Wright Poster #51 76 Fangrui Wu Talk 77 Youli Xia Poster #52 78 X. Sophie Xu Talk 79 Zenghui Xue Poster #53 80 Damian W. Young Talk 81 Melvin Young Poster #54 82 Zhixian Zhang Poster #55 83 Jing Zhang Poster #56 84 Hanzhi Zhang Poster #57 85 Liuliu Zheng Poster #58 86

11

KEY DETERMINANTS IN THE B-LACTAMASE-MEDIATED MECHANISM OF BACTERIAL RESISTANCE TO CEPHALOSPORIN ANTIBIOTICS

Carolyn Adamski, Ana Maria Cardenas, Lori B. Horton, Nicholas G. Brown, Timothy Palzkill

A comprehensive understanding of the enzymes mediating resistance to the widely prescribed cephalosporin antibiotics is necessary to address the growing need for new antimicrobial therapies. The CTX-M-14 β-lactamase enzyme, one of the most common sources of resistance to cephalosporin antibiotics, was used in this study to identify residues that are necessary for hydrolysis of cefotaxime, an important cephalosporin drug. The roles of residues Ser237 and Arg276 in the activity of CTX-M-14 β-lactamase were investigated using site-directed mutagenesis, enzyme kinetic analysis and X-ray crystallography. Ser237 and Arg276 were mutated to alanine and the catalytic activity of the single and double mutant combinations was evaluated. The kinetic analysis revealed that, together, Ser237 and Arg276 residues are key determinants of cefotaxime hydrolysis by the CTX-M-14 β-lactamase. High-resolution crystal structures of the CTX-M-14 variants alone and in complex with cefotaxime illustrated the roles of Ser237 and Arg276 in cefotaxime hydrolysis. When both residues were mutated to alanine several significant changes were seen in the active site including a compromised hydrogen bonding network, a narrower active site, and a 180° rotation in the orientation of the aminothiazole ring on cefotaxime. These results identify Ser237 and Arg276 as key determinants of cefotaxime activity in the CTX-M-14 β-lactamase and elucidate their role in catalysis. Furthermore, these studies will help to inform future drug development studies by highlighting these residues as potential targets for β-lactamase inhibitor design.

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OLIGOMERIC STATE OF PURIFIED TRPM1, A PROTEIN ESSENTIAL FOR DIM LIGHT VISION

Melina A. Agosto, Zhixian Zhang, Feng He, Ivan A. Anastassov, Sara J. Wright, Jennifer McGehee, Theodore G. Wensel

Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON-bipolar cells. TRPM1 likely forms a multimeric channel complex, though almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryo-electron microscopy and single particle analysis without symmetry imposition yielded a model with approximate two-fold symmetrical features. Finally, electron microscopy of TRPM1/antibody complexes revealed a large particle which can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes – one similar in size to the recombinant protein, and one much larger. Since dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway.

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LOCALIZATION OF TRANSIENT RECEPTOR POTENTIAL CHANNEL TRPM1 IN MOUSE RETINA

Ivan Anastassov, Melina Agosto, Theodore G. Wensel

Recently the transient receptor potential channel TRPM1 has been identified as the protein responsible for the sign-inverting response of ON-bipolar cells in the mammalian retina. TRPM1 is believed to be a constitutively open, mostly nonselective cation-permeable channel, which is kept closed in darkness by a G-protein signaling cascade coupled to the glutamate receptor mGluR6. TRPM1 protein expression has been demonstrated at the site of action, i.e. the ON-bipolar cell dendritic tips, but extensive staining is also present in the dendritic processes, the cell soma and in the proximal area of the descending axons. Paradoxically, recent evidence from ultrastructural localization in human retina indicates that TPRM1 is also found in the pre-synaptic photoreceptor terminal. What elements are required for proper TRPM1 function is still elusive and a detailed understanding of protein distribution in native retinal tissue is essential.

Monoclonal antibodies generated to full-length TRPM1 were used to stain mouse retina by conventional immunocytochemistry and the tissue was imaged by SR-SIM and confocal microscopy. TRPM1 seemingly co-localizes with select markers of ON-bipolar cell dendritic tips (RGS7, Gβ5, mGluR6) and entire cells (PKCα). TRPM1 expression is robust at the dendritic tips and a clear dyad of bipolar cell dendrites can be observed with confocal and SIM microscopy, a morphological feature typically only seen with electron microscopy. Staining is completely absent in TRPM1 null mice.

A drawback of the recent evidence showing ultrastructural localization of TRPM1 in the human retina is the lack of data from TRPM1 knockout tissue. In immuno-EM studies, the most reliable way to differentiate between real and background signal is the use of knockout animals. Experiments are currently underway using the above TRPM1 monoclonal antibodies in TRPM1 wild type and knockout retinas to address the question of the suggested TRPM1 localization in the photoreceptor terminal.

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MINIVECTORS DELIVER SPLIT GENES WITH NO CELL TOXICITY

Lirio M. Arevalo-Soliz, Julyana Zelaya, Naresh Pandey, Daniel J. Catanese Jr, Jonathan J. Silberg, Meredith L. Brow, Lynn Zechiedrich

Gene therapy is a promising approach to treat cancer and other human diseases. A major limitation, however, is the lack of a safe and effective nucleic acid delivery vector. Viruses are effective at cell transduction but carry the risk of erroneous integration and are difficult to regulate. Plasmids are safe but their large sizes result in poor transfection for most human cell types. MiniVectorTM; DNA is a non-viral DNA vector as small as ~250 bp, devoid of bacterial sequences, and able to survive human serum and shear forces associated with gene delivery. The goals of this project were to take advantage of the small size of MiniVectors yet deliver entire genes, and to quantify the effect of vector size on cell toxicity and proliferation after transfection. Two fragments of the yellow fluorescent protein (YFP) gene that encode polypeptides that reassemble into a functional YFP in vivo, were cloned into separate pairs of MiniVectors or plasmids. Vectors were transfected at either the same molar concentrations or the same DNA mass into HeLa cells. The percent cells expressing YFP was analyzed by fluorescence microscopy and fluorescence activated cell sorting (FACS). Transfection efficiency of MiniVectors was as good or better as plasmids when delivered at equal mole concentration and required significantly less transfection vehicle. When delivered at equal mass, MiniVectors and plasmids were equally toxic to cells initially but only the cells receiving MiniVectors recovered and proliferated by 72 hours. The toxic effect on cells was plasmid-mediated rather than vehicle-mediated. Furthermore, at equal moles or equal mass, cells grew better when transfected with MiniVectors than with plasmids. In this study, we were able to show that the ability to deliver split genes and the lack of cell toxicity increase the clinical usefulness of MiniVectors.

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EPITHELIAL-MESENCHYMAL TRANSITION METABOLOMIC SIGNATURE (EMS) SIGNIFICANTLY IMPROVES BREAST CANCER OUTCOME PREDICTION

James Arnold, Salil K. Bhowmik, Esmeralda Ramirez-Peña, Nagireddy Putluri, Nathalie Sphyris, Arun Sreekumar, Sendurai A. Mani

Metabolic reprogramming is widely acknowledged as a fundamental hallmark of cancer. The epithelial-mesenchymal transition (EMT) is a latent embryonic program that is aberrantly induced in cancer cells bestowing stem-like and invasive capabilities which are thought to play important roles in metastasis; however the metabolic alterations associated with EMT remains poorly understood. Here we hypothesize that cancer cells undergo metabolic reprogramming concurrent with the onset of EMT and these metabolic alterations can guide the selection of metabolic biomarkers for predicting overall patient survival. The EMT metabolomic signature (EMS) was derived via two-sample t-test of LC-MS generated metabolomic profiles of EMT-induced lines versus vector control (VC). The EMS is composed of all measured metabolites found to be significantly elevated in the EMT-induced group relative to VC. The EMS is primarily composed of glycolytic and TCA intermediates, suggesting cells undergo a shift in energy production following EMT induction which may denote attractive therapeutic targets for targeting metastatic tumors. To test whether the EMS has potential prognostic value we employed multivariate Cox proportional hazards (CoxPH) regression model on a population of 67 breast cancer samples whose metabolomic profiles were available. We created two models for comparison: the first model included common clinical variables including age, stage, grade, ER status, and other factors while the second model contained common clinical variables as well as EMS metabolites. Here we report that the addition of EMS metabolites significantly improved the ability to predict overall patient survival at 3- and 5-year intervals. Furthermore, dividing patients on their relative levels of EMS metabolites is sufficient to derive a statistically significant survival model, suggesting the EMS metabolites do have potential prognostic value. In addition to potential prognostic value, the EMS illuminates the biochemical mechanisms underlying EMT reprogramming, and functional studies are underway to follow up these interesting leads.

16

X-RAY STRUCTURE OF A MAMMALIAN STEAROYL-COA DESATURASE-1

Yonghong Bai, Jason McCoy, Elena Levin, Kanagalaghatta Rajashankar, Ming Zhou

Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl CoAs. Since the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters, and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologs (SCD1 and SCD5), and mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity. Thus, SCD1 is a promising pharmacological target in the treatment of obesity, diabetes, and other metabolic diseases. SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyzes the formation of a cis-double bond between the 9th and 10th carbons of stearoyl- or palmitoyl-CoA. The reaction requires molecular oxygen, which is activated by a diiron center, and cytochrome b5, which reduces the diiron center. To better understand the structural basis of these characteristics of SCD function, we crystallized and solved the structure of mouse SCD1 bound to a stearoyl-CoA molecule at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel suggests the structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The structure reveals a dimetal center coordinated by a unique configuration of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a potential pathway for substrate access and product egress, and a possible route for electron transfer from cytochrome b5 to the diiron center.

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EPITHELIAL-MESENCHYMAL TRANSITION METABOLOMIC SIGNATURE (EMS) SIGNIFICANTLY IMPROVES BREAST CANCER OUTCOME PREDICTION

Salil Kumar Bhowmik, James M. Arnold, Esmeralda Ramirez-Peña, Nagireddy Putluri, Nathalie Sphyris, Sendurai A. Mani, Arun Sreekumar

Metabolic reprogramming is widely acknowledged as a fundamental hallmark of cancer. The epithelial-mesenchymal transition (EMT) is a latent embryonic program that is aberrantly induced in cancer cells bestowing stem-like and invasive capabilities which are thought to play important roles in metastasis; however the metabolic alterations associated with EMT remains poorly understood. Here we hypothesize that cancer cells undergo metabolic reprogramming concurrent with the onset of EMT and these metabolic alterations can guide the selection of metabolic biomarkers for predicting overall patient survival. The EMT metabolomic signature (EMS) was derived via two-sample t-test of LC-MS generated metabolomic profiles of EMT-induced lines versus vector control (VC). The EMS is composed of all measured metabolites found to be significantly elevated in the EMT-induced group relative to VC. The EMS is primarily composed of glycolytic and TCA intermediates, suggesting cells undergo a shift in energy production following EMT induction which may denote attractive therapeutic targets for targeting metastatic tumors. To test whether the EMS has potential prognostic value we employed multivariate Cox proportional hazards (CoxPH) regression model on a population of 67 breast cancer samples whose metabolomic profiles were available. We created two models for comparison: the first model included common clinical variables including age, stage, grade, ER status, and other factors while the second model contained common clinical variables as well as EMS metabolites. Here we report that the addition of EMS metabolites significantly improved the ability to predict overall patient survival at 3- and 5-year intervals. Furthermore, dividing patients on their relative levels of EMS metabolites is sufficient to derive a statistically significant survival model, suggesting the EMS metabolites do have potential prognostic value. In addition to potential prognostic value, the EMS illuminates the biochemical mechanisms underlying EMT reprogramming, and functional studies are underway to follow up these interesting leads.

18

MODAFINIL + LISDEXAMFETAMINE FOR COCAINE USE DISORDER

Alex J. Brewer, James J. Mahoney, Richard De La Garza, Thomas Newton

AIMS: The dopamine transporter (DAT) has been implicated in the subjective and reinforcing effects produced by cocaine. Modafinil has shown some efficacy in reducing these effects. The aim of this study was to determine if the ability of modafinil to reduce the subjective and reinforcing effects produced by cocaine could be enhanced by combination with another drug, lisdexamfetamine, that is also known to interact with the DAT.

METHODS: Non-treatment seeking, cocaine-dependent individuals had a history of cocaine use via the smoked or intravenous route, were 18-55 years old, were not dependent on other drugs of abuse excluding nicotine, and did not have unstable medical conditions or psychiatric diagnoses. Eligible individuals were randomized to one of four treatments: placebo, modafinil (200 mg, p.o.), lisdexamfetamine (30 mg p.o.), or modafinil (200 mg, p.o.) + lisdexamfetamine (30 mg, p.o.) for four days. On the fourth day, individuals participated in two double-blind self-administration sessions for the opportunity to receive cocaine or saline. Upon receiving a contingent dose of cocaine (20 mg, iv) or saline, individuals had the opportunity to make 5 subsequent choices for either another infusion or keep $1 USD. Subjective effects and heart rate and blood pressure were recorded prior to and throughout each session.

RESULTS: Participants (n = 27) were mostly male (74%), mostly African American (78%), and non-Hispanic (85%). Compared to the other treatments, modafinil attenuated subjective ratings of “high”, “desire”, “any drug effects”, “good effects”, and “stimulated” , but the combination of modafinil and lisdexamfetamine did not produce significant attenuations of any subjective or physiological responses induced by cocaine (all p-values > 0.05).

CONCLUSIONS: These data support previous findings that modafinil is effective for treating cocaine dependence, yet it does not appear to be more effective when combined with lisdexamfetamine.

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CYCLIC NUCLEOTIDE RECOGNITION MECHANISM OF CYCLIC GMP DEPENDENT PROTEIN KINASE II

James Campbell, Kevin Y. Li, Gilbert Huang, Albert S. Reger, Banumathi Sankaran, Todd M. Link, John E. Ladbury, Choel Kim

Membrane bound type II cGMP dependent protein kinase (PKG II) is a central mediator of the cGMP signaling cascade, which regulates circadian rhythmicity, intestinal water secretion, bone growth and renal function. PKG II contains a N-terminal regulatory (R)-domain, and a C-terminal catalytic (C)-domain. The R-domain contains tandem cyclic nucleotide binding domains (CNB-A and B) each with different affinities for cGMP, the second messenger that regulates kinase activity of PKG II. Once PKG II is active by cGMP it phosphorylates pharmaceutically relevant protein target, but due in part to a lack of structural information drug discovery efforts on have been slow. While it is known that PKG II needs to be highly selective for cGMP over cAMP to prevent cross-talk little is known about its cyclic nucleotide selectivity mechanism and the allosteric activation of PKG II. We seek to understand the cyclic nucleotide selectivity of PKG II’s CNB domains and their role in the activation of PKG II. In this pursuit, we characterized the CNB domain’s cyclic nucleotide selectivity. Our findings are that CNB-B imparts an almost 500 fold preference for cGMP, while CNB-A only offers a 10 fold preference. We then solved atomic resolution crystal structures of the two CNB domains of PKG II, to gain insight into the molecular basis of the selectivity seen. We have discovered a unique ligand recognition mechanism in CNB-B and tested its role in the activation of PKG II.

20

SIGNALING EVENTS TRIGGERED BY THE HETEROPHILIC CELL ADHESION PAIR, TGRC1 AND TGRB1, DURING DICTYOSTELIUM DISCOIDEUM DEVELOPMENT

Gong Chen, Xiaoqun Xu, Xiangfu Wu, Alexander Thomson, Chi-Hung Siu, Adam Kuspa, Gad Shaulsky

The social amoeba Dictyostelium discoideum expresses three cell adhesion systems, DdCAD-1, csA/gp80 and TgrB1-TgrC1, which play critical roles during development. Among them, only TgrB1 and TgrC1 constitute a heterophilic adhesion system. Knockout of either tgrB1 or tgrC1 results in the arrest of development at the loose aggregate stage. Polymorphic members of TgrB1 and TgrC1 can mediate kin discrimination between strains with different genetic background. The assembly of the TgrB1-TgrC1 adhesion complexes was investigated using FRET and GFP complementation split assays. The results led to a model, in which the formation of TgrC1constituitive cis-homodimers induce TrgB1 cis-homodimerization upon trans-binding with TgrC1. Additionally, the interaction between TgrC1 and TgrB1 induces the phosphorylation of Ser839 and Ser845 in cytoplasmic domain of TgrB1. Mutations in these two residues will result in abnormal cell sorting and decreased spore yield. On the other hand, myosin heavy chain kinase C (MHCKC) has been identified as the cytoplasmic partner of TrgC1. Substitution of the basic residues in the TgrC1 cytoplasmic domain abrogates TgrC1-MHCKC interaction, leading to the activation of MHCKC and the subsequent sequestration of myosin II in the cytoskeleton fraction. In transfection studies, tgrC1- cells expressing TgrC1 carrying mutations in its polybasic cytoplasmic domain give rise to smaller cell aggregates, slimmer slugs and lower spore yield in comparison with wildtype cells. Additionally, impaired interactions between TgrC1 and MHCKC induce abnormal cell sorting between prespore and prestalk cells, as a result of altered cell motility exhibited by the mutant cells. Our studies have highlighted several biological roles for the pair of adhesion receptors TgrB1-TgrC1 and led to the identification of several key components in the downstream signaling cascades that regulate cell differentiation and morphogenesis in the post-aggregation stages of Dictyostelium development.

21

BINDING TO HUMAN ΓD-CRYSTALLIN CAUSES PARTIAL CLOSURE OF CIS-RING AND SYMMETRY-BROKEN FEATURES OF TYPE II CHAPERONIN

Bo Chen, Oksana Sergeeva, Daniel Goulet, Kelly Knee, Jakana Joanita, Wah Chiu, Johnathan King

Chaperonin is a class of protein that plays an essential role in protein folding for all cells from bacteria, archaea and eukaryotic cells. We are interested in understanding the structural mechanism of how type II chaperonin recognizes a substrate with beta-sheet propensity: the γD-crystallin.

Here, cryo-electron microscopy (cryo-EM) single particle analysis method was applied to resolve the structure of Mm-Cpn and human γD-crystallin during their initial recognition step. To resolve the potential conformational heterogeneity problem, we used a multi-model refinement protocol to sort out particle images according to their structural uniformity. Our analysis showed three conformations: 33% of the particles (Subset II) resembled the apo state Mm-Cpn conformation, while 39% of particles (Subset I) had one-ring less open, one-ring open conformation and 28% of the rest particles (subset III) did not yield a reliable map. The control cryo-EM map from the Mn-Cpn showed both ring open which was similar to the map from the subset II of the Mm-Cpn and human γD-crystallin complex.

Based on the Statistical Variance Analysis, we conclude that the subset I corresponds to the Mm-Cpn population with substrate binding. High levels of variances are observed inside the cis-rings. Subsequent symmetry-free reconstructions of subset I particle images converged to one-ring less open and one-ring open conformation. Furthermore, differential conformations of each of the 8 subunits in the less open cis-rings were observed to form a tetramer of dimers while both rings in subset II appeared to have a good 8-fold symmetry. Interestingly the 8-fold symmetries on apical domain and equatorial domain are largely broken, while intermediate domain still maintain good 8-fold symmetry. It seems that apical domain and equatorial domain work cooperatively to recognize the unfolded substrate.

In conclusion, our results demonstrate the conformational changes and symmetry broken features of type II chaperonin upon binding to γD-crystallin.

22

TRAPPING G PROTEIN-COUPLED RECEPTORS IN ACTIVE SIGNALING STATES THROUGH PROTEIN DESIGN

Kuang-Yui Chen, Patrick Barth

G protein-coupled receptors (GPCRs) are one of the largest families of membrane-embedded receptors that transduce extracellular stimuli into cytoplasmic responses. Increasing evidence from structural, mutational and spectroscopic studies on model GPCRs (e.g. rhodopsin, beta-2-adrenergic receptor) indicate that these receptors signal through long-range conformational changes. However, the atomic-level sequence/structure/energetic relationships governing such allosteric transitions in this large receptor family remain poorly understood, thereby hindering the development of more effective therapeutics (i.e. allosteric regulators) targeting these receptors. We have developed an integrated homology modeling/multistate design/experimental approach to reprogram the signaling properties of structurally uncharacterized GPCRs by redesigning conformational switches in transmembrane (TM) and loop regions. The method was applied to switch the function of the structurally uncharacterized dopamine D2 receptor (DRD2) towards the active state. Inactive and active states of DRD2 were modeled by conformational ensembles using the homology modeling mode of RosettaMembrane. By manipulating the receptor conformational energy landscape and rewiring the networks of energetically coupled residues, multistate design of the DRD2 TM region resulted in receptor variants exhibiting up to an eight-fold increase in basal activity compared to wild type receptor. In agreement with the predictions, biophysical and pharmacological characterizations indicate that the designed receptor variants adopt different local conformations exhibiting various levels of basal and ligand-induced activities. To our knowledge, this is the first computational modeling and design approach that can reprogram the signaling properties of structurally uncharacterized GPCRs by rationally designing novel residue conformational switches and energetic couplings between receptor allosteric regions.

23

FROM IN VITRO TO IN VIVO, CURRENT VIEW ON THE ROLE OF CALCIUM-DEPENDENT INTERACTION BETWEEN RECOVERIN AND RHODOPSIN KINASE IN PHOTOTRANSDUCTION

Ching-Kang Jason Chen, Michael Woodruff, Gordon Fain

Recoverin is a myristoylated Ca2+ binding protein that binds three retinal proteins in a Ca2+-dependent manner: interphotoreceptor retinoid binding protein, tubulin, and rhodopsin kinase (RK). The interaction of RK with recoverin inhibits RK-mediated rhodopsin phosphorylation. Photoresponses of recoverin knockout (Rv-/-) rods show an accelerated recovery to dim light and a shortened “time in saturation” under bright light. Recovery of phototransduction is a complicated process involving at least four simultaneous reactions: rhodopsin phosphorylation, GTP hydrolysis by transducin, inhibition of phosphodiesterase, and de novo synthesis of cGMP from GTP. Transducin deactivation is the slowest reaction and it determines the overall rate of rod recovery. By genetically manipulating the rate-limiting step of rod recovery, we have obtained clear evidence that modulation of rhodopsin phosphorylation by recoverin and through RK does occur in mouse rods. We also demonstrate that recoverin is needed for acceleration of phototransduction recovery under background light in intact rods and this is achieved by facilitating PDE inactivation through an unidentified mechanism.

24

CONTROL OF NEURONAL DEVELOPMENT BY THE RAC-GEFS TIAM1 AND TIAM2

Jinxuan Cheng, Sanyong Niu, Karen Firozi, Kimberley R. Tolias

To form a functional nervous system, neurons undergo highly regulated processes, including migration, axonal and dendritic outgrowth, and synapse development. The small RhoGTPases Rac1, RhoA and Cdc42 are vital regulators of nervous system development and perturbations in their signaling are associated with neurological diseases. Rac1 cycles between a GTP-bound activate state and a GDP-bound inactive state. Active Rac1 binds to effector proteins and stimulates signaling pathways that promote neuronal development. Rac1 activity needs to be tightly regulated in space and time. Rac1 is activated by the guanine nucleotide exchanger factor (GEF) Tiam1 and Tiam2.

Tiam1 (T-lymphoma invasion and metastasis 1) is a large multi-domain protein, which has been shown in vitro to regulate neuron development. Tiam2 (STEF, SIF and Tiam1-like exchange factor) is also required for neurite outgrowth and growth cone morphology in cultured neurons.

The expression levels of Tiam1 and Tiam2 are high in the brain during development. Furthermore, Tiam1 and Tiam2 are both components of the evolutionary conserved PAR polarity complex, which is essential for establishing and maintaining cell polarity during processes such as cell migration, axon initiation, and synapses formation. This evidence suggests that Tiam1 and Tiam2 may play important roles in neuron development. However, most of the studies investigating Tiam1 and Tiam2 have been done in vitro, and little is known about their roles in vivo.

Here we describe the generation of Tiam1 and Tiam2 knockout mice and our attempts to use these mice to determine the roles of Tiam1 and Tiam2 in neuronal migration and synapse development in vivo. Our preliminary data indicate that these knockout mice show decreased spine density, defects in astrocytes and interneuron migration. My future work will be using in utero electroporation to study neuron migration in these knockout mice, and characterize the role of Tiam1 and Tiam2 in regulating these processes.

25

ANALYSIS OF ENDOGENOUS EXPRESSION PATTERNS USING HIGH-THROUGHPUT RECOMBINEERING IN DROSOPHILA MELANOGASTER

Audrey Christiansen, Soo Park, Benjamin W. Booth, M. Alejandro Sarrion Perdigones, Jeff Schreifels, Nicholas Abel, Paul Vandeventer, Koen Venken, Roger A. Hoskins

The Drosophila melanogaster genome can be interrogated using P[acman] methodology for high-throughput molecular genetics and genomics by combining recombineering and site-specific integration in the genome. P[acman] can be used to incorporate tags such as GFP to generate fusion proteins that can then be followed in live and fixed tissue or used for biochemical analysis. We have taken advantage of P[acman] genomic DNA libraries to incorporate a protein tagging cassette using high-throughput recombineering, creating genomic fusion constructs for a collection of 400 well-studied Drosophila genes. These constructs were used to generate transgenic fly lines expressing these tagged proteins. We are characterizing the expression pattern of these lines, analyzing them for expression in different tissues in the embryo, larva and adult stages. The resulting expression patterns are then compared with published ones when available for the same genes. To date, 66 of 67 lines reveal expression with 30 of 31 lines recapitulating published expression patterns, showing that this method provide a rapid and reliable method for generating tagged transgenic proteins, allowing us to follow expression at different stages and tissues as well as within subcellular regions of the cell.

26

DECONSTRUCTION OF COMPLEX ENDOGENOUS PROTEIN EXPRESSION PATTERNS THROUGH A SPLICING SWITCH IN DROSOPHILA MELANOGASTER

Audrey Christiansen, Koen Venken

Problem: Protein tagging, relying on either ectopic or endogenous protein expression, allows for the tracking of proteins over time in live and fixed tissue. Ectopic expression via binary activation systems like GAL4/UAS induces expression of a transgenic fusion protein but can have disadvantages: they lead to artificial overexpression of fusion proteins, they are limited to a single protein isoform per transgene, and they don’t always recapitulate the endogenous subcellular expression pattern. Alternatively, protein tags can be introduced through recombineering in transgenic genomic DNA rescue constructs. However, these constructs do not always represent the entire regulatory repertoire and may be lacking regulatory elements that are otherwise present at the endogenous locus. On the other hand, endogenous protein tagging can be done through homologous recombination or transposon protein trapping. Homologous recombination is labor intensive while transposon hopping is random. Although endogenous protein tagging documents the entire expression pattern, it does not allow restricting expression to a limited amount of cells which is useful to deconstruct complex endogenous protein expression patterns into meaningful subsets of expression patterns.

Solution: Here, we present a novel method that addresses these issues. The prototype of the method relies on the repurposing of existing transposon insertion sites with a switchable artificial exon encoding a protein tag that can be deactivated and activated on demand. The current version of the technology uses existing MiMIC transposons that are broadly distributed all over the Drosophila genome. However, ultimately, we would like to extrapolate this system towards a method that is independent of existing transposon insertions.

27

CANDIDATE GENE APPROACHES REVEAL NEW TARGET PATHWAYS FOR TREATING ANTIBIOTIC-RESISTANT GRAM-NEGATIVE BACTERIAL INFECTIONS

Zachary Conley, Kimberly Carlson-Banning, Ashley Carter, Andrew Chou, Richard Hamill, Yongcheng Song, Lynn Zechiedrich

New antibiotics are needed because resistance has rendered many existing drugs ineffective. We discovered that the antifungal drug, ciclopirox, prevents growth of problematic, multidrug-resistant clinical isolates, although the drug target remains elusive. We showed that both sugar metabolism and free iron in the growth medium affect ciclopirox inhibition of E. coli. Additionally, iron-acquiring siderophore production in P. aeruginosa was increased in the presence of ciclopirox, implying a disruption of iron acquisition. We performed a literature search and identified 103 genes involved in specific iron-utilization and sugar metabolism pathways. To test for those that are affected by ciclopirox, we screened gene deletion strains for increased sensitivity to ciclopirox. We filtered out those gene deletions affected by any antibiotic based on the literature. Because ciclopirox can bind free iron, we screened our remaining 29 gene set for increased sensitivity to the iron chelator 1,10-phenanthroline. This stratification resulted in 18 gene deletions with increased susceptibility to ciclopirox alone. Most of these genes encode proteins involved with the synthesis of the surface glycolipid enterobacterial common antigen, consistent with our laboratory’s published work that shows ciclopirox alters glycolipid expression in the cell outer membrane. Other stratified genes encode proteins involved in the uptake of the siderophore enterobactin, a gram-negative-specific siderophore with the highest known iron binding affinity of any siderophore. We hypothesize that ciclopirox drug targets are not restricted to one pathway. Here, we identified the construction of glycolipids that are attached to the bacterial outer membrane as one pathway, and iron uptake through siderophores as another pathway affected by ciclopirox. Perhaps the existence of multiple essential targeted pathways explains why this drug is effective against antibiotic-resistant bacteria. These data are an important step toward developing ciclopirox or new derivatives of ciclopirox, as drugs against multidrug-resistant gram-negative pathogens for which few therapeutic options exist currently.

28

ELUCIDATING THE INTERACTIONS OF MUTANT HUNTINGTIN WITH THE TRIC-LIKE CCT5 COMPLEX

Michele C. Darrow, Oksana A. Sergeeva, Jose M. Isas, Jesus Galaz-Montoya, Jonathan A. King, Ralf Langen, Wah Chiu

Huntington’s disease, a neurodegenerative disorder characterized by loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat, which translates as a polyglutamine repeat in the protein product. 40+ trinucleotide repeats have been described as pathological.

Mutant huntingtin exon 1 with 46 polyglutamine repeats was purified via thioredoxin tag and incubated with a homo-oligomer complex composed of one subunit from TRiC chaperonin (CCT5), markedly slowing mHtt fibril aggregation, as shown using a filter trap assay and cryo-electron microscopy.

Cryo-electron tomography and sub-tomogram averaging was also performed on these samples with the goal of identifying the structure of CCT5 homo-oligomer in complex with mHtt fibrils and oligomers. Results indicate that CCT5 homo-oligomer interacts with mHtt at the tips of the fibrils, and by encapsulating oligomers, acting to slow mHtt aggregation.

This study provides further information about how a complex formed from a single subunit of TRiC chaperonin interacts with mHtt fibrils and oligomers to slow aggregation, identifying a potential target for therapeutics.

29

DISCOVERY OF NOVEL REGULATORS OF SENSITIVITY AND RESISTANCE TO HER-FAMILY TARGETED THERAPIES

Rocio Dominguez-Vidana, Ronald J. Bernardi, Christopher S. Bland, Kathleen A. Scorsone, Thomas F. Westbrook

Background: Targeted therapies exploit the fact that many tumors have genetic drivers that control tumor progression. Tumors rely on these drivers for their continued survival; therefore, inhibition of these genes strongly impairs tumor growth with minimal side effects to normal cells. However, resistance can arise when these drugs are used as single agents. Clinical and experimental data suggest that there is substantial heterogeneity in drug-response between patients and even between cells of the same tumor. However, the mechanisms driving this heterogeneity are poorly understood. The goal of this project is to identify the genetic networks that govern response to the anti-cancer drug lapatinib in Her2+ breast cancers.

Experimental design and methods: We performed pooled RNA interference-based genetic screens in a panel of Her2+ breast cancer cell lines using a short hairpin RNA (shRNA) library with ~9k unique hairpins targeting ~2k genes. We then assessed the effects of these shRNAs in untreated cells and in lapatinib-treated cells. We identified ~300 Lapatinib Sensitivity Regulators (LaSRs); from which we validated ~100 using small interference RNA (siRNA) based assays, and organized in protein networks using public protein-protein interaction (PPI) databases.

Results: We focused on 12 LaSRs that show a combinatorial effect with lapatinib in multiple Her2+ models, and we systematically tested whether they shared a PPI and genetic coregulation. We have identified a potential mechanism in which our LaSRs WEE1, CSNK2A1, and CDC14B deregulate mitotic entry in conjunction with lapatinib, causing cell death.

Conclusion: We developed an unbiased functional screen that identified genes governing sensitivity to lapatinib, and a validated these results using orthogonal methods. We identified WEE1, CSNK2A1, and CDC14B as a network governing lapatinib sensitivity from a systematic analysis of genetic coregulation and PPIs.

30

TRPV STRUCTURE AND FUNCTION INVESTIGATED WITH CRYO-ELECTRON MICROSCOPY AND NOVEL ACTIVITY ASSAYS

Timothy Dosey, Zhao Wang, Guizhen Fan, Zhixian Zhang, Irina Serysheva, Theodore G. Wensel, Wah Chiu

Transient Receptor Potential (TRP) channels are a superfamily of non-selective cation channels conserved in eukaryotes. In mammals, the Vanilloid (TRPV) sub-family is involved in sensory processes including nociception and temperature sensing. However, our understanding of how TRP channel activity is regulated by these diverse stimuli has been impeded by the lack of structure-function information. Here we report that TRPV2 as well as a TRPV4 functional fragment can be purified from a yeast heterologous expression system and we demonstrate that they remain mono-dispersed in detergent-free buffer when stabilized with amphipoles. Further, we have developed cell-based and vesicle-based activity assays to investigate TRPV2 and TRPV4 function and we are utilizing recent advancements in cryo-electron microscopy to achieve high resolution structures without crystallization. With these structure and function techniques, we will be able to elucidate the gating mechanisms of these TRPV channels and gain insights into how other TRP channel families respond to a wide range of stimuli.

31

THE FUNCTION OF EXTRACELLULAR TRAP ASSOCIATED PROTEINS IN DICTYOSTELIUM DISCOIDEUM SENTINEL CELLS

Timothy Farinholt, Olga Zhuchenko, Adam Kuspa

The social amoeba Dictyostelium discoideum is a model for studying bacteria-amoebae interactions that shape bacterial virulence. These initially solitary amoebae feed on bacteria, but when starved, they lose their ability to consume bacteria and enter a 24-hour developmental cycle culminating in a fruiting body. During the migratory slug stage of development we have discovered a specialized population of Sentinel (S) cells that release structures similar to neutrophil extracellular traps (ETs) that ensnare and kill bacteria through an unknown mechanism (Chen et al., Science 317:678-81). ETs are comprised of mitochondrial DNA coated with antimicrobial proteins. We purified ETs and analyzed them by mass spectrometry, identifying several proteins, including CadA. Using available anti-CadA antibodies we confirmed the presence of CadA on ETs. Amoeba lacking CadA protein (cadA-null) displayed variable growth on Klebsiella pneumoniae, which suggests that CadA is necessary for the killing of bacteria during growth and developmental phases of the Dictyostelium life cycle. Additionally, single cadA-null amoeba cells form plaques of widely varying sizes that suggests a deficiency in the first few cell divisions of colony formation. Our lab’s data suggests Dictyostelium kills bacteria extracellularly before phagocytosis. Initial data suggest cadA-null amoebae are deficient in extracellular killing of bacteria. The crystal structure of CadA reveals immunoglobulin-like and gamma-crystallin-like domains connected by a short linker sequence (Lin, Nat. Struct. Mol. Bio. 13(11):1016-22). Gamma-crystallin domains are found in plant antimicrobial proteins (Huchinson, Protein Eng. 6(3):233-45). I am currently testing the potential role of CadA in the killing of bacteria by Dictyostelium discoideum. I will add exogenously purified CadA to bacteria to directly test its antimicrobial function. Purified CadA will be added to our extracellular killing assay to observe if it rescues the killing of bacteria in cadA-null amoebae. We hope to garner new insights into the ET mechanism for killing bacteria in eukaryotes.

32

A SIMPLIFIED SEQUENCE/STRUCTURE ALPHABET FOR MULTIPLE TRANSMEMBRANE HELICES ASSEMBLIES

Xiang Feng, Patrick Barth

TransMembrane Helical (TMH) domains play critical roles in diverse physiological functions and are important drug targets. The lack of structural information, however, hinders our understanding of their functional regulation and prevents the rational design of selective therapeutics. Computational modeling techniques represent important alternative approaches but currently lack the efficiency and accuracy required to consistently predict membrane protein structures at atomic resolution. Understanding the sequence/structure determinants controlling the packing of TMHs is a major step in accurately modeling and designing TMH proteins but such determinants have not yet been characterized for multiple TMH assemblies.

We conducted a bioinformatics analysis of the sequence/structure relationships defining the packing of three TM helices. We generated a library of more than 800 closely packed TMH trimer. Based on structure similarity, we found that more than half of the trimers can be classified into only 6 major clusters with distinct geometrical/topological features. We identified statistically enriched sequence motifs that correlate with the geometric features of each cluster. The motifs were used to train an SVM-based predictor of TMH trimer topologies from sequence that achieved up to 3-fold improvement over random selection, highlighting the significance and specificity of the motifs. Furthermore, enriched sequence/structure motifs share similar interaction patterns in diverse protein families, suggesting the existence of convergent determinants in TMH trimer packing. Analysis of residue conservation or co-evolution and in silico alanine scanning indicates that most motifs create energetically important contacts that are under evolutionary pressure. Structural analysis of atomic contacts mediated by these motifs uncovered novel consensus physical interactions that are unique to TMH trimer interfaces.

Our results indicate that a limited number of local sequence/structure motifs can recapitulate a large fraction of TMH trimer structures. These findings provide important novel insights into the sequence/structure determinants governing the packing of multiple TM helices.

33

SINGLE-MOLECULE CONFORMATIONAL MAPS OF INTRINSICALLY DISORDERED PROTEINS

Allan Chris Ferreon

Single-molecule fluorescence detection methods exhibit enhanced capability in resolving protein structural and conformational heterogeneity, and are suitable to studying high-affinity interactions of aggregation-prone protein systems. Experimentally derived protein conformational maps in combination with phase diagram analysis provide excellent tools for dissecting the complexities and intricacies in the folding, misfolding and binding behavior of intrinsically disordered proteins in particular, and proteins in general. This talk will present conformational maps that link the binding-folding properties of the Parkinson’s disease-linked protein alpha-synuclein to its misfolding-aggregation behavior.

34

ALLOSTERY IN ‘MOLECULAR HUB’ INTRINSICALLY DISORDERED PROTEINS

Josephine Ferreon

Intrinsically disordered proteins (IDPs) are ubiquitous in nature. However, due to experimental limitations, the structures and roles of IDPs in biological processes are poorly characterized. This talk will summarize previous work on two molecular hub IDPs: the tumour suppressor protein p53 and adenoviral oncoprotein E1A, highlighting the role of intrinsic disorder in allosteric interactions in hub protein networks. I will also share some stories behind the science.

35

A NEW PATHWAY FOR CALCIUM MOBILIZATION BY BETA-2 ADRENERGIC RECEPTOR

Monica Galaz-Montoya, Gustavo Rodriguez, Olivier Lichtarge, Theodore G. Wensel

Beta adrenergic receptors (β-ARs) are important for cardiovascular regulation and for physiological responses to the hormones adrenaline and noradrenaline. They are found throughout the body and are the targets of numerous widely used drugs. Their canonical signaling pathway involves activation of adenylate cyclase (AC) and a rise in cyclic AMP (cAMP) levels which activates cAMP-dependent protein kinase (PKA).

My research has recently led to the discovery of a novel signaling pathway downstream of β2-AR. In this pathway there is a robust mobilization of Ca2+ in response to adrenergic agonists. By monitoring Ca2+ mobilization in real time using cultured human cells I have shown that a selective β2-AR antagonist (ICI 118,551) inhibits this response. Additionally, the adrenergic agonists isoproterenol, epinephrine and norepinephrine show activation potencies consistent with β2-AR’s pharmacological profile.

I have also shown that the removal of extracellular Ca2+ (with EGTA) does not block the response, but depletion of intracellular Ca2+ stores (with thapsigargin) or inhibition of phospholipase C (with U73122) and InsP3R (with 2-APB) receptor does. These results indicate that the Ca2+ is mobilized from intracellular stores in a PLC and IsnP3R dependent manner.

Additionally, activation of Gs (with cholera toxin), and activation of PKA (with 8-bromo-cAMP) does not lead to Ca2+ mobilization from intracellular stores. Furthermore, inhibition of AC (with SQ22, 536) and PKA (with H-89 and KT-5720) do not inhibit the response triggered by AR agonists.

Together these findings indicate that activation of β2-AR leads to an increase in cytoplasmic Ca2+ by a previously unrecognized signaling pathway.

The discovery of a new signaling pathway for β2-AR could have serious implications in the use of beta agonists and blockers for the treatment of disease.

36

BIOINFORMATICS-BASED DATA MINING REVEALS TRYPTOPHAN 2,3-DIXOYGENASE (TDO) REGULATES NAD METABOLISM AND PROMOTES BREAST CANCER PROGRESSION

Franklin Gu, James Arnold, Susmita Samanta, Arun Sreekumar

Tryptophan catabolism is linked to malignant progression of breast cancer through the suppression of anti-tumor immune responses. The initial rate-limiting step in tryptophan catabolism is catalyzed by two enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), that share low structural similarity and are encoded on separate chromosomes. Compared to IDO, the molecular functions of TDO in breast tumors, where it is overexpressed (relative to normal breast tissues), are less well characterized. Analysis of breast tumor microarray datasets reveals that TDO is up-regulated in high grade tumors as well as in more aggressive subtypes of breast cancer, such as basal-like breast cancer. In addition, high TDO expression in breast tumors is associated with worse clinical prognosis. A gene signature that could predict high TDO expression in breast tumors showed enrichment in key pathways such as calcium signaling and tryptophan catabolism downstream of TDO that were not present in a gene signature for high IDO expression. As TDO is known to be involved in de novo NAD synthesis via tryptophan catabolism, a possible function of TDO in breast tumors is to replenish NAD levels to promote tumor growth and survival as has been shown in other cancers. Furthermore, high TDO and high IDO expression in breast tumors did not display a mutually exclusive pattern. This finding is further supported by known differences in the transcriptional activation of TDO and IDO, which are primarily induced by glucocorticoids and inflammatory cytokines, respectively. Together, this suggests that TDO and IDO play non-redundant roles in breast cancer that can be elucidated to determine new therapeutic targets for aggressive subtypes of breast cancer.

37

THE ROLE OF TYPE III PHOSPHOINOSITIDE 3-KINASE (PI3KC3/VPS34) IN LIGHT STIMULATED SYNTHESIS OF PHOSPHATIDYLINOSITOL-3-PHOSPHATE AND CELL SURVIVAL

IN RETINAL ROD PHOTORECEPTORS

Feng He, Yan Yin Tse, Melina A. Agosto, Samuel M. Wu, Theodore G. Wensel

Phosphoinositide 3-kinases consist of three classes: PI3K class I (PI3Kr1), PI3K class II and PI3K class III (PI3KC3, Vps34) that can generate different phosphoinositides and play important roles in various cellular functions, including cell survival and intracellular trafficking. The biological significance of PI3K class III in retina is not fully understood, but this enzyme is thought to be the major source PI(3)P in other systems. We are investigating the role of PI3K class III in vertebrate rod photoreceptors by deleting its ATP binding domain.

Mice with an ICre transgene under control of the rhodopsin promoter were bred to mice with a floxed ATP binding domain of Vps34 to generate a conditional functional deletion of Vps34 in rods. A new ultrasensitive ELISA-based assay was used to measure phosphoinositide levels in the extracts of purified mouse rod cell fragments containing the outer segments and much of the inner segments from dark adapted and light-exposed mice.

Light exposure induced a massive increase in rod cell PI(3)P levels, with a slow time course spanning hours. The PI(3P) was localized to discrete puncta of various sizes in the inner segment. Deletion of Vps34 in rods caused aggressive retinal degeneration. The number of photoreceptor cells was decreased by about 50% in 1.5 months and there were almost none left at 3 months, without significant effects on inner nuclear and ganglion cell layers. In contrast, conditional KO of P85alpha in PI3K class I had no effect on retinal structure, and no effect on PI(3)P levels. The level of autophagy marker LC3-II was remarkably increased in Vps34 KO mice after exposure with 90x103 Lux light. Rhodopsin trafficking in Vps34 KO was examined using Vps34 KO mice also heterozygous for a rhodopsin-EGFP knock-in and there was little if any effect of the Vps34 KO on rhodopsin trafficking. Results to date indicate that the Vps34 contributes to light induced synthesis of PI(3)P in rods, and is important for rod cell survival. The mechanisms may involve disruption of authophagy or related pathways. The type I PI-3 kinases do not contribute significantly to PI(3)P production or photoreceptor function and survival.

38

USE OF GENE EDITING FOR GENE THERAPY OF AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA

Tyler Hilton, John Wilson, Theodore G. Wensel

Retinitis pigmentosa (RP) is a neurodegenerative disease that causes progressive loss of vision due to defective retinal cell function. RP is a very heterogeneous disease, with over 60 genes associated with the disease phenotype. Of these genes, rhodopsin is linked mostly to Autosomal Dominant RP (ADRP), and accounts for about 10% of all cases of RP. Mutations in this gene alter rhodopsin function or localization, resulting in progressive degeneration of the rod photoreceptors in the retina. Past experiments in the lab have used zinc-finger nucleases to cause a double strand break in the last exon of the rhodopsin gene, which was found to be repaired predominantly by non-homologous end-joining (NHEJ) and produce an insertion or deletion. If such a frameshift site were inserted upstream of the mutations in rhodopsin associated with ADRP, it could potentially attenuate the effect of the toxic protein products. Combined with a functional copy of the rhodopsin gene resistant to the double-strand break agent, this “kill and replace” gene therapy strategy may be used to treat the ADRP phenotype from the associated rhodopsin mutations. Thus far, we are focusing on using TALENs and CRISPR/Cas9 systems to target sites in the first and second exons of the rhodopsin gene, to introduce a double-strand break that will be repaired by NHEJ in the rod photoreceptors. These double-strand break agents will be tested in HEK 293 cells to determine activity before they will be packaged into rAAV and used in murine models of ADRP.

39

A HIGH RESOLUTION PROTEOMIC PROFILING OF 2I TREATED STEM CELLS

Matthew Holt, Nur Yucer, Sung Yun Jung, Yi Wang, Jun Qin, Christopher Peters, Anna Malovannaya

Recent advances in stem cell research have shown intricate and robust mechanisms which are responsible for the establishment and maintenance of pluripotency: the ability to generate any embryonic germ layer. The addition of only four transcription factors is capable of reprogramming somatic cells into a pluripotent state. Embryonic stem cells lack the capability of significantly contributing to extra-embryonic lineages such as the trophectoderm, and thus are different than totipotent cells which can contribute to both. However, the addition of two inhibitors (CHIR99021 and PD 0325901) has been shown to cause embryonic stem cells to express a trophectoderm marker in conjunction with pluripotency markers and actively contributing both embryonic and extra-embryonic lineages. The primary objective of this project is to understand the key players involved in the response to this inhibitor treatment and which are the key transcription factors involved in totipotency. Transcription factor DNA binding activity and abundance can be determined at the protein level by using transcription factor response elements as an affinity reagent to pull-down DNA binding proteins (catTFRE pull-down). These proteins can be subsequently analyzed by liquid chromatography and mass spectrometry, and through our database software package (iSPEC and Align!) to determine which transcription factors and associated proteins are activated in a totipotent state. We have identified 436 DNA binding transcription factors in pluripotent and totipotent stem cells. Furthermore, distinct transcription factor patterns arise from each individual inhibitor treatment potentially underscoring the transcription factors directly involved in the transition from pluripotency to totipotency.

40

EFFECTS OF DNA SIZE ON TRANSFECTION

Benjamin Hornstein, Lirio Milenka Arevalo-Soliz, Adam Szafran, Michael Mancini, Lynn Zechiedrich

Gene therapy requires the delivery of nucleic acid to replace, regulate, or correct genes to treat diseases. Viral vectors deliver DNA efficiently to cells, but are often unsuitable for therapeutic applications because their integration into the host genome can disrupt essential genes. Nonviral vectors do not have this issue, but many human cell lines are refractory to plasmid transfection. Our laboratory developed minimized nonviral vectors called minivectors. Minivectors are devoid of bacterial sequences, and are unlikely to be silenced. Published data show that minivectors transfect hard-to-transfect cell types, including primary cells without causing toxicity.

During transfection, DNA vectors need to enter the cell, translocate to the nucleus, and be expressed to mediate an effect. Whereas minivectors get into cells extremely well, the observed minivector-mediated knockdown was less efficient than when equal moles of plasmid were transfected. I hypothesized that smaller vectors get expressed less efficiently than larger ones. To test how vector length affects transfection, I created DNA vectors, from 383 to 4,556 bp long, expressing anti-GFP-shRNA. I tested transfection efficiency by measuring GFP-knockdown in HeLa-GFP cells. Electroporation was used to test how vector length affects nuclear localization and expression without the variable of cell entry. Flow cytometry and fluorescence microscopy were used to quantify the data. The smallest vector showed the least knockdown, while the largest three vectors showed similar knockdown. To test directly whether these differences in GFP-knockdown are a consequence of RNA polymerase inhibition, I am quantitatively measuring in vitro transcription. Because clinical application vary, understanding vector size-effects on transfection is necessary for calculating the amount of vector needed to elicit the appropriate expression level for any given application. The goal of this project is to find the optimal minivector size for nonviral gene therapy vectors to maximize expression while minimizing toxicity from transfection vehicle and vector.

41

DETECTING THE POSITIVE SELECTION ON CANCER GENES

Teng-Kuei Hsu, Panagiotis Katsonis, Olivier Lichtarge

Cancer proceeds as a multi-step process involved in the accumulation of genetic alterations that transform normal human cells into highly malignant derivatives. Different from organismal evolution, which is dominated by purifying selection that removes the deleterious mutations that reduce fitness, the cancer evolution is dominated by positive selection of 'driver' somatic mutations that activate oncogenes or inactivate tumor suppressor genes. To detect the positive selection, we shall determine the magnitude of the change in protein fitness induced by a mutation. For this, my lab derived an “Evolutionary Action” equation that defines the overall change in fitness due to a mutation as a novel biological quantity called the Evolutionary Action (EA) of the mutation and that factors in the size of the mutation plus the site at which it occurs. This approach is different than those that have been developed to date to identify the impact of genetic alterations, which are based on structural information, machine learning approaches, and statistical analyses of cross-species conservation. The distribution of EA scores of cancer somatic mutations in cancer genes does reveal unique patterns. Mutations are strongly biased to high EA scores in tumor suppressors and biased to moderate scores in oncogenes. For mutations in the genes unrelated to cancer, the EA distribution is indistinguishable from the simulated random nucleotide changes, suggesting these mutations occur randomly. These data show that the distribution of functional impact of mutations allow us to detect the presence of positive selection on the mutations of a gene, and hence help in determining if this gene is cancer-associated. Identifying novel cancer genes will not only direct research towards new therapeutic targets, but also help in understanding the mechanism and further control or even eradicate the disease.

42

ONCOGENIC MYC INDUCES A DEPENDENCY ON THE SPLICEOSOME IN HUMAN BREAST CANCER

Tiffany Hsu, Lukas Simon, Nicholas Neill, Richard Marcotte, Azin Sayad, Christopher Bland, Tingting Sun, Rocio Dominguez-Vidana, Sarah J. Kurley, Siddhartha Tyagi, Kristen Karlin, Jessica Hartman, Alexander Renwick, Ronald Bernardi, Samuel Skinner, Antrix Jain, Mayra Orellana, Chandraiah Lagisetti, Ido Golding, Joel Neilson, Xiang Zhang, Thomas Webb, Benjamin Neel, Chad Shaw Sung Jung, Thomas F. Westbrook

c-MYC (MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. Like other classic oncogenes, hyperactivation of MYC leads to collateral stresses onto cancer cells, suggesting that tumors harbor unique vulnerabilities arising from oncogenic activation of MYC. Herein, we discover the spliceosome as a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene, and demonstrate that BUD31 is associated with the core components of the human spliceosome. These BUD31-associated core spliceosomal factors (SF3B1, U2AF1, and others) are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total pre-mRNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Importantly, genetic or pharmacologic inhibition of the spliceosome in vivo impairs survival, tumorigenicity, and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.

43

THE FIRST STRUCTURE OF A VIRAL 2H-PHOSPHODIESTERASE – ANTAGONIST OF HOST ANTIVIRAL RESPONSE

Liya Hu, Kristen M. Ogden, Babal K. Jha, Banumathi Sankaran, Susan R. Weiss, Robert H. Silverman, John T. Patton, B.V.V. Prasad

As the major pathogen of gastroenteritis in young children, rotavirus (RV) causes nearly half a million children’s death worldwide annually, with 8 species of rotavirus (A-H) have been found so far. The rotavirus structural protein VP3 is a multifunctional capping enzyme that is also an antagonist of the interferon-induced cellular oligoadenylate synthetase (OAS)-RNase L pathway. In addition to a N-terminal domain, a central guanine-N7-methyltransferase domain, and a guanylyltransferase and RNA 5'-triphosphatase domain required for capping activities, it also contains a C-terminal 2H-phosphodiesterase (PDE) domain that can cleave 2'-5' oligoadenylates (2-5A), signaling molecule that activates RNase L. Due to the lack of structural information on VP3, the mechanism for the enzymatic functions remains unknown. Here we present the crystal structure of PDE domain of rotavirus VP3 at 1.35Å resolution, which is the first structure of a viral 2H phosphoesterase superfamily protein. The VP3 PDE structure exhibits an α/β-fold with three α-helices and seven β-strands, which form an antiparallel, concave, twisted β-sheet. The Hφ(S/T)φ motifs are located adjacent to one another at the base of the positive charged catalytic cleft, establishing a structural platform for binding to the negatively charged 2-5A substrate. To further explore how rotavirus VP3 recognizes its substrate, we carried out molecular docking with 2-5A using AutoDock Vina, which suggests the potential residues involved in 2-5A binding and cleavage. Mutational analysis and fluorescence resonance energy transfer (FRET)-based 2-5A cleavage assays verified the 2-5A substrate binding site. Together, our findings provide new structural and biochemical insights into mechanisms of 2-5A recognition by the rotavirus VP3 PDE, which exemplifies how a viral 2H-phosphodiesterase prevents the host innate immune defenses.

These studies were supported by grants from NIAID and Robert Welch foundation.

44

STRUCTURAL BASIS FOR CYCLIC-NUCLEOTIDE SELECTIVITY AND CGMP-SELECTIVE ACTIVATION OF PKG I

Gilbert Huang, Jeong Joo Kim, Albert S. Reger, Robin Lorenz, Daniela Bertinetti, Friedrich Herberg, Choel Kim

Cyclic guanosine monophosphate (cGMP) is a key secondary messenger that is produced in response to nitric oxide. One of the key mediators of cGMP signaling, cGMP-dependent protein kinase (PKG), is activated upon binding to cGMP and phosphorylates downstream substrates in a process required for important physiological processes such as vasodilation, nociception, and memory formation. PKGs are also known to mediate most effect of drugs that increase cellular cGMP levels, including nitric oxide-releasing agents and phosphodiesterase inhibitors, which are used for the treatment of angina pectoris and erectile dysfunction, respectively. We have investigated the mechanism of cyclic nucleotide selectivity by PKG by determining crystal structures of the cGMP-selective carboxyl-terminal cyclic nucleotide-binding domain (CNB-B) of human PKG I bound to cGMP and in the apo form. Our crystal structure of CNB-B with bound cGMP reveals that cGMP adopts the syn configuration in the binding pocket and is coordinated by a previously unidentified arginine residue. Furthermore, comparison of the cGMP-bound crystal structure of the apo structure suggests a role for a C-terminal tyrosine residue in capping the nucleotide into the binding pocket. The interaction of this tyrosine residue with cGMP appears to result in conformational rearrangement of the C-terminal helix, suggesting a mechanism for kinase activation by cGMP.

45

HIGH-THROUGHPUT DISCOVERY OF NOVEL PHAGE-DISPLAYED PEPTIDE REAGENTS FOR THE DETECTION OF NOROVIRUS

Amy Hurwitz, Wanzhi Huang, Robert Atmar, Timothy Palzkill

Norovirus (NoV) infections cause over 96% of non-bacterial gastroenteritis and lead to about 21 million new cases annually in the United States alone. Existing diagnostics have significant limitations in feasibility for point-of-care applications, so there is a clear need for more reliable, rapid, and simple-to-use diagnostic tools to prevent epidemic outbreaks and to inform appropriate actions. In this study, phage display technology was used to screen libraries of phages displaying random 12-mer peptides for those that bind to NoV virus-like particles (VLPs). Since NoV strains classified in genogroups I and II cause the majority of human outbreaks, we focused initially on the prototypical Norwalk strain within genotype GI.1. After five rounds of library selection against GI.1, we amplified the phage DNA from phage populations eluted from each round for high throughput sequencing. Using an in-house computational algorithm for analysis, a consensus motif was identified from thousands of sequences for binding GI.1. Peptides containing this consensus motif were then validated for their ability to bind GI.1 VLPs in multiple formats. Overall, the work here provides a proof-of-concept for the use of high-throughput phage display sequence analysis for the identification of peptide motifs with specific binding affinity for a target protein of interest.

46

TOWARDS QUANTITATIVE IMAGING OF INTRACELLULAR GLUTATHIONE DYNAMICS

Xiqian Jiang, Yong Yu, Mingkun Zhao, Alexandra J. Matzuk, Jianwei Chen, Xiao Tan, Antons Sizovs, Jin Wang, Meng C. Wang

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispensable tool in current biological studies. In order to minimize the perturbance on the biological system in live cell imaging, the probe concentration needs to be significantly lower than the analyte concentration. Because of this, any irreversible reaction-based GSH probe will exhibit maximum response regardless of the GSH concentration. A reversible reaction-based probe with an appropriate equilibrium constant allows measurement of an analyte at much higher concentration, and thus, is a prerequisite for GSH quantification inside cells. In this contribution, we report the first fluorescent probe CouBro for quantitative imaging of GSH in live cells. CouBro evenly distributes inside cells without organelle specificity. The GSH concentrations measured using CouBro in NIH-3T3, HeLa, HepG2, PANC-1, and PANC-28 cells are well correlated with the values obtained from cell lysates. CouBro imaging can also resolve the changes of GSH concentration in PANC-1 cells upon diethylmaleate (DEM) treatment. Through this study, we demonstrate the importance of reaction reversibility in designing quantitative reaction-based fluorescent probes.

47

STRUCTURAL AND EVOLUTIONARY BASIS FOR SPECIFICITY IN METABOTROPIC GLUTAMATE RECEPTORS

Hye Jin Kang, Angela Wilkins, Kit Menlove, Jianpeng Ma, Olivier Lichtarge, Theodore G. Wensel

Metabotropic glutamate receptors (mGluRs) are important for modulating signaling by glutamate, the main excitatory neurotransmitter in the central nervous system. The mGluRs have been implicated in protection from neuronal excito-toxicity and in learning and memory. To define the upstream and downstream signaling specificities of mGluR, experiments with pertussis toxin and dominant-negative Gαi/o proteins were performed to reveal that mGluR1 couples strongly to TRPC4β through Gαi/o, in addition to coupling to PLC through Gαq/11. Also I found the stringent specificity of group I (mGluR1,5) / II(mGluR2,3) mGluRs for L-glutamate and of group III (mGluR4,6,7,8) for another endogenous ligand, L-serine-o-phosphate (L-SOP). I also identified that L-SOP antagonizes the effects of L-glutamate for group I/II receptors. On the other hand, mGluR7 differs from mGluR4 and other group III mGluR in that L-glutamate and L-SOP activate it with low potency and efficacy.

In order to determine the structural origins of the diverse ligand response specificities among mGluR (group I/II versus group III, mGluR4 versus mGluR7), we have combined enhanced versions of the Evolutionary Trace (ET) algorithm used by my collaborators with mutagenesis and ligand response assays to identify specificity-determining residues in the group I receptor, mGluR1, and the group III receptors, mGluR4 and mGluR7. The residues that when I swapped them between mGluR1 and mGluR4 increased the potency of L-SOP inhibition relative to the potency of L-glutamate activation in mGluR1 mutants, and others diminished the potency and efficacy of L-SOP action on mGluR4 mutants. Combining ET identified swaps from mGluR4 with one identified by computational docking, produced mGluR7 mutants that respond with dramatically enhanced potency and efficacy to L-SOP. These results demonstrate not only how early and late functional divergence within mGluRs occurred but also the power of ET for identifying allosteric determinants of evolutionary importance and functional specificity.

48

UGT2B28 IS A KEY REGULATOR OF ANDROGEN SIGNALING IN PROSTATE CANCER

Akash Kaushik, Rajni Sonavane, Vasanta Putluri, Mohan Manikkam, Stacy Llyod, Jie Gohlke, Shaiju Vareed, Michael Ittmann, Nagireddy Putluri, Nancy Weigel, George Michailidis, Ganesh Palapattu, Arun Sreekumar

Androgen signaling is the central modulator of prostate cancer (PCa) development and progression. UDP-glucuronosyltransferases (UGTs) are the major glucuronidation enzymes known to participate in the inactivation of androgens in prostate cancer. Previously, using an integrative approach combining gene expression and metabolomics we reported higher activity of UGT2B28 in castrate resistant prostate cancer and verified its higher expression in these tumors using RNASeq data. Here we have knocked down (KD) UGT2B28 in AD LNCaP PCa cells. The KD resulted in an increased expression of androgen receptor (AR), elevated levels of testosterone and its precursors as well as elevated levels of prostate specific antigen (PSA), a classical AR regulated gene. Consistent with this, microarray analysis showed increased expression of genes involved in cholesterol-derived androgen biosynthesis. Furthermore, the KD cells exhibited significantly higher rate of proliferation in vitro and formed higher number of xenograft tumors in vivo. Microarray data attributed this tumor promoting phenotype to a bio-energetically favored metabolic rewiring via alterations in mitochondrial activity. This was confirmed using targeted metabolic analysis that revealed elevated levels of citrate, isocitrate, glutamate and glutamine in the KD cells. Intriguingly, the microarray signature for the KD cells, in addition to revealing changes in tumor promoting metabolic processes, also enriched for concepts describing cellular senescence. Androgens, in a dose dependent manner have been known to regulate both proliferation and senescence in prostate cancer cell lines. Clinical trial data also suggest that prostate cancer patients having higher baseline levels of androgen benefit from hormone supplementation. Interestingly enough UGT2B28 KD cells (that have higher levels of testosterone to begin with), unlike controls, are growth arrested by addition of exogenous androgen. From this we infer that UGT2B28 regulates the tumor promoting function of androgen receptor in prostate cancer by fine tuning the intra-tumoral levels of androgens.

49

THE HISTONE VARIANT MACROH2A IS A SUBSTRATE OF BRCA1 UBIQUITIN LIGASE

Beom-Jun Kim, Doug Chan, Sung Yun Jung, Yi Wang, Jun Qin

The breast and ovarian cancer specific tumor suppressor BRCA1 and its heterodimer partner BARD1 have an ubiquitin ligase activity and this activity can be compromised by cancer-associated mutations frequently. However, it remains unclear whether its ubiquitin ligase activity is important for cancer development or BRCA1 function. It is due in part to the lack of bona fide substrates despite extensive efforts to find them. We developed an approach that allows us to enrich ubiquitinated proteins and peptides from BRCA1/BARD1 overexpressed cells by two consecutive enrichment steps and identified a number of candidate substrates by mass spectrometry analysis. We confirmed BRCA1/BARD1-dependent enhancement of ubiquitination of these candidates in vivo and further confirmed that BRCA1/BARD1 specifically ubiquitinates histone variant macroH2A in vitro and in vivo. macroH2A ubiquitination plays a role in cellular senenscence as human fibroblast IMR90 cells underwent delayed senescence when ubiquitination-deficient form of macroH2A was expressed. Our findings show that macroH2A is an in vivo substrate of BRCA1/BARD1 ubiquitin ligase and its ubiquitination plays an important role in cellular senescence.

50

CONSERVED NCRNA STRUCTURES IN PRC2 MEDIATED H3K27 METHYLATION

Brian Kirk, Kit Menlove, Jason Burton, Qinghua Wang

Non-Coding RNAs (ncRNA) have increasingly been found to have functionally significant roles within the cell with a wide range of prescribed mechanisms ranging from miRNA regulation to chromatin rearrangement. One mechanism of particular interest in cancer biology is in the potential for ncRNA directed recruitment of Polycomb Repressive Complex 2 (PRC2) mediated epigenetic silencing. Genome level chromatin rearrangements by PRC2 are of key importance in understanding how cancer cells are reprogrammed and maintained as compared to healthy cells. As such, hundred of ncRNAs have been identified to bind to PRC2 and it is our goal to identify which of these ncRNA’s are the most critical towards altering PRC2 programs in cancer and by what mechanisms this is achieved.

As such, PRC2 binding ncRNAs were selected and then reduced to highly conserved mammalian regions of at least 50 bases. Using RNA structural predictions of secondary and pseudo knot structure for each conserved regions allowed to compare all conserved RNA regions against for statistical enrichment among PRC2 binding ncRNAs. This procedure identified dozens of potentially important structural elements that are over-represented and could be the mechanistic means for PRC2 binding.

Verification of these potential binding elements is being done using an Octet based binding protocol using E. coli expressed PRC2 component protein, as well as in vitro using whole cell lysate to identify if computationally discovered lncRNA regions affect ncRNA binding to PRC2. Successful demonstration of this process will have wide application in novel discovery of ncRNA function within a host of cellular systems. This work is supported by a training fellowship from the Keck Center Computational Cancer Biology Training Program of the Gulf Coast Consortia (CPRIT Grant No. RP101489).

51

NECROTIC CELLS ACTIVELY ATTRACT PHAGOCYTES THROUGH THE COLLABORATIVE ACTION OF TWO DISTINCT PS-EXPOSURE MECHANISMS

Zao Li, Victor Venegas, Yuji Nagaoka, Eri Morino, Prashant Raghavan, Yoshinobu Nakanishi, Zheng Zhou

Cells injured pathologically undergo necrosis, a type of cell death distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells must be swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in necrotic-like cell death of six touch neurons. Necrotic touch neurons are engulfed and degraded inside engulfing cells. It is unclear how necrotic cells are recognized by engulfing cells. Phosphatidylserine (PS) is an important apoptotic cell surface signal that attracts engulfing cells. Using ectopically expressed MFG-E8, a high-affinity PS-binding protein, we observed that PS was present on the surface of necrotic touch neurons. In addition, phagocytic receptor CED-1, whose function is needed for the efficient clearance of necrotic cells, clusters around necrotic cells. The extracellular domain of CED-1 associates with PS as determined in assays for an enzyme-linked immunosorbent assay-like solid-phase reaction and surface plasmon resonance. We further identified a necrotic cell-specific function of CED-7, the worm homolog of mouse ABC1 transporter, in promoting PS-exposure on necrotic cell surfaces. In addition to CED-7, ANOH-1, the C. elegans homolog of mammalian Ca2+-dependent phospholipid scramblase TMEM16F, plays an independent and unique role in promoting PS-exposure on the surface of necrotic but not apoptotic cells. The combined activities from CED-7 and ANOH-1 ensure sufficient PS exposure for necrotic cells to attract their phagocytes. Our work demonstrates that cells killed by different mechanisms (necrosis or apoptosis) expose a common “eat me” signal that attracts their common phagocytic receptor CED-1. Furthermore, unlike previously believed, we discovered that PS is actively exposed onto the outer surface of necrotic cells through two distinct molecular mechanisms, rather than being leaked out passively.

52

EVOLUTION VS. DISEASE: THE CALCULUS OF LIFE

Olivier Lichtarge

The relationship between genotype mutations and phenotype variations determines health in the short term and evolution over the long term, and it hinges on the action of mutations on fitness. A fundamental difficulty in determining this action, however, is that it depends on the unique context of each mutation, which is complex and often cryptic. As a result, the effect of most genome variations on molecular function and overall fitness remains unknown, and stands apart from population genetics theories linking fitness effect to polymorphism frequency. Here, we hypothesize that evolution is a continuous and differentiable physical process coupling genotype to phenotype. This leads to a formal equation for the action of coding mutations on fitness that can be interpreted as a product of the evolutionary importance of the mutated site with the difference in amino acid similarity. Approximations for these terms are readily computable from phylogenetic sequence analysis, and we show mutational, clinical, and population genetic evidence that this action equation predicts the effect of point mutations in vivo and in vitro in diverse proteins, correlates disease-causing gene mutations with morbidity, and determines the frequency of human coding polymorphisms, respectively. Thus elementary calculus and phylogenetics can be integrated into a perturbation analysis of the evolutionary relationship between genotype and phenotype that quantitatively links point mutations to function and fitness and that opens a new analytic framework for equations of biology. In practice, this work explicitly bridges molecular evolution with population genetics with applications from protein redesign to the clinical assessment of human genetic variations.

53

ROLE OF N-TERMINUS, POLYQ AND POLYP REGION IN HUNTINGTIN PROTEIN AGGREGATION

Boxue Ma, Koning Shen, Wah Chiu

Huntington’s disease (HD) is a hereditary neurodegenerative disease. The exon1 fragment of mutated huntingtin protein (mHtt) with polyglutamine expansion forms fibril in neurons. Exon 1 fragment consists three major regions, an N terminus containing 17 amino acids (N17), a polyglutamine (polyQ) region and a polyproline (polyP) region close to the C terminal. To understand the role of each region during aggregation, we expressed various GST tagged mHtt constructs. Once the GST tag been cleaved off, each construct would start forming aggregates. We used cryoEM to observe the morphology of each aggregate after 24hrs incubation. Full length exon1 with 51 glutamine tract (WT-Q51) formed branched bundles of fibrils. The polyQ region (Q50) formed smaller bundles. Exon1 with deletion of polyP and the rest of C terminal (ΔP) formed larger and wider bundles. Exon1 with deletion of N17 peptide (ΔN) formed the smallest bundles. We further examined the ΔN construct by incubating up to 60hrs. CryoEM images showed that the aggregate could not grow any larger even when sufficient time was given. On the other hand, seeding of N17 peptide restored the aggregation of ΔN construct that the morphology of seeded ΔN became similar to the WT-Q51. Therefore, we conclude that the polyQ region of mHtt exon1 alone is sufficient to form fibril aggregate. The N terminus promotes aggregation, while the polyP region inhibits aggregation.

54

EVOLUTION OF ANTIBIOTIC RESISTANCE AMONG CLINICAL VARIANTS OF KPC Β-LACTAMASE

Shrenik Mehta, Kacie Rice, Timothy Palzkill

β-lactam antibiotics are the most commonly prescribed class of drugs for treating bacterial infections. β-lactamase enzymes are the most important source of resistance to these drugs as they hydrolyze the antibiotic and render it inactive. The KPC group of β-lactamases is one of the most threatening groups of enzymes as they are capable of hydrolyzing the carbapenem class of β-lactam antibiotics. The carbapenems currently serve as our last line of defense against bacterial infections. Variants of KPC β-lactamases differing by one-two amino acids have been isolated from patients all over the world. Despite the identification of these enzyme variants from the patients, there is a lack of information about their substrate profiles and catalytic mechanism, which could help in formulating treatment regimens for such infections. In this study, we have characterized nine KPC variants that differ from KPC-2 by one to two amino acids. The results indicate that both single and double mutant enzymes hydrolyze the carbapenem substrates as proficiently as KPC-2. When tested for ceftazidime, a 3rd generation cephalosporin antibiotic, the bacterial cells expressing the single mutants showed a minimum inhibitory concentration (MIC) upto 6-fold higher than KPC-2 while the bacterial cells expressing the double mutants showed a 3-100 fold increase in MIC. The purified double mutant enzymes also exhibited a 7-75 fold increase in catalytic efficiency for hydrolyzing ceftazidime while the single mutants had a catalytic efficiency 2-11 fold higher than KPC-2. 3rd generation cephalosporins are the drug of choice for treating unknown bacterial infections in patients; thus, the evolution of KPC to better hydrolyze ceftazidime is likely the result of selective pressure due to antibiotic therapy. Additionally, this improvement in ability to hydrolyze ceftazidime does not significantly alter their ability to hydrolyze carbapenems. Thus, mutations acquired by KPC-2 in natural isolates of bacteria significantly broaden its substrate profile leading to multi-drug resistance.

55

SEQUENCE DETERMINANTS OF CLASS C BETA-LACTAMASE SUBSTRATE SPECIFICITY AT HIGH RESOLUTION

Rose Mikulski, Michael Courtland, Wanzhi Huang, Ruddy Guerra, Timothy Palzkill

The class C β-lactamase P99 efficiently hydrolyzes extended-spectrum cephalosporins thereby conferring many gram-negative bacteria with resistance. The goal of this study is to identify those amino acid residue positions in the active site of the P99 enzyme that are critical for the hydrolysis of β-lactam antibiotics as well as those positions that control the substrate specificity of the enzyme, i.e., positions whose sequence requirements change based on the β-lactam antibiotic being tested. A total of 21 residues in and around the active site were chosen for determination of their role in catalysis and substrate specificity. Combinatorial libraries were constructed by individually randomizing the codons for the chosen amino acid residues to create collections of all possible substitutions for each position randomized. The libraries were each transformed into Escherichia coli and the cells were spread on agar plates containing one antibiotic (ampicillin, cefotaxime or cephalothin) to select for functional random substitutions that allow the enzyme to hydrolyze the antibiotic being tested. The mutants from each library selected for function against each antibiotic were pooled and the entire collection was processed by next generation DNA sequencing to generate sequences of functional mutants. We assess the sequence requirements for enzyme action for each residue position for each antibiotic tested. Several positions exhibited stringent sequence requirements for each antibiotic, which indicates these residues are critical core positions required for catalysis. Difference maps show that the sequence requirements change depending on the antibiotic, suggesting other positions that control the substrate specificity. In conclusion, deep sequencing of functionally selected combinatorial libraries reveal the sequence requirements for active site residues in the P99 β-lactamase for hydrolysis of three important β-lactam antibiotics. These studies provide insights into the plasticity of an enzyme and the structural and sequence requirements that control substrate specificity, which can facilitate drug design.

56

THE STRINGENT STRESS RESPONSE IS REQUIRED FOR MUTAGENIC REPAIR OF DNA BREAKS

Phillip Minnick, Ryan L. Frisch, Janet L. Gibson, Austen L. Terwilliger, Tyler J. McCue, Michele C. Darrow, Christophe Herman, Susan M. Rosenberg

Mutagenesis mechanisms controlled by stress responses increase mutation rates during stress, accelerating adaptation to stressors such as nutrient limitation, antibiotics and antifungal drugs, in bacteria, yeast and human cancer cells. One stress-induced mutagenesis mechanism in Escherichia coli is mutagenic repair of DNA double-strand breaks (DSBs). Under stress, or if the general (RpoS) stress response is activated artificially, repair of DSBs switches from use of the high fidelity DNA Pol III to use of error-prone Pol IV causing point mutations and gene amplifications. Mutagenic repair of DSBs localizes new mutations both in time (under stress), and in genomic space (near DSBs). Three stress responses promote mutagenic break repair in E. coli: the envelope-protein stress response promotes break formation; the DNA-damage response upregulates Pol IV (promoting point mutagenesis); and the general stress response licenses use of Pols IV, II and I in repair. Here we demonstrate that a fourth stress response must be activated for mutagenic break repair to occur: the stringent (starvation) response. We show that stringent-response-defective ∆relA or ∆dksA cells are defective for both point mutations and gene amplification. Moreover, the stringent response promotes mutagenesis independently of the other three stress responses. First, DksA is still required if DSBs are artificially generated. Thus, the stringent response role is not in formation of DSBs (role of the envelope-protein-stress response). Second, the RelA/DksA role is not in DNA damage-response induction because it is not suppressed by expression of Pol IV (the sole role of the damage response). Third, mutations that allow general-stress-response-independent mutagenesis do not compensate for RelA/DksA, demonstrating a role independent of the general stress response. We conclude that the stringent response constitutes a fourth, independent, stress response input into mutagenic break repair. The stringent response presumably regulates the expression of a currently unknown protein(s), critical to mutagenic break repair.

57

STRUCTURE AND FUNCTION OF STEAP PROTEINS

Sharmistha Mitra, Ming Zhou

STEAP1, Six Transmembrane Epithelial Antigen of Prostate member 1, was first identified owing to its abnormally high expression level in advanced prostate cancer. Three more members of the family, STEAP2-4, were subsequently identified. Since STEAP1 has no or extremely low levels of expression in normal cells, STEAP1 proteins has attracted considerable attention as a potential biomarker for early detection of prostate cancer. However, STEAP1 and 2 have also been found to express at very high levels in other common cancers including breast, colorectal and ovarian cancer, suggesting that these proteins play a fundamental role in cancer cell induction or proliferation. Cell-based studies indicated that the expression of STEAP1 is associated with elevated level of reactive oxygen species (ROS) whereas STEAP2-4 have metalloreductase activities. Combined, these observations led us to propose that STEAP proteins associate with each other to form heterooligomeric complex and thus induce carcinogenesis through their NADPH oxidation or metalloreductase activity. This central hypothesis is novel because the connection between cancer progression and STEAP function has never been studied before. So far we have overexpressed and purified all four members of STEAPs in milligram quantity suitable for structural and functional studies. This is the first time full-length membrane-bound STEAP proteins have been expressed and purified. We aim to crystallize and solve structures of the STEAP family of proteins, and in parallel, we will employ spectroscopic, fluorescence and colorimetric methods to characterize the reaction mechanism. Preliminary studies on STEAP1 proteins have already produced a wealth of new information including the presence of a heme group in the protein and its coordination structure and redox potentials. Successful completion of the project will establish a new avenue of research on how STEAP protein activity affects cancer progression, and thus will lead to novel ways of treating cancer by targeting STEAP protein activity.

58

STRUCTURAL CHARACTERIZATION OF INFLUENZA A VIRUS NON-STRUCTURAL PROTEIN-1 (NS1) BOUND TO CPSF30

Sayantan Mitra, B. V. V. Prasad

Influenza viruses cause acute highly contagious respiratory disease and affect 500 million people annually worldwide. Currently the highly lethal H5N1 strain posing a serious threat for worldwide pandemic, along with recent emergence of influenza virus strains resistant to the available antiviral drugs make it necessary to identify potential new drug targets. One of the promising targets is the non-structural protein 1 (NS1) of influenza virus, which is important for virus replication, spread, and pathogenesis. It consists of an RNA binding domain (RBD) and an effector domain (ED) separated by a flexible linker. It interacts with the F2F3 domain of cleavage and polyadenylation specificity factor (CPSF30) to inhibit 3’ end processing of cellular pre-mRNA including IFN-β pre-mRNA, thus inhibiting host antiviral response. Although the structure of NS1 ED with F2F3 has been determined, the structural characteristics of the full-length (FL) NS1 suggest these structures might not be relevant. To determine the structure of H5N1 FL-NS1 and F2F3 by X-ray crystallography, both the proteins have been purified individually and after forming complex, it has been used for initial crystallographic screening. Currently, optimization of the favorable conditions are been carried out. The binding studies of H5N1 FL-NS1 and H5N1 NS1-ED with F2F3 by Isothermal titration calorimetry (ITC) have also suggested FL-NS1 interacts with F2F3 more strongly than ED alone. This might be due to a different and/or additional set of interactions that allows for increased F2F3 binding affinity with the FL-NS1 than with ED alone. In addition to providing a better mechanistic understanding of how FL-NS1 interacts with F2F3 and what are the characteristics of these interacting regions, they will also provide a rational basis for the design and development of new small molecule drugs that disrupt NS1 interaction with F2F3 and thus the ability of NS1 to antagonize the host antiviral response.

59

PERSISTENT OXIDATIVE LESIONS IN DNA ARE REQUIRED FOR ENVIRONMENTAL STRESS INDUCED MUTAGENESIS IN ESCHERICHIA COLI

Jessica Moore, Ana K. Mojica, Susan M. Rosenberg, PJ Hastings

E. coli under stress such as starvation increase mutation rate transiently under the control of stress responses, until a mutation occurs that allows adaptation. The mechanism of stress response-controlled switch to mutagenic repair of DNA breaks is well characterized. lac frameshift-bearing cells starved on lactose medium acquire compensating frameshift (“point”) mutations, or amplifications of the leaky lac allele to 20-50 copies, which confers sufficient enzyme activity for growth. The mutagenesis requires (spontaneous) double-stranded DNA breaks and their repair. We show that reactive oxygen species, specifically persistent oxidative lesions in DNA that result from them, are required for mutation formation under stress in E. coli. Addition of exogenous reducing agents, use of constitutively activating alleles for the oxidative damage responses, and over-expression of katG to scavenge hydroxyl radicals dramatically reduce mutation formation. We show that reactive oxygen species are not required in stress-induced mutation to oxidize proteins, but to form oxidative lesions in DNA. These lesions are not required for the formation of DNA double-stranded breaks or to titrate out mismatch repair components, and the effect on mutation is lost if lesions are excised from DNA. Together, these data show a novel requirement for unrepaired oxidative base changes in DNA for the formation of environmental stress-induced mutants separately from the requirement for DNA double-stranded breaks and stress response regulation. We suggest that lesions in DNA pause the replicative DNA polymerase, allowing error-prone translesion polymerases to become active, leading to mutation formation.

60

SEQUENCE, STRUCTURE, FUNCTION IN NON-CODING RNAS.

Ilya Novikov, Angela Wilkins, Olivier Lichtarge

Functional non-coding RNAs play a vital role in a variety of subcellular processes including RNA and DNA modification, maintenance of genome stability, and gene regulation. In this work we aim to gain better insight into the evolutionary relationship between sequence, structure, and function of these molecules. In particular, we hypothesize that functionally-important nucleotides in these molecules can be identified by analyzing the patterns of sequence and phylogenetic variation displayed by the molecule through its evolutionary history. We combine this evolutionary information with structural analysis, and discover that the more evolutionarily-important nucleotides tend to form well-defined, non-random clusters on the structure of the molecule. We also show that these clusters often constitute known functionally-relevant regions, such as metabolite and ion binding sites, protein interfaces, and catalytic pockets. This suggests that evolutionarily-important nucleotides in structured ncRNA molecules evolve in a manner that is detectable, and therefore that functional regions in these molecules can be predicted based on sequence and phylogenetic information.

61

ROLE OF STABILIZING MUTATIONS IN THE EVOLUTION OF ANTIBIOTIC RESISTANT ENZYMES

Maha Patel, Bartlomiej Fryszczyn, Timothy Palzkill

The Centers for Disease Control and Prevention reports that 2 million people acquire antibiotic-resistant infections and 23,000 people die as a result each year in the United States. β-lactam antibiotics are the most commonly prescribed antibiotics for Gram-negative bacterial infections. The most common mode of resistance to β-lactam antibiotics in Gram-negative bacteria is the production of β-lactamases. In particular, the CTX-M family of β-lactamases is the most widespread and frequently isolated in clinical samples around the world. CTX-M β-lactamases continually evolve in their ability to hydrolyze extended spectrum β-lactam antibiotics under selective pressure through point mutations in and around their active site. Studying the effect that naturally occurring mutations have on the structure, function and specificity of the CTX-M family of β-lactamases will provide insight into the evolution of these enzymes.

The CTX-M family of β-lactamases is divided into five subfamilies based on amino acid sequence homology. The P167S and D240G substitutions were identified individually in the active site of CTX-M enzymes in several subfamilies upon the introduction of ceftazidime, a third generation cephalosporin antibiotic, into the clinic. These substitutions allow CTX-M β-lactamases to effectively hydrolyze ceftazidime but also result in an overall loss in stability to the enzyme. Stability defects can be offset through the acquisition of secondary mutations. The A77V mutation has been identified in combination with the P167S and D240G substitutions in numerous clinical isolates. The prevalence of the A77V mutation among clinical isolates emphasizes its probable importance in the evolution of CTX-M β-lactamase enzymes. Here we characterize the A77V mutation in the CTX-M-14 model enzyme using kinetic analysis, competition assays and thermostability assays. We hypothesize that the A77V substitution acts as a stabilizing mutation to offset the defects caused by both the P167S and D240G substitutions, allowing the enzyme to retain stability while acquiring the ability to hydrolyze ceftazidime.

62

INSIGHTS INTO THE ROLE OF DISULFIDE BONDS IN THE ACTIVATION OF CGMP-DEPENDENT PROTEIN KINASE IΑ

Liying Qin, Albert Reger, Elaine Guo, Matthew Yang, Peter Zwart, Choel Kim

Type Iα cGMP-dependent protein kinase (PKG Iα) regulates physiological processes such as vasorelaxation and smooth muscle tone, thus plays a crucial role in heart protection and blood pressure regulation. Because PKG Iα is a broad specificity kinase, its interaction with specific substrates is essential for proper substrate phosphorylation and signal transduction. As one of the functionally important substrates of PKG Iα, RhoA is recognized by the N-terminal leucine zipper (LZ) of the kinase. Phosphorylation of RhoA at serine188 by PKG Iα inhibits RhoA from activating Rho kinase, resulting in smooth muscle relaxation. While it is well established that PKG Iα can be activated by either cGMP or autophosphorylation, recent reports suggest that residue cysteine42 (C42) may serve as a redox sensor where its oxidation and the resultant interchain disulfide bond (C42-C42’) formation directly activates PKG Iα independent of cGMP. However, the underlying mechanism of this redox sensing remains unknown.

We hypothesize that the formation of C42-C42 disulfide bond does not directly activate PKG Iα independent of cGMP, but indirectly by increasing the stability of the LZ domain, resulting in a higher affinity for RhoA. Since most of oxidizing regents suffer from partial oxidation and non-specific effects, we constructed two LZ mutants, C42S and C42L, representing the reduced and oxidized states of the wild type LZ, respectively. We then solved the crystal structures of both wild type and C42L LZ domains. The structure of the C42L LZ is highly similar to that of the wild type containing the C42-C42 disulfide with an RMSD of 0.49 angstrom. The C42L LZ also has a lower temperature factor than wild type, indicating a higher thermal stability. To further investigate the stability of the LZ domains, we are in the process of obtaining their melting temperatures using circular dichroism. Ongoing studies include a pull-down assay to test the interaction between wild type and mutant PKG Iα and RhoA and a kinase assay to investigate the phosphorylation of RhoA by PKG Iα in vitro.

63

UNCOVERING RELATIONSHIPS IN DRUG-GENE-DISEASE DATA USING NONNEGATIVE MATRIX FACTORIZATION

Sam Regenbogen, Ben Bachman, Angela Wilkins, Olivier Lichtarge

The explosion of "big data" in recent years has led to an accompanying need for methods to make sense of huge databases. Nonnegative matrix factorization (NMF), a technique that is used by Netflix to recommend movies for users, has been used to identify relationships in biological data as well. I explored how far this could be pushed using the Comparative Toxicogenomics Database (CTD), which contains separate networks of chemical-gene, chemical-disease, and gene-disease associations curated from literature. I showed that not only can NMF uncover hidden interactions within these networks, but also that when any two of these networks are combined, NMF can recover interactions of the third. Furthermore, when all three networks are combined, NMF can predict relationships of a type not present in any of the original data. For example, gene-gene associations predicted this way turn out to match the unrelated protein-protein interaction database STRING. These results suggest that these enormous databases contain even more information than is obvious to human observation, and that methods such as NMF can help to elucidate the complexities therein.

64

STRUCTURE AND DYNAMICS OF THE EIIC SUGAR UPTAKE SYSTEM

Zhenning Ren, Ming Zhou

Phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) is a sugar uptake system unique to bacteria. It is a multicomponent system composed of several cytosolic proteins and a transmembrane protein, EIIC. A sugar substrate is taken up by EIIC from the extracellular side, translocated across the membrane, and phosphorylated before being released into the cytosol. Although there is a crystal structure of an EIIC, the basic mechanisms of sugar transport and phosphorylation remain unknown. I plan to employ a combined approach of X-ray crystallography, biochemical crosslinking, and EPR and fluorescence spectroscopies to unravel the molecular details of the sugar uptake system. Preliminary results on these efforts will be reported.

65

LOCALIZATION OF THE BARDET-BIEDL SYNDROME PROTEIN COMPLEX IN ROD PHOTORECEPTOR CILIA

Michael Robichaux, Theodore G. Wensel

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy characterized by the dysfunction of primary cilia and retinal degeneration of the photoreceptor cells of the retina, which, among other disease outcomes, leads to vision loss and blindness. 8 essential BBS ciliary proteins form the macromolecular BBSome protein complex, which functions as a membrane coat in vitro and is hypothesized to regulate of key trafficking events within the primary cilia of most cell types. Beyond preliminary findings, however, little is known of the macromolecular structure of the BBSome or its precise function in primary cilia, including the rod outer segment (ROS) sensory cilium of the retina. One key limitation to our understanding of the retinal BBSome is the lack of sublocalization data within the small area of the ROS cilia in photoreceptor cells. Thus, I have begun a rigorous sublocalization analysis of the BBSome complex in ROS sensory cilia using high-powered STORM super resolution imaging, which enables the localization of the retinal BBSome proteins to a resolution of approximately 20-30 nm. At such a high-resolution, I have been able to confirm the localization of BBSome proteins near the basal body complex and surrounding the restrictive connecting cilium portion of the ROS cilium. However, to improve this STORM super resolution analysis and complete my analysis of the retinal BBSome, I have developed a method for creating homemade STORM antibodies that are specially conjugated to multiple STORM-specific photoswitching fluorophores. I have demonstrated that this novel immunostaining reagent that I specifically designed for STORM imaging will yield more accurate STORM reconstructions than those generated using commercial reagents. Together, these early and proposed sublocalization findings will greatly inform future functional studies of the BBSome and contribute to our current understanding of Bardet-Biedl syndrome and other crippling retinal ciliopathies.

66

TRIC/CCT CONTRIBUTES TO PROTEIN FOLDING AND FUNCTION OF LEUKEMOGENIC FUSION PROTEIN AML1-ETO

Soung-Hun Roh, David Tweardy, Wah Chiu

AML1-ETO is the most common fusion oncoprotein causing acute myeloid leukemia (AML). It affects the expression of genes critical for myeloid cell development and differentiation by acting as a rogue transcription factor. While known for decades to be the translational product of a chimeric gene created by the stable chromosome translocation t(8;21)(q22;q22), it is not known how AML1-ETO achieves its native and functional conformation or whether this process can be targeted for therapeutic benefit. Here, we show that the folding and biosynthesis of the AML1-ETO protein is facilitated by its interaction with the essential eukaryotic chaperonin, TRiC (or CCT). We also demonstrate that a folding intermediate of AML1-ETO binds TRiC directly, mainly through its β-strand rich, DNA-binding domain (AML1-175) with the assistance of HSP70. In addition, single particle cryo-electron microscopy (cryo-EM) reveals that active AML1-175 associates with adjacent subunits CCT4 and CCT2. Taken together, our results suggest that TRiC contributes to AML1-ETO proteostasis through specific interactions with CCT2 and CCT4, which may be targeted to treat AML.

67

TELOMERE SIGNALING NETWORKS IN AGING AND CANCER

Zhou Songyang

Telomeres are specialized nucleoprotein structures at the ends of linear chromosomes. Telomere dysfunction has been implicated in cancer and aging. In mammalian cells, the telomeres are elongated by the telomerase, and bound by the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. The six telomeric proteins form large protein complexes to recruit the telomerase for telomere length control, and a diverse array of signaling molecules such as DNA repair proteins for protecting telomeres from being recognized as DNA double-stranded breaks. Through proteomic approaches, we have systematically identified proteins that associate with telomeres and modulate the telomerase. The function and regulation of these proteins in telomere maintenance, aging, and cancer will be discussed.

68

IDENTIFICATION OF SUBSTRATE BINDING SITE AND INHIBITION MECHANISM OF A BILE ACID TRANSPORTER.

Vitali Stanevich, Ming Zhou

Apical sodium-dependent bile acid transporter (ASBT, also known as SLC10A2) is an integral membrane protein catalyzing sodium-dependent transport of bile acids from intestine into the cytoplasm of enterocytes. ASBT and Na-taurocholate co-transporting polypeptide (NTCP) are responsible for the reabsorption of 90% of cholesterol secreted with bile. Due to such function ASBT has attracted significant attention as a drug target for treating hypercholesterolemia. Moreover, ASBT is proved to have potential for prodrug approaches aiming to increase drug absorption and bioavailability. Current mechanistic understanding of ASBT function is based on the structures of several bacterial homologs. Our group has reported two structures of an ASBT homolog from Y. Frederiksenii in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved “crossover” region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for sodium ions. Our current efforts are focused on the identification of bile acid binding site and discovering new ASBT inhibitors. We perform screening for substrates and inhibitors of ASBT using electrophysiology approach. To identify binding site we employ biochemical methods (PEGylation of surface-accessible Cys, ITC) and co-crystallization with potential inhibitors and substrates.

69

SUBSTITUTIONS IN THE Ω-LOOP OF TEM BETA-LACTAMASE RESULT IN AN ENLARGEMENT OF THE ACTIVE SITE AND CHANGE THE SUBSTRATE PROFILE OF THE ENZYME

Vlatko Stojanoski, Liya Hu, B.V.V. Prasad, Dar-Chone Chow, Banumathi Sankaran, Timothy Palzkill

Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. Mechanistically, serine β-lactamases are very similar to serine proteases such as chymotrypsin. They both utilize acylation and deacylation of an active site serine in their mechanism of catalysis. TEM, a common plasmid-encoded serine β-lactamase, catalyzes the hydrolysis of early penicillins and cephalosporins. Here we examine a previously identified triple mutant of TEM 165-TyrTyrGly-167 (wild type165-TrpGluPro-167) with switched substrate specificity from ampicillin to ceftazidime. When compared to chymotrypsin, the Glu166Tyr substitution in the TEM triple mutant is analogous to a substitution of the general base His57 in chymotrypsin that results in an enzyme that maintains function. Our findings agree with previous observations of altered substrate specificity of the triple mutant, which displays increased hydrolysis of ceftazidime. Additionally, enzyme kinetic analysis shows that the hydrolysis of ceftazidime follows a branched pathway characteristic of substrate-induced reversible inactivation. Structural analysis of the triple mutant at 1.39 Å resolution reveals enlargement of the active site making it more accessible for larger substrates. Furthermore, in the crystal structure of the triple mutant Tyr166 is within hydrogen bond distance to Ser70 suggesting it acts as a general base to activate the serine in the catalytic mechanism of the enzyme. These findings change the current view that Glu166 is indispensible in the mechanism of serine β-lactamases. More generally, this provides insights into alternate mechanisms for hydrolysis reactions catalyzed by enzymes.

70

EXAMINING THE SEQUENCE REQUIREMENTS FOR METALLO-Β-LACTAMASE CPHA FUNCTION BY DEEP SEQUENCING

Zhizeng Sun, Vlatko Stojanoski, Shrenik Mehta, Timothy Palzkill

CphA is a class B2 metallo-β-lactamase (MBL) identified from the Gram-negative bacterial pathogen Aeromonas hydrophila. Similar to other MBLs, CphA requires divalent metal ion cofactors such as Zn2+ to hydrolyze β-lactam antibiotics. However, CphA differs from other MBLs in that it is stimulated by low concentrations (nM) but inhibited by high concentrations (μM) of Zn2+ and is only active against carbapenem antibiotics. Therefore, to understand the sequence determinants for the function of CphA, randomized single site mutation libraries were constructed for amino acid residues around the active site of CphA. The libraries of CphA random mutants were sorted based on function by selecting for growth of E. coli containing the libraries on agar plates containing the carbapenem antibiotic, imipenem. Imipenem resistant mutants were pooled, PCR amplified, and subjected to Illumina sequencing, which produced 1.8x107 reads in total and 1.4 x105 reads on average for each individual library. Sequence analyses showed that in addition to residues involved in Zn2+ binding, many other residues were also conserved in libraries selected by high levels of imipenem. Consistently, mutation in any of these residues attenuated the function of CphA in conferring imipenem resistance. Biochemical analysis confirmed that this reduction in function was attributed to a decrease in protein stability and/or catalytic activity of the mutant CphA. On the other hand, a few positions that tolerate substitutions under stringent selection conditions were also identified and some mutants even displayed higher imipenem-hydrolytic activity than wild type CphA. This study conclusively identified essential and non-essential amino acid residues for the function of CphA and suggests a new approach for studying sequence determinants for the function of enzymes or other bioactive proteins.

71

A NOVEL APPROACH TO STUDY THE MECHANISMS OF COMBINATION THERAPY

Tingting Sun, Amritha Nair, Rocio Dominguez Vidana, Siddhartha Tyagi, Mayra C. Orellana, Alexander Renwick, Lukas M. Simon, Michael T. Lewis, Chad A. Shaw, Thomas F. Westbrook

Combination therapy is considered an essential strategy in effective and durable cancer treatment. However, a major challenge to rationally combining therapies and improving such strategies is to elucidate the mechanisms by which 2 therapies cooperate in inhibiting tumor cell survival.

We designed and developed an unbiased forward genetic screen (Drug-interaction Genetics, or DiG) to search for the mechanisms by which two anti-cancer drugs cooperatively impair triple-negative breast cancer (TNBC), a common subtype of breast cancer that confers poor prognosis and is refractory to current targeted therapies.

Previous studies from our lab have shown that the combination of two tyrosine kinase inhibitors, Sunitinib and Crizotinib (S+C), elicits cell death and tumor regression in as many as 65% of patient-derived TNBCs. Here we use multiple parallel genetic screens to compare the genetic networks that govern tumor cell response to the single agents and the combination. In addition to canonical growth factor effectors pathways (ex. PI3K and MAPK), our forward genetic approach revealed that the RTKs inhibited by Sunitinib and Crizotinib converge on a TAK1-p38 signaling axis in a cooperative manner. The combination of S+C elicits inhibition of TAK1-p38 and consequent tumor cell death in vitro and in vivo. Inhibition of p38 signaling enhances the effectiveness of S+C therapy in TNBCs. We are currently testing whether constitutive p38 activation confers resistance to S+C, and whether p38 signaling is retained in de novo S+C-resistant TNBCs in the presence of drug.

Collectively, these data suggest that Sunitinib and Crizotinib converge on TAK1-p38 to suppress TNBC tumor growth, and suggest that p38 inhibitors may be used in combination with low dose S+C to improve efficacy and overcome resistance. Moreover, the unbiased and high-throughput “drug-interactiong genetics” (DiG) approach used in this study provides a powerful tool to understand and improve combination therapies.

72

FUNCTIONAL ROLES OF CO-VARYING RESIDUES IN DOPAMINE D2 RECEPTOR

Yun-Min Sung, Angela D. Wilkins, Gustavo J. Rodriguez, Olivier Lichtarge, Theodore G. Wensel

Previous studies of amino acid co-variation in G protein-coupled receptors (GPCRs) mainly focused on predicting structural proximity of residue pairs in order to construct protein 3D structures. However, whether evolutionary amino acid co-variation can be used to detect functional coupling between residue pairs has not been tested. We investigated the functional relationships between evolutionarily correlated residue pairs in GPCR, focusing on dopamine D2 receptors (D2Rs). The D2R ligand binding pocket is predicted to be structurally similar to that of 5-HT2A serotonin receptors (5-HT2ARs); however, D2R responds and binds to dopamine better than serotonin, indicating the ability of D2R to discriminate between the neurotransmitters dopamine and serotonin. We hypothesized that residues may work in pairs or larger groups to maintain functional specificity of D2R. By applying the Evolutionary Trace (ET) algorithm to pairs of residues, we first predicted residue pairs in D2R that were likely to co-vary in evolution and replaced the co-varying ET-residues with the corresponding residues in 5-HT2AR. By comparing the double swaps with individual swaps, we observed non-additive effect on the efficacy of Gαi activation induced by dopamine-stimulated D2R, indicating functional coupling between the ET-residue pairs. We also found dramatic rescue effect on serotonin responses in some of the co-varying ET-residue pairs, suggesting their functional role in discriminating between dopamine and serotonin. Furthermore, we observed that one combined ET-residue swap showed higher binding affinity for serotonin compared to the wild-type, while the individual swaps had no effect on serotonin affinity. The non-additive free energy change upon binding revealed the functional coupling in ligand recognition. Taken together, these findings indicate that key co-varying ET-residue pairs have worked together during evolution to help fine-tune responses to dopamine, whereas other pairs have worked together to help confer selectivity, with the latter property likely more important for conferring selective evolutionary advantage.

73

EVOLUTIONARY TRACE-GUIDED MUTATIONS TO GENERATE D2 DOPAMINE RECEPTOR EFFECTOR BIAS IN THE BETA-ARRESTIN2 PATHWAY

María Elisa Terrón-Díaz, Yun-Min Sung, Angela Wilkins, Theodore G. Wensel, Olivier Lichtarge

Given that one-third of all available medications target G-Protein Coupled Receptors (GPCR), specificity towards ligands and downstream effectors of GPCRs is important for understanding protein function and the development of selective therapeutic drugs with fewer side effects. The serotonin (5-HT2AR) and D2 dopamine (D2R) receptors are both part of the bioamine subfamily of GPCRs and have similar sequence identity. However, they discriminate stringently in their cellular responses between endogenous agonists. Using Difference Evolutionary Trace (ET) analysis, functional determinants were previously identified and rationally targeted by swapping residues from 5-HT2AR to D2R. The sixteen residues swapped altered D2R ligand potency, affinity, and specificity. Beyond the ligand bias, we aim to utilize these sixteen D2R mutants to determine the effector bias with respect to the beta-arrestin2 pathway, involved in receptor desensitization and resensitization, after agonist treatment using a live cell-based assay known as Bioluminescent Resonance Energy Transfer2 (BRET2). The BRET2 assay is being used to detect the proximity between D2R-RLucII and beta-arrestin2-GFP10 at a distance less than 100Å. Five plasmids for the BRET assay have been generated: GFP10 fused to RLucII (positive control), GFP10 (negative control), RLucII (negative control), D2R-RLucII (donor), and beta-arretin2-GFP10 (acceptor). Plasmid titrations are currently being tested and site-directed mutagenesis is being performed on D2R-RLucII to generate 16 point mutations. We hypothesize that one or more of the 16 D2R mutants, that were previously shown to have ligand bias, will also have beta-arrestin2 bias. The beta-arrestin2 bias will be detected in the BRET assay as an increased GFP10/RLucII signal in the mutant D2R after agonist treatment as compared to wild type. This project will shed light as to how allosteric regulation of D2R can influence effector bias aiding in the understanding of the pathway and for improving rational approaches to drug design.

74

THE ROLE OF ADAPTIVE IMMUNE SYSTEM IN THE ESCAPE OF BREAST CANCER CELLS FROM PRIMARY TUMORS

Lin Tian, Thomas Welte, Xiang Zhang

Angiogenesis is one of the hallmarks of cancer. Using the bioinformatic tools, we found that some angiogenesis genes actually correlate with the good prognosis. These angiogenesis genes are involved in the pathways that protect the vascular integrity (vascular normalization). Gene set enrichment analysis (GSEA) on these good angiogenesis shows they are associated with adaptive immune response signaling. Based on these bioinformatics analysis, we hypothesize that adaptive immune system can prevent the cancer cells from entering into the circulation through normalizing the tumor-associated vasculatures.

We utilized four different murine mammary tumor models to test our hypothesis. We found the tumor-associated endothelial cells in immunodeficient mice lack the coverage of perivascular cells (pericytes). Depletion of pericytes in the tumor of immunodeficient mice correlates with leakier tumor-associated vasculature, higher number of circulating tumor cells as well as higher lung metastasis frequency.

We then interrogated which subset of T lymphocytes is the effector cells that mediate vascular normalization. Using antibody depletion and knockout transgenic mice, we found that CD4+ cells play an important role in pericyte recruitment. The mice treated with CD4 antibody or the CD4 knockout mice lack pericyte coverage in the breast tumor.

We also established an in vitro co-culture system to study the molecular mechanism of CD4+ T cell mediated vascular normalization. We found the S1PR1, a key factor that promote pericyte recruitment and proliferation, has higher mRNA expression after co-cultured with CD4+ T cells.

In conclusion, we found that not all the angiogenesis genes are bad. CD4+ T cells may elevate the expression of some good angiogenesis genes, which could further decrease tumor-associated vascular permeability and further decrease distance metastasis.

75

THE STOCHASTIC KINETICS OF E. COLI TRANSCRIPTION AT A SINGLE GENE LOCUS

Mengyu Wang, Jing Zhang, Leonardo Sepulveda, Ido Golding

Gene regulation consists of a series of stochastic, single-molecule events, resulting in substantial randomness in mRNA production between individual cells, and even between the individual copies of the same gene within a single cell. To characterize the stochastic kinetics of transcription in E. coli, we combine fluorescently labelled DNA-binding proteins and single-molecule fluorescence in situ hybridization (smFISH), to simultaneously detect a gene of interest and measure its transcriptional activity, in individual bacteria. Our preliminary results, using the lactose promoter (Plac), indicate that we can reliably detect the site of active transcription. They also indicate that active transcription involves a change in the gene’s spatial position in the cell. Under conditions of strong repression, we find that transcription is more likely to occur close to the time of gene replication. Next, we will use mathematical modeling to map the observed nascent and total mRNA copy-number statistics to the underlying stochastic kinetics of transcription at a single gene locus. We will examine how this stochastic kinetics is modulated by transcription factors that regulate gene expression, and whether the activity of multiple copies of the same gene is correlated within the single cell.

76

DETERMINING THE ROLE OF GPCR SIGNALING PROTEINS ON THE ACTIVITY OF THE METABOTROPIC GLUTAMATE RECEPTOR, MGLUR6

Sara Wright, Theodore G. Wensel

Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCRs) that are responsible for sensing the neurotransmitter glutamate in the brain, retina, and other CNS tissues. In the retina, G protein signaling is crucial for detection and processing of light. Photons are sensed by the GPCR rhodopsin in rod photoreceptors, which leads to a halt in the release of glutamate at the photoreceptor/ON-bipolar cell synapse. ON-bipolar cells sense glutamate through the metabotropic glutamate receptor mGluR6. In the dark, photoreceptor cells release glutamate, which binds to mGluR6 and activates G protein signaling through G alpha o. This pathway is negatively regulated by RGS7 and RGS11, which are members of the R7 family of regulator of G protein signaling (RGS) proteins. It has been recently determined that the membrane localization of RGS7 is dependent on the orphan receptors GPR158 and GPR179. This project involves determining the effects of RGS7, GPR158, and other potential pathway players on the activity of mGluR6. Assays of mGluR6 activity will be done in cell culture using the FLIPR membrane potential assay. This assay detects changes in membrane potential in response to ion channel activation. Previous results have shown that this assay can be used to determine mGluR6 activity. In addition, localization of the pathway players will be studied in these cells using Immuno-fluorescence, as well as in retina and brain slices.

77

CYCLOPROPYLAMINE CONTAINING INHIBITORS OF LYSINE SPECIFIC DEMETHYLASE 1 ARE POTENTIAL THERAPEUTICS FOR MLL-REARRANGED LEUKEMIA

Fangrui Wu, Zizhen Feng, Chao Zhou, Yuan Yao, Liping Wei, Wei Liu, Eric Pai, Shuo Dong, Michele Redell, Yongcheng Song,

Mixed lineage leukemia (MLL) gene translocations are found in ~75% infant and 10% adult acute leukemias, showing a poor prognosis. Lysine specific demethylase 1 (LSD1) has recently been implicated to be a drug target for the subtype of leukemia. Several series of cyclopropylamine compounds were synthesized for medicinal chemistry studies of LSD1 inhibition as well as tumor cell killing. Potent LSD1 inhibitors (IC50: 10-190 nM) exhibit strong activity against MLL-rearranged leukemia cells with EC50 of 10-280 nM, while these compounds are non-cytotoxic, with negligible activity against several other tumor cells. One compound showed significant in vivo activity in a systemic leukemia mouse model without overt toxicities. Mechanistically, the LSD1 inhibitors increased histone-H3 lysine-4 (H3K4) methylation, reduced expression of key leukemia genes, induced apoptosis and differentiation, and blocked self-renewal of the leukemia cells. Moreover, LSD1 inhibition works synergistically with inhibition of DOT1L, a histone-H3 lysine-79 (H3K79) methyltransferase, against MLL-leukemia.

78

ENHANCER SCREEN IN C. ELEGANS TO IDENTIFY NEW GENES FOR THE REMOVAL OF APOPTOTIC CELLS

Youli Xia, Ryan Haley, Henry He, Jaileen Valentín Rivera, Zheng Zhou

During animal development, a large number of cells are eliminated through apoptosis, a genetically controlled cell suicide that commonly occurs in multicellular organisms. The resulting apoptotic cells are swiftly engulfed and degraded by phagocytes. Phagocytic clearance of apoptotic cells helps sculpt organs, maintain homeostasis, eliminate abnormal or non-functional cells and prevent harmful inflammatory and autoimmune responses.

Genetic screens in the soil nematode C. elegans have revealed two partially redundant signaling pathways controlling the clearance of apoptotic cells. We are now performing a forward enhancer screen aiming to identify new genes involved in this particular process. We use mutagen EMS to induce mutations under a sensitized genetic background and screen worms that contain persistent cell corpses. We have screened 14,000 haploid genomes and have found 53 mutations so far, from which 20 are embryonic lethal.

Meanwhile we are doing genetic analyses including mapping and complementation tests to 14 homozygous viable mutations. Results indicate that 12 mutations are in ced-1, which encodes a membrane receptor that recognizes apoptotic cells. These mutations could be useful in the study of CED-1 protein function. In addition, new genes will be recovered from the mutant collection.

79

CLONAL EVOLUTION OF THE HER2 L755S MUTATION LEADS TO ACQUIRED HER-TARGETED THERAPY RESISTANCE THAT CAN BE REVERSED BY THE IRREVERSIBLE HER1/2

INHIBITOR AFATINIB

X. Sophie Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura M. Heiser, Nicholas Wang, Kyle R. Covington, Edward Chen, Alexander Renwick, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne A. W. Fuqua, Gary C. Chamness, Chad Shaw, Marilyn M. Li, David A. Wheeler, Joe W. Gray, Susan G. Hilsenbeck, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff

Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but intrinsic/acquired resistance (R) commonly occurs. To discover genetic aberrations of acquired resistance and how they evolve during treatment, we completed genomic profiling of 10 HER2+ BC cell line models with acquired resistance to anti-HER2 therapy (LR/TR/LTR) and 17 pre-treatment tumors from our neoadjuvant LT trial TBCRC006.

Methods: Whole exome sequencing/AmpliSeq/RNA-seq/mutant-specific Q-PCR.

Results: We found the HER2 L755S mutation in the BT474ATCC-LTR/BT474AZ-LR lines, in which HER2 was reactivated for resistance. The two HER2 mutant R lines share more similar SNV profiles compared to other BT474 R lines. Mutation-specific Q-PCR found statistically higher levels of HER2 L755S expression in two BT474 parentals compared to other parentals, yet much lower than the two R lines. Sequencing of BT474AZ-LR single cell clones found the mutation in each clone. These data suggest that the L755S subclone shared by two BT474 P lines was selected by L to become the dominant clone in the two R lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of two mutant R lines. Sequencing of TBCRC006 baseline found the HER2 L755S mutation in 1/17 subjects, who did not achieve pCR. The variant was present in 2% reads on both Illumina/Ion AmpliSeq platforms, suggesting a subclonal event.

Conclusion: Acquired resistance in our BT474 models evolves from pre-existing HER2 L755S subclones in parental cells. Subclonal detection of this mutation in BT474 parental and HER2+ BC baseline tumor suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC.

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BACK-TO-BACK MECHANISMS DRIVE ACTOMYOSIN RING CONTRACTION DURING DROSOPHILA CELLULARIZATION

Zenghui Xue, Anna Marie Sokac

While Myosin-2 motor activity is required for actomyosin ring contraction during cytokinesis in some cell types, recent findings suggest that motor activity is dispensable in other cell types, including mammalian cultured cells and budding yeast. One simple possibility is that different cell types use different mechanisms of actomyosin contraction during cytokinesis. Another possibility is that Myosin-2 dependent and independent mechanisms act in the same cell type, but make different contributions to contraction. In this work, we address the contribution of Myosin-2 motor activity during the cytokinetic event of cellularization in early Drosophila embryos.

During cellularization, actomyosin rings contract to build the bottom of newly forming epithelial cells. By quantitative live cell imaging, we find that ring contraction proceeds in two morphologically and kinetically distinct phases. In Phase 1, hexagonal rings become circular, and the contraction rate is slow. In Phase 2, rings get smaller, and ring contraction is fast. F-actin and Myosin-2 levels increase or stay constant, respectively, during Phase 1, but both decrease during Phase 2, suggesting that the Phases are mechanistically distinct. Indeed, using genetic mutants and drug strategies to manipulate Myosin-2 motor activity and F-actin polymerization dynamics, we find that contraction during Phase 1 depends on Myosin-2 motor activity, while Phase 2 does not. Instead, Phase 2 contraction depends on F-actin depolymerization.

Taken together, our work shows that two back-to-back mechanisms drive distinct phases of actomyosin ring contraction during cellularization. Our data supports a model whereby Myosin-2 dependent and independent mechanisms conspire to drive ring contraction, within the same cell type, and even during the same cytokinetic event. Cellularization now provides a unique opportunity to compare distinct contraction mechanisms in the same cell type, and to understand what molecular components control the switch between Myosin-2 dependent and independent mechanisms.

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SYNTHESIS AND SCREENING OF NOVEL FRAGMENTS DERIVED FROM DIVERSITY-ORIENTED SYNTHESIS

Damian W. Young

Fragment based drug discovery (FBDD) has been demonstrated to be an effective strategy toward the generation of therapeutic lead compounds. However, fragment chemical space has largely been explored in the context of commercially available highly sp2 atom enriched aromatic compounds. As a result, it may be concluded that an equivalently narrow biological target space has been probed. Toward the goal of modulating a wider range of biological targets, we hypothesized that a broader sampling of fragment structures was needed. Specifically, we aimed to synthesize fragment libraries containing variable sp2:sp3 carbon atom ratios to study the relationship of shape diversity in 3-dimensions to the binding to various types biological targets. Diversity-oriented synthesis served as the key strategy leading to the preparation of fragments that sample underexplored regions of fragment chemical space. The lecture will cover aspects related to the design and execution of DOS-inspired chemical pathways leading to novel low molecular weight, shape- diverse, and highly soluble fragments to be deployed in FBDD. The lecture will further describe our current experiments aimed at comparing the binding of DOS- derived fragments to traditional commercial fragments over several biological targets.

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COMPUTATIONAL MODELING AND DESIGN OF ORTHOGONAL GPCR-MEDIATED SIGNALING COMPLEXES

Melvin Young, Michael Chen, Patrick Barth

G protein-coupled receptors (GPCRs) commonly exhibit an inherently high level of promiscuity in ligand and effector binding and activation that hinders accurate understanding of their regulation and specific role in diseases. Consequently, many cell and gene therapy strategies are limited by the inability to reprogram receptor signaling properties without affecting endogenous cellular signaling pathways. We hypothesize that orthogonal, highly specific, GPCR-mediated signaling complexes will allow for the properties of specific receptor and downstream effector systems to be finely tuned without perturbing the function of related receptors and alternative pathways.

A combination of Rosetta’s multi-state design (MSD) and docking protocols have been used to evolve and select in-silico novel and highly specific binding interfaces unrelated to native GPCR/G protein complexes. The MSD protocol does not allow for a flexible backbone, limiting the available sequence space. To compensate for this limitation, we use the docking protocol to produce a larger diversity of GPCR/G protein binding interfaces that allows access to a substantially larger sequence space than MSD alone. The most promising computationally designed orthogonal GPCR/G protein complex has been experimentally cross validated by monitoring agonist-induced specific activation of design and WT complexes using cell-based assays. Our activation assay shows that the change in membrane potential with the designed GPCR and designed G protein pair compares favorably with the WT/WT pair, while both design/WT pairs have drastically lower activation as intended by the design process. This suggests that our methodology should greatly aid in the creation of highly specific GPCR-mediated signaling complexes.

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THREE-DIMENSIONAL STRUCTURES ASSOCIATED WITH PHOTORECEPTOR CILIA BY CRYO-ELECTRON TOMOGRAPHY

Zhixian Zhang, Feng He, Michael F. Schmid, Theodore G. Wensel

The light-sensing portion of rod and cone photoreceptor cells, known as the outer segment, is a modified primary cilium. Specialized cellular machinery is needed to assemble and maintain its structure, and to carry out highly specific and active transport and sorting of molecular components between the inner segment, where most biosynthesis occurs, and the outer segment, where those components are utilized for phototransduction. A narrow sub-cellular highway connecting the inner and outer segment, known as the connecting cilium, is the site of action of numerous proteins encoded by genes implicated in inherited diseases known as ciliopathies. At the heart of the connecting cilium is a nine + zero microtubule-based axoneme that emerges from a centriolar structure termed the basal body complex. The evolutionarily conserved features of the basal body-ciliary organelle include the transition fibers, and transition zone with its associated ciliary necklace. We are using cryo-electron tomography of isolated rods to determine the structures of the connecting cilium and associated machinery in wildtype mice. In recent work we have used computational sub-tomogram averaging to determine the three-dimensional structures of the basal body ciliary region and the transition zone. We have observed 9-fold Y-link structures which appear embedded between the cilium membrane and central microtubules (axoneme) from the basal body (Y-link connecting the microtubule triplets to the membrane) toward the rod outer segment (Y-link connecting the doublets to the membrane). These structural features in wildtype connecting cilium will help us to understand the normal mechanisms and functions of the many proteins essential for cilium function and photoreceptor survival, and shed light on the diseases, known as ciliopathies, that arise from defects in those proteins.

84

QUANTIFYING TRANSCRIPTION KINETICS IN INDIVIDUAL E. COLI CELLS

Jing Zhang, Mengyu Wang, Ido Golding

Gene expression and its regulation are traditionally studied by averaging over large cell populations. These “bulk” measurements miss significant cell-to-cell differences, as well as all spatial information within the single cell. To overcome these limitations, we plan to quantify transcription kinetics at the level of individual gene copies in individual E. coli cells. For that purpose, the gene of interest will be labeled using the Fluorescent Repressor Operator System (FROS), which combines fluorescently-tagged DNA binding proteins with a synthetic array of cognate binding sites. The transcriptional activity of the gene will be tracked using an analogous method, where the MS2 RNA-binding protein labels an array of binding sites on nascent mRNA. We have constructed these two reporter systems separately, and are now optimizing single-cell imaging and data acquisition in a microfluidic device. Next, we will combine both reporters to simultaneously follow the individual copies of the gene, and their transcriptional activity, in individual cells. We will examine whether transcription is coupled to cellular events such as gene replication or cell division, and whether transcription is accompanied by changes in the spatial position of the gene. We will also ask whether the transcriptional activity of two gene copies in the same cell is correlated. In the future, we will extend the work to examine the dynamics and correlation of two different genes in the same cell.

85

MECHANISM OF GATING IN THE TRKH K+ CHANNELS

Hanzhi Zhang, Ming Zhou

A high intracellular K+ concentration is required for many physiological processes like setting the membrane potential, enzymatic activity and osmoregulation. To carry K+ through the lipid bilayer membrane, organisms must express K+ transport proteins, such as proteins in the Superfamily of K+ Transporters (SKT). TrkH, a member of SKT, is required for bacteria growth in low external K+ environments. Previous studies showed that TrkH is an ion channel and ATP increases channel activity through an associated cytosolic protein, TrkA. However, how TrkH is gated, and how ATP controls gating of TrkH are still not clear. I hypothesize that ATP induces a conformational change in TrkA that is transmitted to TrkH to open the ion conduction pore. I plan to use cryo-electron microscopy, X-ray crystallography, electrophysiology, and biochemistry to provide direct evidence for this hypothesis.

86

A MATERNAL-ZYGOTIC MODULE STABILIZES F-ACTIN TO PROMOTE ROBUST MORPHOGENESIS

Liuliu Zheng, Heng Xu, Leonardo A. Sepúlveda, Rhonald C. Lua, Olivier Lichtarge, Ido Golding, Anna Marie Sokac

Robustness is a property built into biological systems to ensure that the outcome of an event is reliable even when inputs, like gene dosage and environment, vary. During development, robustness safeguards embryos against mistakes that underlie structural and functional birth defects. Yet, our knowledge of how robustness is achieved during mechanical events like embryonic morphogenesis is very limited.

Here we show that the tissue-building event of Drosophila cellularization is made robust by mechanisms targeting F-actin. We find that F-actin levels are significantly reduced in cellularizing embryos upon high temperature perturbation. This F-actin reduction leads to failures in cellularization and embryos that do not hatch. Using a genetic strategy, we identified two novel members of the Vinculin/α-Catenin Superfamily that work together to reinforce F-actin against such perturbations (Zheng et al., 2013). We find that zygotically-expressed serendipity-α (sry-α) and maternally-loaded spitting image (spt) are paralogs that share an overlapping, actin-stabilizing activity during cellularization. spt alone is sufficient for cellularization at optimal conditions, but both spt plus sry-α are required at high temperature and when the actin cytoskeleton is genetically compromised by a reduced dose of the actin accessory protein Profilin.

Strikingly, we find that Sry-α and Spt expression is precisely controlled, and that there is crosstalk between them. Specifically, Spt provides pre-cellularization functions that Sry-α cannot provide; and expressing elevated levels of Spt alone is not an option because it induces gain-of-function phenotypes. In addition, reducing maternal spt dosage triggers a compensatory up-regulation of zygotic sry-α transcription.

We suggest that Sry-α and Spt represent a robustness-promoting module for cellularization: Cross-talk between maternal spt and zygotic sry-α demonstrates that “paralog interactions” can span between maternal and zygotic genomes to promote robustness. In addition, we show for the first time that specific cytoskeletal-based mechanisms promote robust morphogenesis.

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