INDM 4003Lecture 10

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How does the cell 'know' where to send a protein??

I cell disease, lysosomal proteins are secretedinstead of being directed to the lysosome

Failure to transport CFTR protein to theplasma membrane causes cystic fibrosis

Failure to transport proteins to peroxisomescauses Zellweger syndrome

What do WE care????

New applications, e.g. protein display on cell-surfaces

Production of biologically active hormones, enzymes etc

Disease

Biotechnology

Eukaryotes

Proteins are synthesised in the cytoplasm by 'free' ribosomes. Protein transport occurs after translation is complete

Direct targetting to organelles:

MitochondriaChloroplastsPeroxisomesNucleus

Examples

Proteins are synthesized by ribosomes associated with the endoplasmaticreticulum

Secretory pathway

Examples Integral membrane proteins (not from the above organelles)Secreted proteinsRough ERGolgi complexLysosomes

Overview of the secretory pathway

Observation........ all ribosomes are the same!

Free ribosomes and those associated with the ERare indistinguishable

Endoplasmatic ReticulumTransport across the ER membrane

Endoplasmatic ReticulumTransport across the ER membrane

Endoplasmatic ReticulumTransport across the ER membrane

Endoplasmatic ReticulumTransport across the ER membrane

Bip protein prevents denaturation or aggregation of proteins by binding to hydrophobic segments

BIP binds to stretches containing bulky hydrophobic amino acids,which are normally in the interior of the folded protein

Proteins preventing denaturation or misfolding are known as chaperones

Endoplasmatic ReticulumPost-translational modification in the ER

Formation of disulfide bonds catalysed by glutathion onlyoccurs in the ER

Folding Prevention of aggregation by BipFormation and rearrangement of disulfide bonds

The correct disulfide bonds are not immediately formed. Rearrangement of disulfide bonds is catalysed by Protein Disulfide Isomerase, a chaperone

Formation of correct disulfide bonds is critical to obtain active compounds.e.g., hormones, anticlotting agents. These are produced usingmammalian expression systems

INDM 4003Lecture 11

Endoplasmatic ReticulumPost-translational modification in the ER

Formation of disulfide bonds catalysed by glutathion onlyoccurs in the ER

Folding Prevention of aggregation by BipFormation and rearrangement of disulfide bonds

The correct disulfide bonds are not immediately formed. Rearrangement of disulfide bonds is catalysed by Protein Disulfide Isomerase, a chaperone

Formation of correct disulfide bonds is critical to obtain active compounds.e.g., hormones, anticlotting agents. These are produced usingmammalian expression systems

The golgi complexTransport from ER to the Golgi

ER proteins are retrieved from the golgi by a KDEL receptor protein

This binds to a C-terminal KDEL sequence in e.g., BIP

ER

cis golgi

Transport vesicle

The golgi complexTransport from ER to the Golgi

Proteins that are misfolded in the ER are retained and subsequentlydegraded in the ER

Emphysema and cystic fibrosis may be caused by by improper folding and transport failure

Trans region

Medial region

Cis region

Post translational modification in ER and golgi

Post translationial modifications in the golgi and ER includeglycosylation:

the addition of sugars residues to serine/threonine: O-linkedasparagine: N-linked

The ER and cis, median, trans golgi contain different enzymes, catalysing modifications specific for a particular compartment

Protein glycosylation is required folding sorting recognition by other proteins stability

Overview of the secretory pathway

The formation of transport vesicles

This process solves two problems: how are vesicles formed? what to include?

vesicles formed following budding from trans golgi or cell membraneare coated by proteins: clathrin

Clathrin is made up of two proteins which form a three-legged strcuture.

These 'triskelions' assemble into 'cages'

Clathrin pinches off a vesicle

The clathrin 'cage' dissassemblesinvolving a chaperone

The formation of transport vesicles

Clathrin cages

Clathrin coated pit

For a movie see: http://www.hms.harvard.edu/news/clathrin/

The formation of transport vesiclesWhat to include in the vesicle?

Lysosomal proteins are produced and glycosylatedin the ER

The sugar residues become phsophorylated by aphospho transferase in the cis golgi.

Example targeting to the lysosome

The sugar-phosphate groups are recognisedby a membrane boundmannose-6-phosphate receptor

The mannose-6-phosphate receptorbecomes internalised in a clathrin coatedpit

The formation of transport vesiclesWhat to include in the vesicle?

Clathrin coated vesicles become'uncoated' and fuse with the lateendosome also known as CURL

(compartment uncoupling receptorand ligand)

CURL has a low pH which causesdissociation of the sugar-phosphategroup from the receptor.

A vesicle containingthe receptor buds of and recycles to thegolgi.

A transport vesicle containing the lysosomal protein fuses with the lysosome.

Lysosome

Transport vesicle

The lysosomal protein is dephosphorylated

How does the vesicle ‘know’ that it has to include the mannose-6-phosphate receptor?

Assembly proteins (Ap) bind to proteins that become internalised in clathrin coated pits.

AP-1 and AP-2 recognise golgi and plasmamembrane proteins, respectively

INDM 4003Lecture 12

Overview of the secretory pathway

Cholesterol esters in lipid monolayer

A short sequence (Asp-Pro-X-Tyr) in thecytosolic domain of the LDL receptor is recognised by AP-2

LDL receptor binds to apoB

Uptake of cholesterol by cell

The LDL particle isinternalised and transported tothe lysosome

In the CURL vesicle the receptor isseparated from the receptor.

Receptors regenerate to the surface

Transport from CURL to the lysosome,LDL is degraded

Mutations in the recognition sequence result in the failure to internalise the LDLparticle resulting in heart attacks

Overview of endocytososis

Transport to mitochondria

Localisation of mitochondrial proteins

Chaperones (Hsp70) related to Bip keep the proteins in an unfolded form to facilitate transport

There are a number of destinations in the mitochondrion. The 'address'label is contained within the N-terminal part of the protein

Signal sequences and Destinations

Translocation accross the mitochondrial membranes

Translocation across the mitochondrial membrane

DHFR folds in the presence of theinhibitor. Translocation is blocked

Experiments like this revealed important aspects of translocation of proteinsinto mitochondria

1) proteins are only transported in an unfolded state2) proteins are transported through pores which span the outer and inner membrane3) energy is required for transport

Energy is required for translocation

Translocation requires a proton motive force ATP hydrolysis in cytosol and matrix

INDM 4003Lecture 13

How are proteins translocated to the intermembrane space

Experiments altering or deleting the second targeting sequence fail toreach their destination and accumulate in the matrix

Targeting to matrix

Targeting to innermembrane space

Two models are proposed to account for this:

conservative sortingnonconservative sorting

Matrix targeting signalis cleaved off.

Protein interacts with Hsp70

intermembrane spacetargeting signal iscleaved off,protein folds

Conservative sorting

A sequence of ~22 hydrophobicamino acids may prevent translocationacross the mebrane:

a stop-transfer membrane anchor

Non-conservative sorting

Intermezzo: a few molecular tools for the biochemist toolkit

Tools to study protein localisation

Some jelly fish are fluorescent which is caused by a protein:Green Fluorescent Protein (GFP).

Advantage: no need to disrupt cells, fluorescence is visible in living cells

GFP expression can be used to select cells using FACS, fluorescent activated cell sorting

Cell expressing GFP

Very powerful method to select cells expressing/not expressing certain genes, e.g, mutant selection

See Brock or http://www.icnet.uk/axp/facs/davies/sort.html for background on FACS

Purification of proteins

Many proteins of interest are present at low quantities in the cell.

-> overexpression

E. coli BL21 E. coli BL21 pETR401

transketolase

Purified proteins are required to study biochemical processes

Classical approach: selective binding and release of proteins to solid matrices selective precipitation

Binding of negatively charged protein to positively charged column

Elution of bound protein with Cl- ions

Ion exchange columns

Affinity chromatography is a powerful purification method:the protein binds specifically to a ligand.

Protein specifically binds to a ligand for which it has a high affinity.

Ligand is covalently bound to the column

Elution of protein with unbound ligand

Affinity purification

The combination of molecular biology and biochemistry allowsthe purification of almost any protein

ligand binding domains: maltose binding protein gluthathione-S-transferase

What if you want a native protein??

Cleavage with site specific proteasesallow generation of native protein.

Expensive, unique sites not always presentCleavage does not always occur

Smallest ligand binding domain: His-His-His-His-His-His His-tag

Column His-tagged protein

His-tags usually do not influence activity of protein, no need for removal

Allows purification in large quantities

Simple to construct

Antibodies against the His-tag are available: detection of protein without need for specific antibodies

Advantages

INDM 4003Lecture 14

Advantages of fusion proteins

Purification via affinity chromatography

Elution under non-denaturing conditions

Marker for gene expression

Targeting to cell compartments

Disadvantages

Native protein is not produced -> lower/no activity incorrect folding

Protease cleavage not always 100% specific

Expensive on scale up

Peroxisomes in the yeast Hansenula polymorpha

Electron micrographs of peroxisomes of H. polymorpha grown on methanol

Alcohol oxidase

Peroxisomes are organelles containing a single membrane and no DNA

The organelles are involved in a variety of metabolic activities

oxidation of fatty acids and lipids purines, amino acids biosynthesis of bile acids and cholesterol removal of peroxide

The composition of peroxisomes is highly variable:

Pulse-chase studies have shown that peroxisomal proteins are incorporated into the peroxisome after (20 min) synthesis in the cytosol

Peroxisomes

Initial experiments showed that a carboxy terminal SKL sequence isimportant and sufficient for import in peroxisomes

However, the SKL sequence is not present in some other proteinswhich are imported into the peroxisome.

These proteins have an amino terminal sequence of ~26-36 AA whichis removed following translocation across the membrane

At least one protein is known which has both pts-1 and pts-2

The analysis of protein import is hampered by the inability toset up an in vitro import system.

Genetic approach is used

mutants have been isolated which fail to take up pts-1 proteins fail to take up pts-2 proteins fail to take up both

Yeast mutants:

growth on substrates requiring peroxisomes is impaired other substrates is normal

Assembly of alcohol oxidase in cytosol

Genes involved in PTS-1 uptake include a homolog of a protein whichis responsible for Zellweger syndrome

A gene for PTS-2 uptake has a human homolog responsible forchondrodysplasia punctata, causing shortened limbs and cataracts

These observations have led to attempts to complement yeastperoxisome mutants with human c-DNA libraries

Interesting observation:Early observations show that monomers of catalase associate withthe membrane of peroxisomes before uptake takes place

Tetramerisation takes place after uptake in the peroxisome ??

Experiments with fibroblasts of Zellweger syndrome patients show thatpre existing tetrameric catalase can be taken up.

Detection of alcohol oxidase (a) and catalase (b) in peroxisomes using immunogold labeling

Antibodies are linked to gold particles.

These show up as black dots following electron microscopygold

antibodyprotein

Protein localisation with immunogold labeling

Studies using antibodies directed against monomeric and tetramericcatalase showed that oligomerisation takes place before uptake

Antibody interacting with monomerAntibody interacting with tetramer

Unfolding does not take place in latter situation

Similar observations were made with pts-2 targeting signals

Cross-linked proteins, and gold particles conjugated to pts-1 targetingpeptides are also imported!

Data suggest that peroxisomes import oligomeric structures.

Monomers without pts1 ‘piggyback’ on monomers which do have the signal sequence: assembly takes place before import

Export of bacterial proteins(overview)

SecB (a chaperone) binds to the preprotein and prevents it from folding

SecA binds to bothSecB and the signal sequence

SecA has a high and low affinity binding site

SecA is both a cytosolic and a membranebound proteinProperties of SecA

The complex ofSecA (ADP) SecB andthe preprotein bind tothe translocase (SecYEG)

SecA inserts in the membrane andbrings the preprotein in contact withthe translocase

The preprotein is partially translocated

ADP in the high affinity site is replacedby ATP

SecB is released

ATP hydrolysis in the high affinity ATPbinding site causes the release of thepreprotein from SecA

Hydrolysis of ATP in the low affinityATP binding site causes a conformationalchange resulting in a compactSecA

SecA releases from the membrane

The proton motive force'pulls' the preprotein acrossthe membrane

Folding of the protein may provide anadditional driving force

The positively charged signal peptide is cleaved by signal peptidase

INDM 4003Lecture 15

Cell surface display of protein on Gram-positive bacteria

Gram-positive bacteria have many cell-wall anchored proteins to interactwith their environment.

Examples: attachment to the Extra Cellular Matrix

fibronectin elastin collagen

protein A (binds antibodies)

proteases

Comparison of the amino acid sequence of a cell-surface proteins revealeda common motif in the C-terminus of these proteins

Pulse chase experimentLabel cells with 35S-methionineChase with excess cold methionine

take samples after 0,1,5, 20 minutes

Purification of the MalE fusion using affinity chromatography

N-terminal sequencing revealed that the first amino acid = Gly

Cleavage of the LPXTG motif occurs between Thr and Gly

To what is the protein linked?

Expression of the fusion proteinDigestion of the cell-wall with specific enzymesPurification of MalE using affinity chromatographyAnalysis of attached cell wall components

Cell wall anchored proteins are linked to the glycine cross-bridge of the peptidoglycan layer

Protein

Peptidoglycanlayer

1) Protein secretion using followed by cleavage of signal sequence2) Cleavage between Thr and Gly. C terminus anchors in membrane3) Covalent link between Thr and Gly of peptidoglycan layer

The ability to express proteins on the surface of bacteria has arange of applications.

Both Gram-negative and gram-positive bacteria are used.

Advantages of Gram-positive bacteria

G+ proteins are more permissive for insertion of foreign sequences G+ have a conserved mechanism for cell wall targeting G+ require only one protein translocation step to direct a protein to the cell-surface G+ bacteria are more sturdy due to their cell-wall

Vaccine development

The ability to express antigens on the surface of bacteria allows thedelivery of live oral antigens in for example feed of cattle

Bacteria (G+) used for this purpose

Staphylococcus carnosus used as starter cultures for meat fermentationStaphylococcus xylosus

Streptococcus gordinii Mouth commensal

Mycobacterium bovis Attenuated pathogen

Vaccine development

Antigens used, e.g., :

HIV-1 papiloma virusmalariaBurrelia burdorferiTetanus toxin

Antigens introduced between signal sequence and LPXTG motifare directed to the cell wall

These antigens evoked an immune response in many cases when surface exposed, but not when secreted, or intracellular

Bacteria presenting antigens may persist in the gut or stayresident for a long time (over 70 hours)

Oral immunization resulted in increases of IgG (serum) and IgA (lungs and saliva)

Surface display of enzymes

Surface display is an inexpensive method to produce immobilised enzymes

Production is cheapEnzymes can be regenerated by providing the cells with nutrientsExamples: lipase, beta-lactamase

Surface display of antibodies and peptide libraries

Selection of epitopes of monoclonal antibodies

Selection of antibodies The larger size of bacterial cells compared to phages make FACS (flluorescence activated cell sorting) possible (faster than phage selection procedures)

Environmental Applications

Production of cell-surface receptors for pollutants

INDM 4003Lecture 16

Genomic sequencing is a huge effort resulting in a stretch of3-6 million basepairs (bacterial)

How to make sense of all this?

Annotation is the process of identifying functional regions (e.g. genes)and assigning a function to them

The first step is to identify Open Reading Frames

Additional information is needed: the sequence upstream of a gene isNOT random

This helps, but is not sufficient:

poorly expressed genes (eg regulatory proteins) usually have poor ribosome binding sites

What else can we do?

The distribution of nucleotides inside a gene is NOT random.

For example many species have a preference for the use one codonover the other: codon bias

Non random distribution of nucleotides is used by programsto predict probable coding regions.

Some ORF's are identical to Expressed Sequence Tags (EST)These are transcribed and therefore are functional genes

Some ORF's resemble known genes from other organisms

We have identified all putative genes

Now what?

Analyse the sequence !

Many genomes transcribe their genes (especially highly expressed ones) in the same direction as the genome is replicated

Identification of putative promoter sequences:

non-random distribution of nucleotides

Identification of binding sites of transcriptional regulators

Identification of anomalies in GC content

The GC content of a genome is NOT homogenous.

These are often derived from other sources

Pathogenicity islands often have different G+Ccontens

Lets have look at the genes themselves

First question asked: does it look like anything we know?

Most popular method: Basic Local Alignment Search ToolBLAST (http://www.ncbi.nlm.nih.gov/)

BLAST compares an unknown sequence to all known sequences(protein or nucleotide) in GenBank

The use of BLAST provides insight into a possible function for genes identified in genomic sequencing.

Hmmmm…. could we be dealing with an iron repressor gene??

Subsequent biochemical analysis: YES!

Sequence information directs biochemical and microbiological experiments!

Two types of related sequences are recognised:

Paralogs: sequences which share a common evolutionary history within a given organism. Frequently the result from gene duplication

Orthologs:sequences which share common evolutionary history in two or more organisms.

WARNING!!!!Although sequences share a common history, they may have different functions

Many genes (up to 40%) are not similar to any known gene.

Hopeless?? Go home??? I think not

Analysis of the amino acid sequence provides clues to function,structure and localisation of a protein.

Hydrophobicity plots reveal stretches of hydrophobic segments in a protein which could indicate that it is an integral membrane protein

Other secondary structure (alpha helices, beta sheets) can be predicted

A protein on the Rhodococcus equi virulence plasmid is not similar to anything. Hopeless?

Signal sequences are conserved, allowing programs to identify these e.g. http://www.cbs.dtu.dk/services/SignalP/

>Sequence Prediction:

Signal peptide

Signal peptide probability: 1.000

Max cleavage site probability: 0.627 at 32

This prediction is 100% accurate! VapA is excreted, and N-terminalsequencing showed that cleavage occurs at residue 32

Can we do more?

Proteins are organised in domains which may be involved in a particularfunction

e.g, binding of ATP, NADH binding to DNA (helix-turn-helix)

These domains are conserved among proteins, even if the functionis totally different.

Programs like 'Blocks' search databases for the occurrence of sequence motifs associated with a function

>IPB001939 AAA-protein (ATPases associated with various cellular activities)

IPB001939B <->B (21,2314):77 SP5K_BACSU|P27643 92 ALHMMFKGNPGTGKTTVARLIG ||| | ||||||||||| gi|72905| 78 TLHMaFTGNPGTGKTTVALKMA

Analysis of the VapA protein with Blocks

Recognition of a Walker motif: GXXGXGKT/S

Based on analysis like this we know that the protein is

a) an extracellular proteinb) is cleaved at a Threonine residuec) is a potential ATPase

INDM 4003Lecture 17

Genome sequencing projects have provided an overwhelming amount of data

Analysing gene sequences gives insight in for example:

the metabolic capabilities of an organism

similarities/differences between virulent avirulent strains

how did genomes evolve

how many genes does it take to make an organism

It does not answer questions such as

which genes are required for specific metabolic functions

which genes are activated in specific organs

what mutations are involved in oncogenesis

which genes are required for virulence or cell differentiation

Functional analysis required

Northern Blot

Apply mRNA to an agarose gelBlot the separated mRNA molecules ontoa nylon filter Hybridise mRNA with radioactive probe

Northern blot of mRNA isolated from acetate grown R. equi

2.8 kb (mRNA-1)

1.6 kb (mRNA-2)

Gives information on transcript length abundancyDrawback: labour intensive Only one probe per hybridisation

Quantitative PCR

Depends on the observation that the amount of productfrom a PCR reaction is dependent on the amount of template

PCR is run with primersannealing to the gene ofinterest

AND an internal control

Real time PCR depends on monitoring the formation of a PCR productduring the reaction.

SYBR green bindsto the minor groove ofDNA

is only fluorescentwhen bound to DNA

Gene disruption

An antibiotic resistance marker is inserted into the gene of interest Mutants will be come resistant to the antibiotic The gene of interest is disrupted

This method is invaluable to determine whethera gene plays a role in a particular cellular process

Other methods include the use of reporter genessuch as GFP to determine whether a gene istranscribed

Drawback: only a handful of genes can be analysed Time consuming and labour intensive

Bacterial genomes have ~4000 genes Yeast genomes have ~6000 genes Nematodes have ~16.000 genes Humans have ~40.000 genes

How can gene expression and mutation be analysed in these complexsystems????? Any of the current methodologies is inadequate

Comparison of cell lines: what makes a cell different?

Normal cell Cancer cellresting macrophage activated macrophageuninduced cell hormone stimulated cell

Question: are there specific genes which are up or down regulated in these cells?

Global analysis of gene expression required

Infection of macrophages by Rhodococcus equi

Virulence plasmid ofR. equi

Example: Research on Rhodoccocus equi

The lungs from an infected foal and human

Central questions: which macrophage genes are up or down regulated by R. equi which R. equi genes are responsible

how does R. equi reprogram the macrophage ?

The expression of thousands of genes need to be analysed

Principle: each gene is represented by a short nucleotide sequence on a solid substrate

A sample containing labelled mRNA or DNA fragments is hybridised to these probes

label is detected

The earliest gene arrays were manually made and contained 10 to a few hundred probes.

New methodologies are required to analyse genomic sequencesDevelopment of gene array systems

Spot probes representing genes on a nylon filter

Make cDNA from an mRNA poolLabel the cDNA pool with [32P]dATP

Hybridize the labeled cDNA pool to the filterAnd analyse it with a phosphor imaging system or X-ray film

Hybridizing cDNA

Nylon membranes have only limited capacity. Demands for higher throughput resulted in new, automated techniques

Create a library of the cell line of interest. Most widely usedtechnology is suppression subtractive suppresive hybridisation

cDNA library is cloned into a vector. Insert DNA is amplified by PCR.

This involves: robotic arraying of colonies and DNA isolation high throughput PCR (~20.000 colonies)

PCR fragments are spotted on a 1 x 3 inch glass slide by a robotic spotter(~ 20.000 spots)

Why glass?

Glass provides optically flat and highly uniform surface

Allows covalent attachment of cDNAs and oligonucleotides.

Low intrinsic fluorescence of glass allows the use of fluorescent instead of radioactive labels.

Glass microarrays allow very small (1-40 µl) volumes

Analysis

Make cDNA from control and experiment cell lines

Label with fluorescent dyeCy3 Cy5

Hybridize to DNA probes on glass slide

cDNA competes for hybridization to a particular probe

scan of a microarray

This type of array provides quantitative information of the relative expression levels of a gene in test and control strain

Affymetrix uses a different system to build a genechip.

Photoactivation of solid substrate

Photoactivation of nucleotide

Chemical linkage between nucleotide and solid substrate

Attachement of second nucleotide

Repetition of the photoactivation&linkage cycle allowsone to 'build' an oligonucleotide

www.affymetrix.com

How to do this on a large scale??

a mask allows specific photoactivationof spot on a glass wafer

A nucleotide will be added to thisposition

Repitition with different masks allowsthe assembly of different nucleotideson a single glass wafer

1.400.000 different oligonucleotides can be incorporated on a single DNA chip

Advantage: very high troughputdisadvantage: custom arrays are very expensive therefore not as flexible as the glass slide method

INDM 4003Lecture 18

APPLICATIONS FOR GENECHIP TECHNOLOGY

1) Detection of mutationsCancers, e.g., breast cancer (P53 and BRCA1 genes)Genetic diseases e.g., AlzheimersHIV drug resistance

2) Detection of polymorphismsBasis for IQ, behaviour (e.g., criminal behaviour, alcoholism etc)

Affymetrix and its collaborators have identified thousands of Single Nucleotide Polymorphisms (SNPs) and mapped them to their chromosomal locations. Using the information from these discoveries, we have developed GeneChip® genotyping assays to analyze thousands of SNPs on a single probe array, expediting the process of linking polymorphisms with disease

How about screening individuals forInsuranceInherited diseases (prenatal)Job applications

From www.affymetrix.com

3) Analysis of gene expression In response to growth conditionsDrugs à novel method for identifying new therapeutics

4) Identification of pathogensIdentification of species and resistance factors

These applications are only possible because:

· a very large number of sequences can be analysed · Low cost· Rapid (automation)· Custom designed chips

Analysis of the Mycobacterium tuberculosis genome has provided a wealth of information.

Blast searches revealed two interesting ORFs:

similar to Tyrosine phosphatases

cell growth differentiation mobility metabolismsurvival

Reversible phosphorylation of tyrosine residues is a key mechanism for the transduction of signals that regulate:

of EUKARYOTIC cells.

WHAT IS THIS PROTEIN DOING IN M TUBERCULOSIS??????

Key questions: is it expressed? is it secreted is it a tyrosine phosphatase?

Protein expressed as GST fusion

GST Tyrosine phosphatase gene

Proteins are purified with affinity chromatography

Using artificial substrates it was shown that the twoTyr phosphatases indeed dephosporylate P-Tyr but NOTP-Ser or P-Thr

Proteins are present in filtrate of M. tuberculosis cultures

Detection of proteins using western blots

What is its role in virulence?With what macrophage proteins does it interact?What proteins are modified by these phosphatases?

Some of these questions can not be answered by transcriptomicsAnalysis of proteins is required: Proteomics.

Proteome: ensemble of all proteins in the cell

Note: the genome is fixed, the proteome is not!

Expression profiling using DNA arrays

shows which genes are expressed/regulated

Does not show the effect of a stimulus on protein modification Does not reveal alternative splicing Does not reveal effects of stimuli on protein stability

Proteomics dependent on two dimensional gel electrophoresis

1st dimension is separation of isoelectric point: that pHat which a protein has no net electrical charge

Carrried out in a small tube containing a gel with a pH gradient

For the second dimension the tube containing the proteinsare transferred to a denaturing polyacryl amide gel

Separation on size of the protein

The resulting pattern is very complex.Analysis of 2D gels is done with image analysis usingfor example a BioRad Multiimaging system

2 dimensional gels can be used to examine the effect of a stimulus(e.g. a pathogen) on the protein expression and modification

This can in principle be done with expression profiling,although RNA expression levels do not always correspondto protein expression levels!!!

However, transcript profiling does not reveal post translational processing

Dramatic effects on protein catalytic activity, biological activitystability,interaction with other proteins

Common modifications areadenylationphosphorylationmethylationglycosylation

Protein modifications change charge, mass, isoelectric point of a protein, whichwill therefore change position in a 2D gel

Transcript analysis reveals no difference in expression levels, yet effects oncell function is dramatic

A B

2-D gels may reveal modification of proteins

HOW DO WE DIFFERENTIATE BETWEEN SITUATION A AND B????

Identification of spots is required

Traditionally using radioactive labels, epitope tagging, westernsor N-terminal sequencing

However, none of these methods is suited for high throughput analysis.

Method used is mass spectrometry: determines the molecular weightof molecules within 1-2 Da!!!

In situ digestion withprotease yields protein fragments

Spots are cut out of gel (automatically) and protein mix is analysed

Size of the fragments is compared with predicted MW derivedfrom genomic information

Identity of protein

Combination of proteomics and genomics is extremely powerful

The combination of genomics, transcriptomics and proteomicslead to the creation of databases describing all known informationabout a protein

-Function-Known orthologs/paralogs-When expressed-Where on the protein map-Modification-Interaction with other proteins

Never before in the history of biology has so much data beengenerated in such a short time

There are unparalled opportunities for discovery in all fieldsof biology