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IDENTIFICATION OF PROTEIN-PROTEIN INTERACTIONS Yeast Two Hybrid System DEPARTMENT OF BIOTECHNOLOGY Speaker - Hina Ojha

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IDENTIFICATION OF PROTEIN-PROTEIN INTERACTIONSYeast Two Hybrid System

DEPARTMENT OF BIOTECHNOLOGY

Speaker - Hina Ojha

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Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells.

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Critical aspects required to understand the function of a protein include:•Protein sequence and structure–used to discover motifs that predict protein function•Expression profile–reveals cell-type specificity and how expression is regulated•Post-translational modifications (e.g., phosphorylation, acylation, glycosylation, ubiquitination)–suggests localization, activation and/or function•Intracellular localization–may allude to the function of the protein•Interactions with other proteins-function may be extrapolated by knowing the function of binding partners.

Functional proteomics

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Biological effects of protein-protein interactions

Alter the kinetic properties of enzymes, which may be the result of subtle changes in substrate binding or allosteric effects

Allow for substrate channeling by moving a substrate between domains or subunits, resulting ultimately in an intended end product

Create a new binding site, typically for small effector molecules Inactivate or destroy a protein Change the specificity of a protein for its substrate through the

interaction with different binding partners; e.g., demonstrate a new function that neither protein can exhibit alone

Serve a regulatory role in either an upstream or a downstream event

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Common Methods to Analyze Protein-Protein Interactions

Biochemical and biophysical approaches• Affinity chromatography• Coimmunoprecipitation• Fluorescence Resonace Energy Trasfer (FRET)• Surface Plasma Resonance• Atomic Force Microscopy (AFM)• X-ray Diffraction

Molecular genetic approaches• Yeast Two-hybrid

R0 = 4.9 nm

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Why Yeast Two-Hybrid system

The yeast two hybrid system has a clear advantage over classical biochemical or genetic methods

It is an in vivo technique that uses the yeast cell as a living test-tube.

It bears a greater resemblance to higher eukaryotic systems than a system based on a bacterial host.

With regards to classical biochemical approaches, which can require high quantities of purified proteins or good quality anti-bodies, the two hybrid system has minimal requirements to initiate screening, since only the cDNA of the gene of interest is needed.

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In signaling cascades, weak and transient interactions are often very important. Such interactions are more readily detected with the two hybrid system since the reporter gene response often leads to significant amplification.

The two hybrid assay is also useful for analysis of known interactions, which can be achieved by modifying important residues or modules and observing this effect on binding.

Interactions can be measured semi-quantitatively using the two hybrid system, allowing discrimination between high, intermediate, and low-affinity bindings, the power of which correlates with that of in vitro approaches

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Two hybrid screens are sometimes termed "functional screens", since if at least one of the proteins screened has a known function in a well-defined pathway, it might provide a functional hint in the current interaction.

Although there are certain disadvantages involving the two hybrid assay, the most convincing argument for its use is the speed and ease by which the molecular mechanisms of many signaling cascades have been defined using this technique.

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An Introduction to Yeast Saccharomyces cerevisiae is one of the most

intensively studied eukaryotic model organisms in molecular and cell biology, much like E.coli as the model prokaryote.

Saccharomyces cerevisiae cells are round to ovoid, 5–10 micrometers in diameter. It reproduces by a division process known as budding

S. cerevisiae was the first eukaryotic genome that was completely sequenced. (April 1996)

The genome is composed of about 13,000,000bp and 6,275 genes, although only about 5,800 of these are believed to be true functional genes.

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The classical yeast two-hybrid system

The early yeast two-hybrid systems were based on the finding that many eukaryotic transcription factors have separable DNA-binding and transcription activationdomains.

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•The protein of interest, the “bait”, is fused to a DNA-binding domain.

•Proteins that bind to bait, the “fish” or “prey”, are fused to a transcription activation domain.

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•Proteins that do not bind to the bait will not activate the transcription of the reporter gene (HIS in this case)

•Any protein that binds to the bait will activate the transcription of the reporter gene(a) No binding

(b) Binding between protein and bait

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The first step is to construct a bait plasmid and a library. Each type of plasmid contains a selectable marker such as an essential amino acid.

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Overall summary of Yeast two-hybrid experiment

• Yeast two-hybrid experiments yield information on protein protein interactions

• GAL4 Binding Domain

• GAL4 Activation Domain

• X and Y are two proteins of interest

• If X & Y interact then reporter gene is expressed

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Major applications of classical system Used to determine whether two known proteins interact

with one another Used to identify unknown proteins, encoded by a cDNA

library, that interact with a protein of interest

Powerful tool for investigating the network of interactions that form between proteins involved in particular biological processes

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Selection of host strain The yeast strains used for two-hybrid experiments carry mutations in a

number of genes required for amino acid biosynthesis, such as TRP1, LEU2, HIS3 and URA3.

If these amino acids are omitted from the growth medium the yeast strain will fail to grow.

Many of the two-hybrid plasmids carry genes that complement these mutations and allow for selection of the transformant yeast.

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Reporter Genes LacZ reporter - Blue/White Screening HIS3 reporter - Screen on His+ media (usually need to add 3AT to increase

selectivity) LEU2 reporter - Screen on Leu+ media ADE2 reporter - Screen on Ade+ media URA3 reporter - Screen on Ura+ media (can do negative selection by adding

FOA)

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Modifications of the Yeast Two-Hybrid system

The hSos recruitment system Three protein system The dual-bait system The reverse two-hybrid system

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The hSos recruitment system

•Plasmid encoding X fused in-frame to (Ras GEF) hSos•Plasmid encoding Y fused in-frame with a v-Src myristylation signal

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Procedure Two plasmids are constructed, one encoding protein X fused in-frame to

the human Ras guanyl nucleotide exchange factor (Ras GEF) hSos, the second encoding protein Y fused in-frame with a v-Src myristylation signal.

The plasmids are co-transformed into a temperature-sensitive yeast mutant containing a lesion in the Cdc25 gene which encodes a yeast Ras GEF.

The protein Y fusion is targeted to the yeast plasma-membrane. Interaction between proteins X and Y recruits hSos to the yeast plasma membrane where it complements the cdc25 mutation by activating the Ras signalling cascade.

Interactions are detected through growth of yeast cells at the restrictive temperature (37 ◦C).

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Applications

Cytoplasm-based yeast two-hybrid system Do not rely on a transcriptional readout

Transcriptional repressors Auto-activation of reporter genes by bait constructs The problem of certain proteins not localizing to the yeast cell nucleus

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Three protein system

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Procedure This system is based on the classical yeast two-hybrid system. Proteins X

and Y are expressed in-frame with a transcription factor DNA-binding domain and transcription activation domain, respectively.

A third protein, Z, is expressed with a nuclear localization signal, without any added domains, in the yeast nucleus.

Protein Y may only interact with X in the presence of Z. (i) A domain formed through the interaction between X and Z may provide

an interaction interface for protein Y. (ii) Alternatively, protein Z may act as a bridge between proteins X and Y

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The dual-bait system X1 fused to DNA-

binding domain LexA X2 fused to DNA-

binding domain λcI

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Procedure Two test proteins (X1 and X2) are fused to two different DNA-binding

domains (LexA and λcI, respectively). The two fusion constructs are co-expressed in the same yeast cell and

tested for interaction with proteins fused to the B42 transcription activation domain.

Interaction with X1 induces expression of LexA-dependent reporter genes (lacZ and LEU2).

Interaction with X2 induces expression of λcI-dependent reporter genes (LYS2 and gusA)

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Applications

Two test proteins can be analyzed for protein-protein interaction partners in a single library screening

To test the specificity of a protein-protein interaction amongst evolutionarily conserved proteins

To identify domains or residues required for interaction with one partner but not another

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The reverse two-hybrid system

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Procedure This system is based on the classical two-hybrid system except that

expression of the reporter gene is toxic to the yeast cells under certain conditions.

In this example, the reporter gene URA3 allows for selection of protein-protein interactions between X and Y on media minus uracil and counter-selection of disrupted protein-protein interaction between X and Y on media containing 5FOA.

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Applications This system can be used to identify residues required for protein-protein

interaction by making use of a mutagenised copy of the cDNA encoding one of the proteins.

cDNAs encoding proteins no longer able to interact can be sequenced to reveal amino acids essential for interaction.

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What is the yeast two-hybrid system used for? Identifies novel protein-protein interactions Can identify protein cascades Identifies mutations that affect protein-protein binding Can identify interfering proteins in known interactions (Reverse Two-

Hybrid System)

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Advantages Immediate availability of the cloned gene of the interacting protein Only a single plasmid construction is required Interactions are detected in vivo Weak, transient interactions can be detected Can accumulate a weak signal over time Protein purification not necessary No antibodies requries

Two hybrid systems - > to uncover unanticipated interactions

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Disadvantages False positives are the largest problem with the yeast two-hybrid

system. Can be caused by the ability of bait to induce transcription without interaction with the bait

Possible incorrect protein folding in yeast gene encoding target protein must be available failed to detect some know interactions

Elimination of False Positives Sequence Analysis Retransformation of both strain with bait plasmid and strain without

bait plasmid Test for interaction with an unrelated protein as bait

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Examples of Uses of the Yeast Two-Hybrid System

Identification of caspase substrates Interaction of Calmodulin and L-Isoaspartyl Methyltransferase Genetic characterization of mutations in E2F1 Peptide hormone-receptor interactions Pha-4 interactions in C. elegans

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WAOOW !! This is my favorite protein

Yakkkk!I don't

like that at

all.

My bad day!

When my protein

will come?

THANK YOU