Yeast n hybridsystems

• yeast has been used widely as a host to discover and characterize protein–protein interactions. protein–protein interactions regulate biological processes and are fundamental for cell functions.

• The yeast two-hybrid system originally established by Fields and Song in 1989 has become an invaluable tool for deciphering protein interaction networks and for understanding the function of novel, uncharacterized proteins.

• Following the acceptance and widespread use of the yeast two-hybrid system, a number of variations were developed to detect other macromolecular interactions, i.e., DNA–protein (one hybrid), RNA–protein (RNA-based three-hybrid) and small molecule-protein interactions (ligand-based three-hybrid).

• These versions are usually summarized under the term ‘‘n-hybrid systems’’.

Introduction

The Yeast Two-Hybrid system

• Principle of the yeast two-hybrid system• The yeast two-hybrid assay is based on the modular nature of eukaryotic transcription

factors.• Transcription factors are structurally composed of domains:1. The DNA-binding domain (BD),which binds DNA sequences.2. The activation domain (AD) which is responsible for recruitment of the transcription

machinery.• Both domains together are sufficient to initiate transcription, and it has been established

that they do not need to be present within the same protein in order to function, although they have to be in close proximity.

The classical yeast two-hybrid system

• The protein of interest X, is fused to the DNA binding domain (DBD), a construct called bait.

• The potential interacting protein Y is fused to the activation domain (AD) and is called prey.

• The bait, i.e. the DBD-X fusion protein, binds the upstream activator sequence (UAS) of the promoter. The interaction of bait with prey, i.e. the AD-Y fusion protein, recruits the AD and thus reconstitutes a functional transcription factor, leading to further recruitment of RNA polymerase II and subsequent transcription of a reporter gene

Reporter genes, activation domains and DNA-binding domains used in yeast two-hybrid experiments

Larger scale yeast two hybrid system

• Larger scale two hybrid approaches typically rely on interaction mating. In this method the protein fused to DBD (the bait) and the protein fused to AD (prey) are expressed in two different haploid yeast strains of opposite mating type (mat a and mat ) and the strains are mated to determine if the two proteins interact. Result in fusion of the two haploids to form a diploid yeast strain.

• Primary advantage of this technique is that it reduces the number of yeast transformations needed to test individual interactions.

Choosing the right strategy: Available Y2H systems and their advantages

Alternative yeast two hybrid systems

Nuclear Two-Hybrid Systems RNA Pol III system RTA system Ras signalling based Y2H at the plasma membrane SRS Y2H RRS Y2H rRRS Y2H G-protein signalling-based Y2H at the plasma membrane G-protein fusion Y2H Split ubiquitin based Y2H systems Split ubiquitin Y2H MbY2H CytoY2H Split-protein sensor yeast two hybrid Split-Trp Y2H ER Y2H system SCINEX-P Y2H

Nuclear Y2H systems

• RTA Y2H• Pol III Y2H

• All require protein recruitment and bait-prey interaction at nuclear DNA. The classic Y2H and RTA Y2H both engage RNA polymerase II transcription either by its activation or its inhibition. By contrast, the Pol III Y2H, involves RNA polymerase III transcription.

RTA yeast two hybrid

• In the repressed transactivator (RTA) system, inversely to the classic Y2H, the bait-prey interaction represses transcriptional activation of reporter genes.

• The protein of interest X fused to the DBD of Gal4 is transactive, e.g. a transcription factor. If it interacts with another protein Y fused to the repression domain (RD) of a transcription repressor (e.g. Tup1p), the transcription of the reporter gene is repressed.

• This system has been extended to screen for molecules which inhibit protein-protein interaction e.g. for potentially novel therapeutic compounds acting as inhibitors of a given protein-protein interaction.

RNA Pol III system• The RNA polymerase III based two-hybrid system is another alternative to

screen for interaction partners of transcription factors activating RNA polymerase III-based transcription.

• RNA Pol III transcribes genes that encode untranslated RNA molecules such as rRNA, tRNA, and other small RNAs.

• As in the classic Y2H, a protein X is fused to a Gal4-DBD (bait), and this bait is able to bind DNA due to the Gal1 upstream activating sequence, located downstream of the reporter gene. Interaction of the bait with a prey protein (Y) fused to the τ138 subunit of transcription factor III C (TFIIIC) brings the RNA polymerase III holoenzyme to reporter gene.

• This strategy was later used to discover the interaction between the A. thaliana transcriptional regulators FIL and NZZ.

Pol III Y2H

Ras signalling based Y2H at the plasma membrane

• SRS Y2H• RRS Y2H• rRRS Y2H

• The SRS Y2H, RRS Y2H, and rRRS Y2H are all based on protein recruitment to the plasma membrane via bait-prey interaction and subsequent activation of MAPK downstream signalling. While in the SRS and RRS Y2H the prey constructs harboring protein Y are anchored at the membrane via myristoylation to analyze interactions with cytosolic bait constructs harboring protein X, the rRRS is used to analyze interactions between soluble preys containing protein Y and partner X being a membrane protein

SRS Y2HIn the SOS recruitment system , a soluble protein X is fused to mammalian SOS. If the SOS-X fusion interacts with a prey localized in the membrane (e.g. via myristoylation), SOS stimulates guanyl exchange on yeast Ras (yRas) and promotes downstream signalling

RRS Y2H

In the Ras recruitment system, the soluble protein X is directly fused to constitutively active mammalian Ras (mRas). Already active, this Ras only requires membrane location, bypassing the activity of Ras guanyl exchange factors (Cdc25 or SOS). The mRas-X fusion is recruited to the membrane by interaction with a membrane associated prey.

rRRS Y2H

Specifically for the use of membrane localized baits, the reverse Ras recruitment system has been developed. Conversely to the RRS, the prey is the Ras fusion protein, and the bait is membrane-anchored or itself a membrane protein. Exclusion of membrane and membrane-associated proteins also represents serious limitation as compared to other more recent Y2H techniques.

G-protein signalling-based Y2H at the plasma membrane

• G-protein fusion Y2H• The G-protein fusion system allows,

similar to the rRRS, to study the interaction between integral membrane bait and a soluble prey. The latter is a fusion protein with the γ-subunit of a heterotrimeric G-protein. If the prey interacts with the membrane-located bait, it will sequester G protein β-subunits, thus disrupting formation of heterotrimeric G-protein complex and subsequent downstream signalling.

• G-protein Y2H may identify drugs disrupting protein-protein interactions like RTA.

Split ubiquitin based Y2H systems

• Split ubiquitin Y2H• MbY2H• CytoY2H

• Split ubiquitin based Y2H systems involve reconstitution of ubiquitin from two domains upon bait-prey interaction. Their subcellular localization depends on the nature of interacting proteins X or Y, and on the reporter proteins used. The Split ubiquitin Y2H uses non-transcriptional reporting of protein interactions in the cytosol, but can also be used for membrane proteins. The MbY2H is used for interaction analysis with membrane baits and thus occurs at the membrane location of protein X, e.g. the plasma membrane. The CytoY2H is used for membrane anchored cytosolic baits and occurs close to the ER membrane

Split ubiquitin Y2H• The Split-ubiquitin system allows detection of protein-protein interactions occurring between

cytosolic and membrane proteins. • Ubiquitin is a small protein important for the turnover of cellular proteins. Proteins are

labelled for proteasomal degradation by covalently attaching a poly-ubiquitin chain. This chain is then cleaved off prior to protein degradation by ubiquitin specific proteases (USP).

• The split ubiquitin Y2H technique is based on separation of ubiquitin into two independent fragments: an N-terminal (Nub) and a C-terminal half (Cub) and that these two parts retain a basic affinity for each other, thus allowing spontaneous reassembly of quasi-native ubiquitin. This spontaneous reassociation of Nub and Cub is abolished by point mutations in Nub (NubG, NubA). In these mutants, efficient association is only observed if the two moieties are brought into close proximity by interaction of two proteins fused to NubG/A and Cub respectively. Reconstituted split-ubiquitin is recognized by USPs, which then cleave off any reporter protein fused to the C-terminal end of Cub.

• In the membrane transactivator split-ubiqitin (MbY2H) system, an artificial transcription factor (LexA-VP16) has been used as a cleavable reporter protein to analyse interactions between membrane-bound bait X and prey Y. Once ubiquitin is reassembled, LexA-VP16 is released to the nucleus, where it activates reporter gene transcription (i.e. HIS3, LacZ).

• This system was successfully used to detect interactions involving different kinds of membrane proteins. Split-ubiquitin based systems have become quite popular and have been successfully applied for cDNA library screens and large scale matrix approaches.

Mb Y2H

CytoY2H• In Cyto yeast two hybrid system the bait construct contains both Cub and the

transcription factor and is anchored to the ER membrane thanks to a fusion to the ER membrane protein Ost4p. This allows screening for interaction partners of a soluble protein among membrane and/or soluble proteins, as well as for proteins that are transcriptional activators or otherwise self-activating in nuclear Y2H.

Split-protein sensor yeast two hybrid

• Split-Trp Y2H• The Split-Trp yeast two hybrid is

used to assay cytosolic bait-prey interactions based on reconstitution of an enzyme in tryptophan synthesis (Trp1p), allowing for non-transcriptional reporting.

• Interaction between fragments lead to Trp1p reconstitution and allow trp1 deficient yeast strains to grow on medium lacking tryptophan.

ER Y2H system

• SCINEX-P yeast two hybrid• The screening for interactions between extracellular proteins system

allows the analysis of protein-protein interactions in the oxidizing environment of the ER, based on protein dimerization in unfolded protein signalling.

• This Y2H system was successfully used to analyze the interaction between the proteins involved in protein folding in the ER.

Limitations of Y2H systems

• Its relative methodical simplicity, its diversity, and its high-throughput capacity make the Y2H system the most popular analytic and screening method for interactomics.

• Nevertheless, all Y2H methods face the problem of false negatives and false positives.

• Also The two-hybrid system suffers from two major drawbacks:• It can detect only a subset of the complete interactome,• It can provide only very limited information on the kinetics or dynamics

of a PPI.

False negatives

• False negatives in Y2H are protein-protein interactions which cannot be detected due to limitations of the screening method.

• Reasons : Choose wrong Y2H sterategy Degradation and instability of proteins The fused yeast reporter proteins or anchors may cause steric hindrance that

impedes interaction, thus causing false negatives. Be different or lacking post-translational protein modifications in the yeast system

when analyzing interactions between proteins of higher eukaryotes. Toxicity of some fusion proteins that could affect the viability of transformed

cells.

• The rate of false negatives in Y2H can be reduced by either taking a combinatorial approach or choosing a different experimental approach. For example, using combinations of different Y2H vectors

False positives• False positives in Y2H are physical interactions detected in the screening in yeast

which are not reproducible in an independent system.• Two distinct types of false positive results:• Technical false-positive interaction, the reporter gene is activated without an

interaction between bait and prey.• Biological false positive, is an actual two-hybrid interaction that has no biological

relevance.

• Reasons:1. the bait and the prey do indeed interact in the context of the Y2H assay, but not in

the normal in vivo context, where the two proteins are not expressed at the same time or in the same tissue or subcellular district.

2. Transcriptional activity occurs independently of any protein–protein interaction, due to aspecific activation by BD- or Ad fusion proteins, plasmid rearrangements or copy number changes that generate such auto-activators, alterations at a reporter gene that result in constitutive expression.

3. Proteins which allow yeast to overcome nutritional selection when overexpressed are also often scored as false positives.

4. A third partner can bridge the bait and the prey, that aren’t able to interact directly.

Validation methods

Reverse Two-Hybrid Systems

• the yeast two-hybrid system has been adapted to screen for inhibitors of protein–protein interactions. The selection mechanism is reversed, such that a defined interaction between a bait and its cognate prey leads to activation of a toxic reporter gene, which kills the yeast cell when grown on a special selective medium.

• Addition of an agent which inhibits the protein interaction (e.g., a competing protein or protein domain, a peptide or a small molecule) prevents expression of the toxic gene and restores yeast growth on the selective medium.

• Also this system is capable of selecting mutations that interfere with a protein–protein interaction.

• When proteins X and Y interact the expression of the reporter gene promotes cell death. When any variation in partners occurs (i.e. a mutation) that inhibits the interaction, the transcription factor is not reconstituted, toxic gene is not expressed and cells can grow

Reverse Two-Hybrid Systems

• Reverse two-hybrid systems can be used to Identify proteins able to disrupt a specific interaction Screen randomly generated mutant proteins that are unable to

bind to their molecular partner Screen cDNA libraries in order to identify proteins that

regulate a specific protein–protein interaction Screen for drugs that abolish specific protein–protein

interactions.

Application of reverse Two-Hybrid Systems

Three-Hybrid Systems

• The three-hybrid assay is a genetic method that is based on the yeast two-hybrid system, in which three chimeric molecules are expressed in yeast cells, and the interaction between these molecules induces the activation of a reporter gene.

• Three-hybrid systems rely on the intervention of a third component for PPI detection.

• There are two types of three-hybrid assays:1. The RNA-based Y3H system 2. The Ligand- based Y3H system

The RNA-based Y3H system

• RNA–protein interactions play an essential role in the maturation and regulation of RNAs within eukaryotic organisms. The three-hybrid system provides a powerful means to study RNA–protein interactions.

• The RNA-based three-hybrid system is characterized by two-hybrid protein molecules and a hybrid RNA molecule. The first hybrid protein consists of a DNA binding domain (for example, LexA-BD or Gal4-BD) that is present in multiple copies upstream from the reporter gene and is linked to an RNA binding domain(MS2-coat protein or Hiv-1 RevM10) that binds to a short stem-loop RNA sequence (RNA X). The second hybrid protein is composed of an RNA binding protein (protein Y) and Gal4-AD. The two fusion proteins may be linked by a hybrid RNA molecule. The hybrid RNA is composed of the binding site for the first RNA binding protein (MS2or RRE) and the RNA sequence of interest (RNA X). The interaction between RNA X and protein Y promotes functional transactivation of the reporter gene

The ligand-based Y3H system

• Organic small molecules participate in many biological processes, such as metabolic pathways, signal transduction mechanisms, and developmental programs, in which they often play important, sometimes decisive roles.

• the Y3H comprises three-hybrid components: (1) the hook, (2) the bait, and (3) the fish

1. The hook is a hybrid protein comprising two functional domains, the DNA-binding domain (DBD) and the ligand-binding domain. various proteins have been fused to create the hook, including the glucocorticoid receptor (GR), FK506-binding protein 12 (FKBP12), and dihydrofolate reductase (DHFR). The common feature of these proteins is high-affinity binding of their ligands, dexamethasone (Dex), FK506, and Mtx, respectively.

2. The bait is a hybrid ligand in which an “anchor” moiety is covalently connected via a linker to a compound of interest. Two kind of linkers are: polyethylene glycol (PEG) linker and aliphatic linker.

3. The fish, a transcriptional activation domain fused to a protein from a cDNA library

The ligand-based Y3H system

Yeast One-Hybrid System• The yeast one-hybrid screening system is a versatile and efficient method to identify

transcription factors (or other DNA-binding proteins) that can bind and regulate a given gene-of-interest.

• In this method, multiple copies of the DNA sequence of interest (DNA bait e.g., cis-regulatory DNA elements or gene promoters) are cloned in a reporter plasmid upstream of a reporter gene (e.g., HIS3 or LacZ ).This reporter plasmid is stably integrated into the yeast genome, generating a “DNA bait strain” that can be selected and isolated. After integration, baits are examined for self-activation. The DNA bait strain is transformed with a library containing cDNAs that encode proteins (protein Y e.g., TFs) fused with a strong transcriptional activation domain, usually the GAL4 activation domain (GAL4-AD). The reporter gene is expressed when the fusion protein interacts with the DNA bait, and thus yeast colonies can grow and be selected. The cDNAs are then isolated and sequenced to identify the encoded protein that binds the DNA bait.

Thank you