Two-hybrid screening

[[Image:Two hybrid assay.svg|thumb|300px| Overview of two-hybrid assay, checking for interactions between two proteins, called here Bait and Prey.

A. The Gal4 transcription factor gene produces a two-domain protein (BD and AD) essential for transcription of the reporter gene (LacZ).

B,C. Two fusion proteins are prepared: Gal4BD+Bait and Gal4AD+Prey. Neither of them are usually sufficient to initiate transcription (of the reporter gene) alone.

D. When both fusion proteins are produced and the Bait part of the first fusion protein interacts with the Prey part of the second, transcription of the reporter gene occurs.

]]

Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.

The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS). For two-hybrid screening, the transcription factor is split into two separate fragments, called the DNA-binding domain (DBD or often also abbreviated as BD) and activating domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for the activation of transcription. Since then, the same principle has been adapted to describe many alternative methods, including some that detect protein–DNA interactions or DNA-DNA interactions, as well as methods that use different host organisms such as Escherichia coli or mammalian cells instead of yeast.

Basic premise

The key to the two-hybrid screen is that in most eukaryotic transcription factors, the activating and binding domains are modular and can function in proximity to each other without direct binding. This means that even though the transcription factor is split into two fragments, it can still activate transcription when the two fragments are indirectly connected.

The most common screening approach is the yeast two-hybrid assay. In this approach the researcher knows where each prey is located on the used medium (agar plates). Millions of potential interactions in several organisms have been screened in the latest decade using high-throughput screening systems (often using robots) and over thousands of interactions have been detected and categorized in databases as BioGRID. This system often utilizes a genetically engineered strain of yeast in which the biosynthesis of certain nutrients (usually amino acids or nucleic acids) is lacking. When grown on media that lacks these nutrients, the yeast fail to survive. This mutant yeast strain can be made to incorporate foreign DNA in the form of plasmids. In yeast two-hybrid screening, separate bait and prey plasmids are simultaneously introduced into the mutant yeast strain or a mating strategy is used to get both plasmids in one host cell.

The hybrid expression phagemids can be electroporated into E. coli XL-1 Blue cells which after amplification and infection with VCS-M13 helper phage, will yield a stock of library phage. These phage will each contain one single-stranded member of the phagemid library. In the split-ubiquitin system, two integral membrane proteins to be studied are fused to two different ubiquitin moieties: a C-terminal ubiquitin moiety ("Cub", residues 35–76) and an N-terminal ubiquitin moiety ("Nub", residues 1–34). These fused proteins are called the bait and prey, respectively. In addition to being fused to an integral membrane protein, the Cub moiety is also fused to a transcription factor (TF) that can be cleaved off by ubiquitin specific proteases. Upon bait–prey interaction, Nub and Cub-moieties assemble, reconstituting the split-ubiquitin. The reconstituted split-ubiquitin molecule is recognized by ubiquitin specific proteases, which cleave off the transcription factor, allowing it to induce the transcription of reporter genes.

Fluorescent two-hybrid assay

Zolghadr and co-workers presented a fluorescent two-hybrid system that uses two hybrid proteins that are fused to different fluorescent proteins as well as LacI, the lac repressor. The structure of the fusion proteins looks like this: FP2-LacI-bait and FP1-prey where the bait and prey proteins interact and bring the fluorescent proteins (FP1 = GFP, FP2=mCherry) in close proximity at the binding site of the LacI protein in the host cell genome. The system can also be used to screen for inhibitors of protein–protein interactions.

Enzymatic two-hybrid systems: KISS

While the original Y2H system used a reconstituted transcription factor, other systems create enzymatic activities to detect PPIs. For instance, the KInase Substrate Sensor ("KISS"), is a mammalian two-hybrid approach has been designed to map intracellular PPIs. Here, a bait protein is fused to a kinase-containing portion of TYK2 and a prey is coupled to a gp130 cytokine receptor fragment. When bait and prey interact, TYK2 phosphorylates STAT3 docking sites on the prey chimera, which ultimately leads to activation of a reporter gene.

Two-hybrid screening by sequencing

A number of strategies have been developed in which the two-hybrid positives are identified by DNA sequencing, usually after selection using a reporter gene. A variant of this approach was recently described as Liquid Y2H-Seq.

Following the demonstration by Stanley Fields and colleagues that transcriptional activation assays could be utilized to detect protein-protein interactions and screen libraries for novel binding partners, the method's practical utility was quickly established. In 1993, Vojtek et al. performed the first successful two-hybrid screen to identify a novel interaction, discovering that the Ras GTPase physically binds to RAF kinase—a protein previously known to function genetically downstream of Ras. A subsequent milestone occurred at Johns Hopkins University, where Anton Yuryev identified two novel protein families that interact with the C-terminal domain (CTD) of RNA polymerase II. These families were characterized by highly homologous CTD-interacting domains (CIDs), providing structural evidence for the functional significance of the interactions. Since these proteins also showed homology to known splicing factors, this discovery suggested that the CTD functions to physically link gene transcription and mRNA processing in eukaryotes. Yuryev later expanded the methodology by proposing a technique to identify functional interactions through the selection of isoform-specific binding partners, demonstrated using the three isoforms of RAF kinase.

One-, three- and one-two-hybrid variants

One-hybrid

The one-hybrid variation of this technique is designed to investigate protein–DNA interactions and uses a single fusion protein in which the AD is linked directly to the binding domain. The binding domain may be constituted by a library and thus can be selected for proteins binding a desired target sequence (which is inserted in the promoter region of a reporter gene). In a positive-selection system, a binding domain that successfully binds the UAS and allows transcription is thus selected.

Three-hybrid

thumb|350px|Overview of three-hybrid assay.

RNA-protein interactions have been investigated through a three-hybrid variation of the two-hybrid technique. In this case, a hybrid RNA molecule serves to adjoin together the two protein fusion domains—which are not intended to interact with each other but rather the intermediary RNA molecule (through their RNA-binding domains). Yeast cells often do not have the same post translational modifications, have a different codon use or lack certain proteins that are important for the correct expression of the proteins. To cope with these problems several novel two-hybrid systems have been developed. Depending on the system used agar plates or specific growth medium is used to grow the cells and allow selection for interaction. The most common used method is the agar plating one where cells are plated on selective medium to see of interaction takes place. Cells that have no interaction proteins should not survive on this selective medium.

S. cerevisiae (yeast)

The yeast S. cerevisiae was the model organism used during the two-hybrid technique's inception. It is commonly known as the Y2H system. It has several characteristics that make it a robust organism to host the interaction, including the ability to form tertiary protein structures, neutral internal pH, enhanced ability to form disulfide bonds and reduced-state glutathione among other cytosolic buffer factors, to maintain a hospitable internal environment. and AH109, both produced by Clontech. Yeast strains R2HMet and BK100 have also been used.

Candida albicans

C. albicans is a yeast with a particular feature: it translates the CUG codon into serine rather than leucine. Due to this different codon usage it is difficult to use the model system S. cerevisiae as a Y2H to check for protein-protein interactions using C. albicans genes. To provide a more native environment a C. albicans two-hybrid (C2H) system was developed. With this system protein-protein interactions can be studied in C. albicans itself. A recent addition was the creation of a high-throughput system.

E. coli

Bacterial two hybrid methods (B2H or BTH) are usually carried out in E. coli and have some advantages over yeast-based systems. For instance, the higher transformation efficiency and faster rate of growth lends E. coli to the use of larger libraries (in excess of 108). Transiently transfected mammalian cells are used in this system to find protein-protein interactions.

Using a mammalian cell line to study mammalian protein-protein interactions gives the advantage of working in a more native context.

It is also possible with the mammalian two-hybrid system to study signal inputs.

Another big advantage is that results can be obtained within 48 hours after transfection.

Aplysia californica

The sea hare A californica is a model organism in neurobiology to study among others the molecular mechanisms of long-term memory. To study interactions, important in neurology, in a more native environment a two-hybrid system has been developed in A californica neurons. A GAL4 AD and BD are used in this system.

Bombyx mori

An insect two-hybrid (I2H) system was developed in a silkworm cell line from the larva or caterpillar of the domesticated silk moth, Bombyx mori (BmN4 cells). This system uses the GAL4 BD and the activation domain of mouse NF-κB P65. Both are under the control of the OpIE2 promoter.

Applications

Determination of sequences crucial for interaction

By changing specific amino acids by mutating the corresponding DNA base-pairs in the plasmids used, the importance of those amino acid residues in maintaining the interaction can be determined.

By using a selection gene with the desired target sequence included in the UAS, and randomising the relevant amino acid sequences to produce a ZFP library, cells that host a DNA-ZFP interaction with the required characteristics can be selected. Each ZFP typically recognises only 3–4 base pairs, so to prevent recognition of sites outside the UAS, the randomised ZFP is engineered into a 'scaffold' consisting of another two ZFPs of constant sequence. The UAS is thus designed to include the target sequence of the constant scaffold in addition to the sequence for which a ZFP is selected. or bioinformatics techniques. The latter test whether interacting proteins are expressed at the same time, share some common features (such as gene ontology annotations or certain network topologies), have homologous interactions in other species.

References

External links

Detail on sister technique two-hybrid system
Science Creative Quarterly's overview of the yeast two hybrid system
Gateway-Compatible Yeast One-Hybrid Screens
Video animation of the Yeast Two-Hybrid System
Yeast Two-Hybrid
BioGrid Database with protein-protein interactions