Target discovery using the yeast two-hybrid system

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Target discovery using the yeast two-hybrid system Robert Hollingsworth and Julia H.White A major goal of molecular biology is to understand protein interactions and how interaction networks form the functional circuitry of cells.This goal is also relevant to drug development, as molecular target discovery and validation require an understanding of the function and disease relevance of proteins. One of the best approaches available to achieve this goal is the yeast two-hybrid system and its variants.This powerful tool can be used not only to identify and characterize individual drug targets, but also to dissect whole pathways and map protein interactions on a proteomic scale.

Robert Hollingsworth Julia H.White Pathway Discovery Genomics and Proteomic Sciences GlaxoSmithKline e-mails: [email protected] and [email protected]

▼ The arrival of the genomic era has presented exciting new opportunities to understand human biology and discover new medicines by analyzing the function of every human gene and studying its potential as a drug target. Genome-scale sequence analysis has identified comprehensive lists of gene families known to be tractable drug targets. DNA microarrays, or ‘gene chips’, have enabled us to study the expression of many genes simultaneously and thus elucidate expression networks, transcriptional responses to drug or ligand treatment and differential patterns of expression between diseased and normal cells. Ultimately, however, the functions of gene products are of most direct relevance to drug discovery. These functions invariably depend on interactions with other gene products and thus tools that can rapidly dissect protein interactions are of enormous value. The race to mine the human genome for lucrative new disease targets will probably be won by those that employ the swiftest tools for protein interaction mapping and functional validation. We review here the use of the yeast two-hybrid system and its variants for protein interaction analysis and its relevance in the pharmaceutical setting.

A powerful but limited tool The yeast two-hybrid assay permits analysis of protein associations in an intracellular setting

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and is an easy way to screen large numbers of potential interactions. The assay involves the expression of the protein of interest fused to the DNA binding domain (BD) of a transcription factor (the ‘bait’), and a single target protein or library of such proteins fused to a transcriptional activation (AD) domain (the ‘prey’) (Figure 1a). Association of the bait with a prey protein reconstitutes a functional transcription factor and is detected via the expression of reporter genes controlled by a promoter bearing the cognate DNA BD site. Two main versions of the yeast two-hybrid system exist, differing primarily in the transcription factor components and reporter genes used. The first of these, the GAL4 system, was originally described by Fields and Song [1] and uses the BD and AD of the yeast GAL4 transcription factor, with lacZ and HIS3 as the reporter genes. Expression of the HIS3 gene product permits selection of cells harboring interactors by growth on media lacking histidine. Addition of 3-aminotriazole, a competitive inhibitor of the His3 protein, to the media increases the stringency of the screen. The second version, the ‘interaction trap’ developed by Brent and colleagues [2], instead uses the bacterial BD LexA and the herpes simplex virus VP16 or E. coli B42 AD. These non-yeast transcription factor components were chosen to reduce the number of false positives that might otherwise arise. lacZ and LEU2 genes are used as reporters, and an inducible promoter permits prey cDNAs to be expressed only during the interaction test, thus minimizing growth inhibition by toxic prey proteins. As with any technology, the yeast two-hybrid assay has advantages and disadvantages compared to other approaches (Box 1). On the one hand, the assay tests protein interactions in living cells, does not require isolated protein (only the gene) and is fairly easy to perform. It can also be scaled up and automated

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Box 1. Advantages and limitations of the yeast two-hybrid system Advantages • Cheap, simple and versatile • In vivo protein interaction analysis • Capable of detecting weak or even transient interactions • Can be used to identify novel interactors by cDNA library screening • Several variations allow multiple applications • Can be scaled up through automation for genomic-scale protein interaction mapping

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Limitations • Yeast cell environment may not fully mimic mammalian cells (e.g. post-translational modifications may not be replicated in yeast) • Interactions are assayed in the yeast nucleus rather than the correct cellular compartment • Membrane-bound proteins and transcription are often not suitable (since Y2H forces proteins into the nucleoplasm and relies on transcriptional activation as a read-out). • False positives and false negatives can occur

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Figure 1. The basic yeast two-hybrid system. Bait proteins fused to a DNA binding domain (BD) are tested for interaction with either a single prey protein fused to a transcriptional activation domain (AD) or to a prey library. Bait and prey fusions are either co-transfected into the same yeast cells or by mating MATa bait-containing cells with prey-containing MATα cells. Interaction of the bait and the prey reconstitutes transcription factor activity, which then activates expression of reporter genes. Usually two reporter genes are used, one that permits a colorimetric readout (lacZ), and one that permits growth selection (LEU2 or HIS3).

for high-throughput screening. Because of the signal amplification afforded by the use of enzymes as reporters, the system is also very sensitive and can detect weak protein– protein interactions. On the other hand, this is obviously an artificial system in which the proteins under study are chimeric, being fused to the two transcription factor domains, and are localized to the nucleus of yeast cells. Because some proteins have intrinsic transcriptional activation activity or are inherently sticky (‘promiscuous interactors’), false positives are common, and mechanisms for eliminating or obviating these are important. Thus, as with most scientific tools, it is imperative that results derived using the yeast two-hybrid technique are confirmed by separate methods. Despite its limitations, the yeast two-hybrid assay is one of the most fruitful tools for discovering and dissecting the function of proteins. It is estimated that over half of the protein interaction discoveries reported in the literature

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originate from yeast two-hybrid experimentation [3]. Likewise, pharmaceutical target discovery and validation has benefited from this versatile assay. For example, we have described the discovery that the GABAB-R1 G-protein-coupled receptor functions as a heterodimer in association with a previously unidentified close relative, GABAB-R2 (Figure 2) [4]. Association of GABAB-R2 with GABAB-R1 is necessary for full pharmacological function and proper localization of the receptor. The discovery of receptor heterodimerization was not only instrumental in developing this pharmaceutical target, but also helped to identify dimerization as an important aspect of G-protein-coupled receptor biology. Results from yeast two-hybrid screening have also advanced our understanding of the function of the AMPA ion channel [5]. This channel mediates most neuronal fast excitatory synaptic transmission and is a target for various psychiatric conditions. Interactors that have been identified are implicated in the regulation of post-translational modification, subcellular localization, trafficking and anchoring and thus provide useful information on the function of this target. Another area where yeast two-hybrid technologies have proved invaluable for pharmaceutical target discovery and validation is in the identification and characterization of nuclear receptor cofactors. The ability of nuclear receptors to regulate the expression of specific genes is mediated by their association with protein cofactors, and the variable composition of these complexes elicits different effects.

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RESEARCH FOCUS Identification of co-factors and elucidation of their action by yeast twohybrid analysis has been instrumental in the design of specific drugs aimed at promoting beneficial effects while reducing adverse effects [6]. In addition to dissecting the function of drug targets, yeast two-hybrid screening can reveal links between disease genes and tractable, or ‘druggable’, protein pathways. A study of the schizophrenia susceptibility gene, G72, by Chumakov and colleagues [7] illustrates this application. G72 is expressed in the brain of primates only, but its function and in particular its role in schizophrenia were previously unknown. Yeast two-hybrid screening using a G72 bait identified the enzyme D-amino acid oxidase (DAAO) as a tightly associated protein. DAAO is expressed in the brain and is involved in the activation of the NMDA glutamate receptor. Hence, yeast two-hybrid studies have linked a disease susceptibility gene to a pathway that is tractable to drug development.

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Figure 2. Elucidation of the heterodimeric GABAB receptor complex. Functional GABAB receptors were found to consist of a heterodimer between two related G-protein coupled receptors (GPCRs), GABAB -R1 and GABAB-R2, by yeast two-hybrid analysis.A yeast two-hybrid screen using the GABAB-R1 C-terminus, including its pronounced coiled coil domain, as bait against a Human brain cDNA library revealed the C terminus of the highly related GABAB-R2 receptor as a pronounced interactor. From this original observation, the heterodimeric nature of the functional receptor was deduced and subsequently functionally validated in mammalian cells. The same yeast two-hybrid screen identified other interactors around the GABAB heterodimer including the transcription factors CREB2 and ATFx [39] and 14-3-3 [40].

High throughput yeast two-hybrid systems The yeast two-hybrid system has been modified for largescale protein interaction analysis, which can enable both the rapid study of many different proteins and the mapping of complex interaction networks [8–10]. Towards this end, several drawbacks of the conventional technique have been overcome. The most significant limitation of the conventional approach is the requirement for co-transformation of the bait and prey library into yeast for each experiment, which is laborious and quickly depletes precious prey library stocks. As an alternative, yeast mating can be used to combine cells harboring the bait with cells containing the prey library [11]. In this approach, termed interaction mating, bait plasmid and a library of prey plasmids are transformed independently into opposite mating types, and then brought together into the same diploid by mating. In addition to obviating the need for retransformation of the prey library for every experiment, this method is simple, efficient and more easily automated than the conventional system. To further facilitate automation, we have developed an all-liquid interaction-mating format that can be performed in microtiter plates using highprecision liquid-handling robots [12].

For many proteins, a thorough understanding of their function and how this relates to particular pathologies comes only from dissecting their role in biochemical complexes or metabolic networks. High-throughput yeast two-hybrid systems have become powerful tools for this purpose. As an example of the use of high-throughput interaction mating for dissecting an important regulatory pathway, we have worked out the protein interaction map for numerous proteins involved in the regulation of DNA metabolism [12], a portion of which is diagrammed in Figure 3. Among other things, this study revealed that the human Cdc7 protein kinase functions in several aspects of DNA metabolism, including the initiation of DNA replication and of DNA repair, two pathways relevant to oncology. In addition, comparison to interaction maps produced for S. cerevisiae [9,10] revealed that this regulatory network has been conserved but modified throughout eukaryotic evolution (Figure 3). Interaction-mating-based yeast two-hybrid systems have also been used to study protein interactions on a proteomic scale. In 2000 two groups published the first large-scale interaction maps for an eukaryotic organism using yeast two-hybrid interaction mating, and it is fitting that the

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RESEARCH FOCUS become one of the most widely used approaches for these. In this case, a UMP-CMP Orc6 Cdc47 Centrin fusion between an AD and either an kinase individual candidate DNA-binding protein or a library of such proteins is Cdc45 RAP1 Mcm3 Cdc45 Mcm5 XPAC expressed in yeast harboring a reporter linked to a target DNA sequence, for Spindlin example a transcription factor response Mcm2 NHP2L1 Cdc7 element. If the prey binds to the target Orc2 Drf1 DNA, the AD activates expression of Tel2 Cdc7 the reporter. Several response-elementUMP Cdc53 specific transcription factors have been MSH3 Dbf4 synthase discovered in this manner. Analyzing mutations of either the DNA binding Dbf4 Cdc5 protein or the cognate DNA sequence GANP Mcm3 has also facilitated mapping the key Drug Discovery Today: TARGETS contacts and recognition sequences Figure 3. Interaction network mapping by yeast two-hybrid.Yeast two-hybrid interaction mapping for these interactions. By switching to of yeast and human proteins involved in cell cycle control was conducted [9,10,12]. Cdc7, Dbf4, using a known BD fused to a prey liand Cdc45 from each organism were used as baits (red), and similar interaction networks were revealed. Several interactors known to be involved in either DNA replication (green) or DNA brary, the one-hybrid assay has also repair (blue) implicate the bait proteins in these processes.The human Cdc7 regulators, Dbf4 and been enlisted to find and characterize Drf1, had not been discovered previously. Comparison of interaction maps from different protein domains capable of transcriporganisms permits extrapolation of knowledge from one to the other. tional activation. In the pharmaceutical setting, this has been particularly useful for studying regulators of transcription factor tarproteome studied was derived from the yeast Saccharomyces gets, including the nuclear receptors (for a recent example, itself [9,10]. More recently, it has been used for the rapid see [20]). A common cell-based compound screen for nudissection of the interactions of >4,000 C. elegans proteins clear receptor ligands consists of this configuration of the [13] and of >10,000 Drosophila proteins [14]. Such highly one-hybrid assay established in mammalian cells [21]. efficient, high-throughput approaches now make largescale protein interaction mapping a practical proposition Three-hybrid for any organism, including humans. Several groups have developed systems that depend on the presence of a third component: a bridging protein [22], a Variations on the theme modifying enzyme [23], RNA [24] or a small-molecularThe versatility of the yeast two-hybrid concept was quickly weight compound [25]. Although each of these methods realized, and several related assay systems have been develhas proved useful, the latter scenario has particular applioped to answer different questions. In addition to the incation to drug discovery in that it can be used to identify troduction of two-bait systems to facilitate the discriminathe target(s) of an orphan drug. In the yeast three-hybrid tion of interactions with two proteins [15,16], several technique (Figure 4b), a chemical compound whose target alternative formats permit the study of protein–DNA interis unknown is coupled to a small molecule that binds to a actions, the analysis of bridging proteins or compounds, known BD protein. In other words, the chimeric compound the incorporation of modifying enzymes, and the selection becomes the ‘third hybrid’, and its interaction with the BD of entities that disrupt protein interactions. Several excelprotein effectively converts the assay to an in vivo affinity lent reviews have been written about these variations trap for prey protein that binds to the compound bait. In [17–19], and we will describe here the systems most relevant the initial description of the yeast three-hybrid system, to pharmaceutical research. glucocorticoid receptor coupled with a dexamethasoneFK506 conjugate was used to capture FK506 binding proOne-hybrid teins [25]. However, despite its utility and advantages over Understanding both protein–DNA interactions and the conventional affinity capture techniques, the yeast threefunctions of transcription factors are important aspects of hybrid system has been seldom used for drug development. pharmaceutical target discovery, and simplified, one-hyThe reasons for this are varied, and include the paucity of brid versions of the two-hybrid concept (Figure 4a) have Homo sapiens

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Figure 4. Variations of the yeast two-hybrid system. Several versatile modifications to the basic two-hybrid system have been developed. (a) In the onehybrid system, proteins capable of selectively binding to a specific DNA sequence can be identified by screening a library of random cDNA-AD. Additionally, the one-hybrid system can be used to study the effects of mutations, ligands, and regulatory proteins on the function of a particular transcriptional activation. (b) Compound-based yeast three-hybrid can discover unknown target proteins of a compound.The glucocorticoid (GR) ligand binding domain is used as an anchor to tether a chimera the compound and dexamethasone upstream of the reporter genes.A prey library can then be screened for those that bind specifically to the target compound. (c) The reverse yeast two-hybrid system, which employs counter-selectable reporter genes, allows direct selection of entities that disrupt a particular protein interaction. If a particular bait and prey interact, the counter-selectable reporter is expressed and the yeast cells are unable to grow or are killed. For example, expression of URA3 in the presence of the drug 5-FOA produces a toxin, thus killing the yeast cells. In the presence of an entity that disrupts the interaction, such as a small molecule, the counter-selectable reporter is no longer expressed and the yeast are then viable. (d) The split-ubiquitin membrane yeast two-hybrid system provides analysis of protein interactions at the cell membrane. Bait proteins are fused with the C-terminus of ubiquitin (Cub) and a transcription factor. Potential interacting proteins are then coupled to the N-terminus of ubiquitin (Nub). If the bait and prey proteins interact at the membrane, then ubiquitin is reconstituted, triggering release of the transcription factor to activate the reporter genes.

orphan compounds, the need for yeast membrane permeability, and the difficulty of maintaining compound activity when conjugated.

Reverse two-hybrid Another modification of the yeast two-hybrid system, the reverse two-hybrid system (Figure 3c), has been developed to identify mutations, small molecules or peptides that disrupt protein–protein interactions [26,27]. Reverse twohybrid systems employ reporter gene products for which a

counter-selection is available to give cytostatic or cytotoxic responses in the yeast cell. Generally, one of two counterselection genes has been used for the reverse yeast twohybrid system: the URA3 gene in the presence of 5 FOA (5′fluoro-orotic acid), or the CYH2 (cycloheximide) sensitivity gene. Growth on media containing 5 FOA or cycloheximide kills cells harboring a two-hybrid interaction, whereas cells lacking interacting proteins or in which the interaction is disrupted continue to grow and form colonies. In the pharmaceutical industry, reverse two-hybrid screening has been

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RESEARCH FOCUS used to identify small molecules that specifically disrupt protein interactions. Similar to the three-hybrid variation, application of the reverse two-hybrid assay has not been widely reported beyond the initial proof-of-concept reports using known protein interaction disrupters [28]. In this case, the primary reason is that targeting protein–protein interactions with small molecules has been difficult, although significant progress has been made recently (see [29] for review). As one example, Young, et al. [30] used a CYH2 reverse two-hybrid assay as a high-throughput screen to identify compounds that interfere with the interaction between the human α 1B and β 3 subunits of the N-type calcium channel. This channel is involved in neurotransmitter release in the central and peripheral nervous systems, and a small molecule capable of blocking subunit interaction would effectively inhibit channel activity. A diverse chemical library of over 156,000 compounds was screened and lead compounds were found to possess channel-inhibiting activity in primary neurons.

Non-nuclear two-hybrid Another major limitation to the conventional yeast twohybrid system is that it is unable to accommodate protein interactions involving transmembrane proteins, other than by screening defined intracellular domains. Transmembrane proteins make up ~50% of the current known drug targets, and so this represents a serious shortcoming from the pharmaceutical perspective. Indeed, genome-wide yeast two-hybrid screens have shown that the coverage of transmembrane domain proteins is poor [9,10]. Recently, several yeast two-hybrid systems have been developed for the identification of proteins associated with integral membrane and cytoplasmic proteins. These include the reverse Ras recruitment system, the G-protein fusion approach, and the split-ubiquitin membrane yeast two-hybrid system [18,31]. Although the first two technologies have been shown to be capable of detecting protein interactions at the membrane, neither is suitable for screening a library of candidate interactors. On the other hand, the split ubiquitin membrane yeast two-hybrid system (Figure 3d) has been successfully adapted for prey library screening and has very recently been shown to recover novel interactors of the ErbB3 protein kinase [32]. If this technique proves as versatile the conventional yeast two-hybrid system, it represents a significant step forward for dissecting membrane protein biology.

Beyond yeast Although yeast-based assays have the distinct advantages of efficient transformation and rapid cellular growth, the ability to study protein interactions in other cell types,

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particularly mammalian cells, is important for pharmaceutical research. Mammalian cell assays may be used to validate results derived from yeast two-hybrid assays, and provide the opportunity to test interactions that may require post-translational modifications or cofactors not available in yeast cells. Several approaches for doing this have been developed. The standard mammalian two-hybrid assay uses the same principles as its yeast counterpart, but is adapted for mammalian cell protein expression, transcriptional activation and reporter gene detection [33]. The inefficiency of transformation, however, has prevented screening of libraries of candidate interactors in mammalian cells, and has restricted the use of mammalian two-hybrid assays to hypothesis-driven experiments. Alternative systems depend on fusing test proteins to two proteins that themselves reconstitute an reporter protein, thus eliminating the need for a secondary reporter event. These include β-galactosidase complementation and other forms of protein fragment complementation assays [34,35] and resonance energy transfer techniques (FRET and BRET) [36]. Because there is no need for a separate reporter system, these techniques can be performed in many different cell types and are not restricted to particular compartments within the cells.

Future prospects The ease, versatility, and productivity of the yeast two-hybrid system and all its various derivations will continue to have an increasing impact on biological research and pharmaceutical target discovery. In particular, its applications in linking disease pathways to tractable targets, elucidating the function of targets, and refining strategies to modulate protein function will accelerate pharmaceutical research and development. Protein interaction studies will provide predictors of toxicity and may be used to develop biomarkers as fingerprints for particular disorders or cellular responses. New two-hybrid approaches will continue to come into use to address questions not easily handled by the current formats. One of the key areas for innovation is further development of two-hybrid methods suitable for transmembrane proteins, which constitute a major portion of current pharmaceutical targets. Systems that employ the reporter as the fusion partner domains, such as the β-galactosidase complementation system, will also bring increasing utility as a result of their function in different organisms and at different subcellular locations. In particular, these systems will be useful to confirm and further explore the interactions between two particular proteins. The utilization of high-throughput versions of the yeast two-hybrid system has been perhaps the most exciting

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RESEARCH FOCUS recent step in its evolution, and these versions will serve as key tools for the fast determination of the functions of the plethora of new proteins being identified by genome sequencing projects. Like other genomic technologies, highthroughput yeast two-hybrid systems are permitting faster dissection and a more holistic comprehension of cellular function and malfunction [37,38]. The production of comprehensive protein interaction maps for entire organisms is now possible [9,10,13,14]. In addition, the comparison of protein interaction maps from different organisms will improve our ability to extrapolate knowledge derived from one species to another, for example from model organisms to humans. Integrating such data with results derived from other genomic technologies enables synergistic knowledge gains. Clearly this information will greatly improve our understanding of the pathways underlying disease and will reveal new targets for pharmaceutical exploitation, and in this way the yeast two-hybrid will help translate the fruits of the genomic revolution into important new medicines.

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