Daniel Auerbach

The post-genomic era of interactive proteomics: Facts and perspectives

The availability of completed genome sequences of several eukaryotic and prokaryotic species has shifted the focus towards the identification and characterization of all gene products that are expressed in a given organism. In order to cope with the huge amounts of data that have been provided by large‐scale sequencing projects, high‐throughout methodologies also need to be applied in the emerging field of proteomics. In this review, we discuss methods that have been recently developed in order to characterize protein interactions and their functional relevance on a large scale. We then focus on those methodologies that are suitable for the identification and characterization of protein‐protein interactions, namely the yeast two‐hybrid system and related methods. Several recent studies have demonstrated the power of automated approaches involving the yeast two‐hybrid system in building so‐called “interaction networks”, which hold the promise of identifying the entirety of all interactions that take place between proteins expressed in a certain cell type or organism. We compare the yeast two‐hybrid system with several other screening methods that have been developed to investigate interactions between proteins that are not amenable to conventional yeast two‐hybrid screenings, such as transcriptional activators and integral membrane proteins. The eventual adaptation of such methods to a high‐throughput format and their use in combination with automated yeast two‐hybrid screenings will help in elucidating protein‐protein interactions on a scale that would have been unthinkable just a few years ago.

Yeast-based functional genomics and proteomics technologies: the first 15 years and beyond

Yeast-based functional genomics and proteomics technologies developed over the past decade have contributed greatly to our understanding of bacterial, yeast, fly, worm, and human gene functions. In this review, we highlight some of these yeast-based functional genomic and proteomic technologies that are advancing the utility of yeast as a model organism in molecular biology and speculate on their future uses. Such technologies include use of the yeast deletion strain collection, large-scale determination of protein localization in vivo, synthetic genetic array analysis, variations of the yeast two-hybrid system, protein microarrays, and tandem affinity purification (TAP)-tagging approaches. The integration of these advances with established technologies is invaluable in the drive toward a comprehensive understanding of protein structure and function in the cellular milieu.

Yeast split-ubiquitin-based cytosolic screening system to detect interactions between transcriptionally active proteins.

Interactions between proteins are central to most biological processes; consequently, understanding the latter requires identification of all possible protein interactions within a cell. To extend the range of existing assays for the detection of protein interactions, we present a novel genetic screening assay, the cytosolic yeast two-hybrid system (cytoY2H), which is based on the split-ubiquitin technique and detects protein-protein interactions in the cytoplasm. We show that the assay can be applied to a wide range of proteins that are difficult to study in the classical yeast two-hybrid (Y2H) system, including transcription factors such as p53 and members of the NF-kappaB complex. Furthermore, we applied the cytoY2H system to cDNA library screening and identified several new interaction partners of Uri1p, an uncharacterized yeast protein. The cytoY2H system extends existing methods for the detection of protein interactions by providing a convenient solution for screening a wide range of transcriptionally active proteins.

Analysis of Membrane Protein Complexes Using the Split-Ubiquitin Membrane Yeast Two-Hybrid System

Recent research has begun to elucidate the global network of cytosolic and membrane protein interactions. The resulting interactome map facilitates numerous biological studies, including those for cell signalling, protein trafficking and protein regulation. Due to the hydrophobic nature of membrane proteins such as tyrosine kinases, G-protein coupled receptors, membrane bound phosphatases and transporters it is notoriously difficult to study their relationship to signaling molecules, the cytoskeleton, or any other interacting partners. Although conventional yeast-two hybrid is a simple and robust technique that is effective in the identification of specific protein-protein interactions, it is limited in its use for membrane proteins. However, the split-ubiquitin membrane based yeast two-hybrid assay (MYTH) has been described as a tool that allows for the identification of membrane protein interactions. In the MYTH system, ubiquitin has been split into two halves, each of which is fused to a protein, at least one of which is membrane bound. Upon interaction of these two proteins, the two halves of ubiquitin are reconstituted and a transcription factor that is fused to the membrane protein is released. The transcription factor then enters the nucleus and activates transcription of reporter genes. Currently, large-scale MYTH screens using cDNA or gDNA libraries are performed to identify and map the binding partners of various membrane proteins. Thus, the MYTH system is proving to be a powerful tool for the elucidation of specific protein-protein interactions, contributing greatly to the mapping of the membrane protein interactome.

Drug Discovery Using Yeast as a Model System: A Functional Genomic and Proteomic View

Drug discovery is a complex process that includes the identification of biological targets as well as the identification of leads that aim at altering or inhibiting the function of a particular target. The budding yeast Saccharomyces cerevisiae has long been recognized as a valuable model organism for studies of eukaryotic cells since many of the basic cellular processes between yeast and humans are highly conserved. In this review, we highlight emerging yeast-based functional genomic and proteomic technologies that are advancing the utility of yeast as a model organism in the drug-discovery process. These approaches include the utilization of yeast deletion strain collection, synthetic genetic array combined with chemical genomics, variations of the yeast two-hybrid system, yeast biosensor assay, and protein microarrays. Although still at an early stage, these technologies show promise as novel and useful methods for development of target-specific therapeutic approaches.

Yeast Genetic Methods for the Detection of Membrane Protein Interactions

Due to the pivotal role of membrane proteins in many cellular processes, their direct link to human disease and their often extracellular accessibility towards drugs, an understanding of membrane protein function is desirable. However, the hydrophobic nature of membrane proteins often results in insoluble proteins which makes protein isolation difficult and therefore hinders the determination of protein complex composition and protein function. Recently, several yeast genetic techniques have made the characterisation of interactions among membrane proteins more feasible. Techniques such as the guanine-nucleotide binding protein fusion assay, the reverse Ras recruitment system and the split-ubiquitin system have been fruitful in monitoring known protein interactions and uncovering novel interactions. Since many disease states have altered membrane protein function, one can use these systems to recreate interactions involving disease causing membrane proteins. Once established, screens for small molecules, peptides and/or single chain antibodies which disrupt such interactions can provide insight into the biology of the interaction and thus help guide therapeutical research. In this review, we speculate on the feasibility of using inhibitors of protein interactions as drugs and the adaptation of these techniques to select for inhibitors of defined protein interactions.

Systematic protein-protein interaction mapping for clinically relevant human GPCRs

G‐protein‐coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR‐mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two‐hybrid (MYTH) approach and identified interacting partners for 48 selected full‐length human ligand‐unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5‐HT4d, and adenosine ADORA2A receptors. Our data represent the first large‐scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.

GENETIC APPROACHES TO THE IDENTIFICATION OF INTERACTIONS BETWEEN MEMBRANE PROTEINS IN YEAST

ABSTRACT The recent sequencing of entire eukaryotic genomes has renewed the interest in identifying and characterizing all gene products that are expressed in a given organism. The characterization of unknown gene products is facilitated by the knowledge of its binding partners. Thus, a novel protein may be classified by identifying previously characterized proteins that interact with it. If such an approach is carried out on a large scale, it may allow the rapid characterization of the thousands of predicted open reading frames identified by recent sequencing projects. Currently, the yeast two-hybrid system is the most widely used genetic assay for the detection of protein–protein interactions. The yeast two-hybrid system has become popular because it requires little individual optimization and because, as compared to conventional biochemical methods, the identification and characterization of protein–protein interactions can be completed in a relatively short time span. In this review, we briefly discuss the yeast two-hybrid system and its application to large scale screening studies that aim at deciphering all protein–protein interactions taking place in a given cell type or organism. We then focus on a class of proteins that is unsuitable for conventional yeast two-hybrid systems, namely integral membrane proteins and membrane-associated proteins, and describe several novel genetic systems that combine the advantages of the yeast two-hybrid system with the potential to identify interaction partners of membrane-associated proteins in their natural setting.

Identification of PDE6D as a molecular target of anecortave acetate via a methotrexate-anchored yeast three-hybrid screen.

Glaucoma and age-related macular degeneration are ocular diseases targeted clinically by anecortave acetate (AA). AA and its deacetylated metabolite, anecortave desacetate (AdesA), are intraocular pressure (IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid activity but with an unknown mechanism of action. We used a methotrexate-anchored yeast three-hybrid (Y3H) technology to search for binding targets for AA in human trabecular meshwork (TM) cells, the target cell type that controls IOP, a major risk factor in glaucoma. Y3H hits were filtered by competitive Y3H screens and coimmunoprecipitation experiments and verified by surface plasmon resonance analysis to yield a single target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding protein with additional function outside the PDE6 phototransduction system. Overexpression of PDE6D in mouse eyes caused elevated IOP, and this elevation was reversed by topical ocular application of either AA or AdesA. The identification of PDE6D as the molecular binding partner of AA provides insight into the role of this drug candidate in treating glaucoma.

Proteomic approaches for generating comprehensive protein interaction maps

Abstract The availability of complete genome sequences of numerous model organisms has initiated the development of new approaches in biological research to complement conventional biochemistry and genetics. In this context, high-throughput methods for detecting protein interactions, such as mass spectrometry and yeast two-hybrid assays, have produced vast amounts of data that can be exploited to infer protein function and regulation. In this review, we explore different genome-wide protein interaction studies and comment on their extrapolation towards understanding protein functions. It is likely that improvements of these approaches, together with more sophisticated databases and the invention of novel technologies, will help to decipher the complex interactions among proteins and to integrate interacting proteins into existing and novel cellular pathways.