Area 1: Protein Networks Regulating Membrane Transport and Cell Signaling

Protein-protein interactions play a crucial role in every living organism and information about protein(s) associated with a specific protein of interest (so called “interactors”) largely contributes to our understanding of cellular processes at the molecular level. A wide range of fundamental biological processes such as cell signaling, transport of membrane-impermeable molecules, cell-cell communication, and cell adhesion are mediated by membrane proteins. Due to their pivotal role in many cellular processes, their direct link to human diseases and their extracellular accessibility to drugs, the identification of proteins associated with integral membrane proteins is desirable. As such, there is a strong demand from both academic researchers and biotech/pharma companies to gain further insight into pathways and interactions involving integral membrane proteins. However, due to their complex chemical properties, membrane proteins are very hard to manipulate, making the study of their corresponding interactors even more challenging.

Previously, the Stagljar lab developed the Membrane Yeast Two-Hybrid (MYTH) system, a powerful tool for identifying the interactors of membrane and membrane-associated proteins in an in vivo setting, using the model organism Saccharomyces cerevisiae as a host. An overview of MYTH is shown in Figure 1. The system is based on the ‘split-ubiquitin’ principle, wherein the ubiquitin protein can be split into two stable moieties, an N-terminal fragment called Nub (shown in green) and a C-terminal fragment called Cub (shown in cyan). The wild-type Nub (referred to as NubI) is capable of spontaneous reassociation with Cub to form a full-length ‘pseudo-ubiquitin’ molecule (Figure 1A). Mutation of isoleucine 13 to glycine in the Nub moiety (producing a fragment called NubG) prevents this spontaneous association (Figure 1B), and allows the fragments to be adapted for use as a ‘sensor’ of protein-protein interactions.  A Bait protein of interest (Figure 1C, shown in red) is fused to a Cub moiety linked to a Transcription Factor (TF, shown in dark blue in Figure 1C), while a Prey protein (Figure 1C, shown in purple) is fused to the NubG fragment. If the Bait and Prey do not interact, the NubG and Cub moieties remain separate (Figure 1C, Part I). However, if an interaction between the Bait and Prey occurs, the ubiquitin moieties are brought into close proximity, allowing for pseudo-ubiquitin formation (Figure 1C, Part II). This pseudo-ubiquitin is recognized by cytosolic deubiquitinating enzymes (DUBs, represented as scissors) which cleave off the transcription factor, allowing it to enter the nucleus and activate a reporter system consisting of the HIS3, ADE2 and lacZ genes (Figure 1C, Part III). The use of appropriate selective media allows for sensitive detection of cells expressing interacting Bait-Prey pairs.

There are currently two major forms of MYTH; tMYTH, where tagged baits are expressed ectopically from a plasmid, and iMYTH, where baits are endogenously tagged in the yeast chromosome. iMYTH is particularly useful as it maintains the expression of baits under the control of their natural promoters, and thus avoids problems associated with protein overexpression. However, it can only be employed when studying proteins of yeast origin. When studying proteins from a different organism, tMYTH is the method of choice.

A unique advantage of MYTH is that it detects protein interactors of full-length integral membrane proteins in a high-throughput screening format. Since its development, we and others have successfully applied this genetic method to study protein interactions among various membrane proteins from yeast, plants, fly, worm and humans.

Our current efforts are directed to identify and characterize protein interactors of all (23) yeast integral membrane ABC transporters, 100 selected pharmacologically important human G-protein coupled receptors (GPCRs) as well as all (58) human receptor tyrosine kinases (RTKs) in an effort to understand complex biological processes such as cell signaling and membrane transport at a systems level.

In particular, we are focusing on the following projects:

(i) The Interactome of Saccharomyces cerevisiae ABC Transporters

ABC Binding Cassette (ABC) proteins comprise one of the largest known protein superfamilies, utilizing the power of nucleotide binding and hydrolysis to mediate a diverse range of cellular functions. A major class of ABC proteins are the integral membrane ABC transporters, which are responsible for the movement of a wide variety of substances across cellular membranes. ABC transporters are of clinical interest because of the key roles they play in the multidrug resistance of tumour cells and pathogenic microorganisms, as well as the observation that mutations in these proteins are associated with a range of hereditary diseases in humans. Acquiring a greater understanding of these proteins and the systems in which they are involved is therefore of critical importance.

To this end, our lab has set about using the MYTH technology to map the ABC transporter interactome of the model organism Saccharomyces cerevisiae, which contains a total of 22 ABC transporters, 19 of which are suitable for screening using MYTH. To date, all 19 ABC transporters have been MYTH-tagged at their C-termini, and most have been successfully validated using fluorescence microscopy and the NubG/I control test to ensure that they are properly expressed, localized and functional in MYTH, and screened against both cDNA and genomic libraries. Interactors detected in the screens were examined using a combination of manual and computational approaches, and then tested for specificity of interaction using a control bait strain. The final resulting data was assembled into a comprehensive ABC interactome map, revealing a range of novel and interesting interactions. Functional validation of these interactions is currently underway, and promises to greatly increase our understanding of the cellular role and regulation of ABC transporters.

(ii) The interactome of the human G-protein coupled receptors (GPCRs)

The G-protein coupled receptors (GPCRs) form the largest family of integral membrane proteins with over 1,000 members, and are involved in all aspects of signal transduction. They are involved in the recognition and response to stimuli as diverse as hormones, neurotransmitters, autacoids, calcium, odorants, gustatory signals, or light. Their key roles in cell signaling have made them the target for more than 60% of all currently prescribed drugs. Some of the important diseases and medical conditions treatable using GPCR-directed therapeutic approaches include heart failure, chronic pain, obesity, neuropsychiatric disorders, and carcinomas. In order to design successful treatments for these diseases, it is essential to increase our detailed understanding of the molecular events occurring during GPCR-mediated signal transduction, and identify all of the proteins that are associated with a particular GPCR relevant for human health. However, despite the abundance of biochemical, cellular and genetic studies of human GPCRs, the hydrophobic nature of these receptors has reduced our ability to detect protein interactors of full-length GPCRs in their natural membrane environment and in a scaleable screening format.

We have been applying MYTH together with advanced bioinformatics and biochemical/molecular/cellular biology approaches to both map and investigate the biological significance of the PPIs of 100 selected human GPCRs relevant to human health and disease. We expect to discover novel proteins associated with these selected human GPCRs, and to uncover new biochemical pathways that, when defective, contribute to various human diseases. Our work will provide important insight into the disease-causing mechanisms associated with GPCRs and facilitate the discovery of promising targets for therapeutic treatment.

(iii) The interactome of the human Receptor Tyrosine Kinases (RTKs)

Receptor tyrosine kinases (RTKs) are cell surface receptors for many polypeptide growth factors, cytokines, and hormones. Of the 90 unique tyrosine kinase genes identified in the human genome, 58 encode RTK proteins (Figure 4). RTKs are central components of cell signaling networks and have been shown not only to be key regulators of normal cellular processes, but also to have a critical role in the development and progression of many types of cancer. Along this notion, several innovative drugs that target various RTKs (e.g., Herceptin, Cetuximab, Tarceva, Iressa, and Gleevec) have been approved by regulatory agencies in the last few years. These new drugs can be very potent and exert minimal adverse clinical effects in a well-defined group of patients, indicating the great therapeutic potential of RTK targeting. Nevertheless, there is a lack of in-depth understanding of RTK networks because of their complex biochemical features, enormous complexity and multiplicity. This is a major obstacle for designing improved and more targeted therapies, and, importantly, understanding the biology of receptor deregulation, leading to tumorigenesis. The application of interactive proteomics is a promising approach for improving our understanding of RTK networks, however, it requires developing sophisticated PPI technologies to probe interactomes of the full-length human RTKs.

To address this daunting task, we have recently modified the original MYTH system to make it amenable for screening of protein interactors of the full-length human RTKs. We then used it to search for previously unknown epidermal growth factor receptor (EGFR)-interacting proteins (Lissanu Deribe et al. (2009)). We identified 87 proteins that bound to the EGFR in a ligand-independent fashion and validated the complete set with bioinformatics and a subset by immunoprecipitation. Among these EGFR-interacting proteins was the cytoplasmic lysine deacetylase HDAC6. We also found that HDAC6 deacetylates α-tubulin in stimulated cells—a process known to slow transport through the secretory pathway—and identified a feedback mechanism where EGFR inactivates HDAC6 through phosphorylation, increasing α-tubulin acetylation. These results lead to a model where HDAC6 binding to EGFR allows for immediate downregulation though acetylation of microtubules after ligand stimulation (Lissanu Deribe et al. (2009)).

Our current research activities are directed to the application of MYTH and LUMIER protein interaction assays, coupled to bioinformatics, to derive a complete map of PPIs linked to all remaining human RTKs. Our systematic approach offers an unbiased systems level view that may identify novel drug targets and contribute to therapeutic research.

(iv) the Sho1p-interactome project

Sho1p is a yeast integral membrane protein that is comprised of four transmembrane domains and a cytoplasmic SH3 domain at its C-terminus (Figure 5). Sho1p is activated in the High Osmolarity Glycerol (HOG) MAPK pathway, a stress response pathway that ensures yeast cells adapt and remain viable in conditions of high osmotic stress. In addition, Sho1p has been implicated in filamentous growth and cell wall integrity pathways.

Through the use of both iMYTH and MYTH assays we identified 45 Sho1p interactors in an attempt to further understand the diverse functions of Sho1p. Many of these 45 Sho1p interactors have not been previously characterized. A subset of putative Sho1p interactors identified from the MYTH screening process was further characterized through a series of additional protein interaction assays and functional analyses. By characterizing novel Sho1p-interactors our goal is to gain a better understanding of the signal transduction capabilities of this multifunctional protein and the mechanisms that dictate its signaling specificity.

Selected Publications

Petschnigg J., Snider J., and Stagljar I. (2011) Interactive proteomics research technologies: recent applications and advances. Curr Opin Biotechnol 22, 50-58.

Gfeller, D., Butty, F., Wierzbicka, M., Verschueren, E., Vanhee, E., Huang, H., Ernst, A., Dar, N., Stagljar, I., Serrano, L., Sidhu, S.S., Bader, G.D., and Kim, P.M. (2011) The multiple specificity landscape of modular protein domains,Mol Systems Biol7, 484-493.

Lee, M.E., Singh, K., Snider, J., Shenoy, A., Paumi, C.A., Stagljar, I., and Park, H-O. (2011) The Rho1 GTPase in budding yeast is involved in cellular response to oxidative stress, Genetics 188, 859-870.

Jin, J., Kittanakom, S., Wong, V., Reyes, B.A., Van Bockstaele, E.J., Stagljar, I., Berrettini, W., Levenson, R. (2010) Interaction of the mu-opioid receptor with GPR177 (Wntless) inhibits Wnt secretion: potential implications for opioid dependence, BMC Neurosci 11, 33-48.

Snider, J., Kittanakom, S., Damjanovic, D., Curak, J., Wong, V., Stagljar, I. (2010) Detecting interactions with membrane proteins using a membrane two-hybrid assay in yeast, Nat Protoc 5, 1281-93.

Lissanu, Y., Schmidt, M., Chandrashaker, A., Curak, J., Milutinovic, N., Buerke, L., Fetchko, M.J., Schmidt, P., Kittanakom, S., Brown, K., Jurisica, I., Blagoev, B., Zerial, M., Stagljar, I.*, and Dikic, I. * (2009) Regulation of epidermal growth factor receptor trafficking by lysine deacetylase HDAC6. Sci Signal 2, ra84. (* co-corresponding authors).

Paumi, C., Chuk, M., Snider, J., Stagljar, I.*, and Michaelis, S.* (2009) Yeast ABC transporters and their Interactors: New Technology Advances Yeast MRP (ABCC) Biology. Microbiol Mol Biol Rev 73, 577-593 (* co-corresponding authors).

Paumi, C.M., Chuk, M., Chevelev, I., Stagljar, I.,* and Michaelis, M.* (2008) Negative Regulation of the Yeast ABC Transporter Ycf1p by Phosphorylation within its N-Terminal Extension, J Biol Chem 283, 27079-27088 (* co-corresponding authors).

Gisler, S.M., Kittanakom, S., Fuster, D., Radanovic, T., Wong, V., Bertic, M., Hall, R.A., Engels, K., Murer, H., Biber, J., Markovic, D., Moe, O.W., and Stagljar, I (2008) Monitoring protein- protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system, Mol Cell Proteomics 7, 1362-1377.

Paumi, C.M., Menendez, J., Arnoldo, A., Engels, K., Iyer, K., Thaminy, S., Georgiev, O., Barral, Y., Michaelis, S., and Stagljar, I. (2007) Mapping Protein-Protein Interactions for the Yeast ABC Transporter Ycf1p by Integrated Split-Ubiquitin Membrane Yeast Two-Hybrid (iMYTH) Analysis, Mol Cell 26, 15-25.