Igor Stagljar

MY RESEARCH OVERVIEW (GO TO SCIENTIFIC OVERVIEW)

MYTH and MaMTH - new technologies allow the study of major disease pathways for the first time

Unlocking the immense complexity of the cell is a major goal of modern biological science. In recent years, large-scale studies of proteins have generated complex ‘interactome’ maps, which describe how cellular proteins are connected to each other. These maps have provided an unparalleled global view of the interplay between different processes within the cell.

Our lab focuses on elucidating the interactomes and functions of a specific class of proteins – membrane proteins – that play a wide variety of roles in the cell. We examine how these proteins interact with each other as well as with proteins not bound to the cell membrane, and try to understand how impaired protein-protein interactions (PPIs) lead to numerous human diseases. Proteins involved in disease-related PPIs are potential targets for new therapeutics, and we are also involved in screening possible drugs, to see if they can work either to prevent harmful PPIs or to enhance beneficial PPIs. 

For the study of membrane proteins and to aid in drug discovery, we have developed two unique and powerful technologies: the classic Membrane Yeast Two-Hybrid (MYTH) and the newly created Mammalian Membrane Two-Hybrid (MaMTH). Before that, the study of membrane proteins and related diseases was very difficult, causing this important area of research to be neglected. The diseases we are currently studying using these technologies include lung cancer, pancreatic cancer and cystic fibrosis. 

SCIENTIFIC RESEARCH OVERVIEW

Research in our lab is focused on membrane protein-protein interactions (PPIs), with a particular interest in disease progression due to altered signalling pathways. To this end, the lab has developed two new technologies to enable research in this notoriously difficult environment.

The first was the Membrane Yeast Two-Hybrid (MYTH) system. Based on the split-ubiquitin principle, 'bait' and 'prey' proteins are fused to Cub (C-terminal ubiquitin domain) plus a transcription factor (TF) and NubG (N-terminal domain, I13G mutation), respectively. A bait and prey interaction brings the two domains of ubiquitin into close proximity, forming pseudo-ubiquitin. This is recognized by deubiquitinating enzymes, which cleave off the TF. The TF enters the nucleus and activates a reporter system, allowing detection of interacting proteins.

MYTH has been successfully used in a number of studies, including building a comprehensive map of the yeast ABC transporter interactome (Snider et al. [2013] Nature Chemical Biology 9:565).

Mammalian Membrane Two-Hybrid (MaMTH) is a powerful evolution of MYTH, developed for use in mammalian cell lines. The MaMTH approach enables quantitative measurement of dynamic protein-protein interactions (PPIs) in vivo in the natural membrane environment of human cells. The system addresses a currently challenging area of transmembrane protein analysis, which is of keen interest to the pharmaceutical market, as membrane proteins are the major class of drug targets.  We believe that our MaMTH technology will provide a means of both increasing our basic knowledge and promoting drug-discovery, thereby making significant contributions to human health care and research.

The development of MaMTH, results of MaMTH screening assys and the potential of the system as a drug screening platform were recently published in Nature Methods (Petschnigg et al. [2014] Nature Methods 11:585)

Current projects in our lab include the following:

1. Adapting MaMTH technology for use in new high-throughput PPI and drug screening formats

These new variations of MaMTH are currently being used to map the complete interactome of all human ABC transporter proteins, as well as to screen small molecule libraries for compounds targeting PPIs associated with diseases such as cancer. This work should provide powerful new tools for proteomics research, as well as valuable information to improve our understanding of fundamental cellular processes and aid in the development of novel therapeutic approaches.

2. Adapting MaMTH for identification of PPIs with one membrane and one cytosolic protein partner

This will widen the application of MaMTH and allow a greater number of disease pathways to be studied by this method.

3. Identification of PPIs involved in cystic fibrosis

Cystic fibrosis (CF) is the most common lethal genetic disease in Caucasian populations, caused by loss-of-function mutations on the Cystic Fibrosis transmembrane Conductance Regulator (CFTR) gene. Treatments for CF patients are currently symptom-based and only alleviate the conditions of the disease without restoring the underlying protein folding defects. Studying the dynamic CFTR interactome, which takes into account the change in protein-protein interaction (PPI) that occurs in the presence of drugs, will provide insight in to the molecular machinery affecting CFTR in CF, and will be of great value in the development of future therapeutics. We propose utilizing the MaMTH system to build a comprehensive and dynamic protein interactome of both full-length wildtype and F508del mutant CFTR. Using the MaMTH assay, we are searching for novel interacting protein partners of CFTR and further investigating the mechanisms of protein interactions affected by existing drug compounds. ​

4. Identification of PPIs involved in receptor tyrosine kinase signalling

Receptor tyrosine kinases (RTKs) play essential roles in many cellular processes. Malfunction of RTKs lead to multiple diseases such cancer and development disorders. Signalling initiated by RTKs is a complex process including multiple steps such as engagement to ligands, RTK dimerization, autophosphorylation, recruitment and phosphorylation of downstream signalling molecules and downregulation by dephosphorylation or degradation. MYTH and MaMTH allow us to investigate RTK signalling at the systems level. Current RTK research in the Stagljar lab focuses on 1) indentifying protein phosphatases involved in RTK signalling, 2) RTK-RTK interaction and 3) RTK-downstream target protein interaction. These studies systematically examine RTK signalling events from different angles and will reveal new mechanisms governing RTK signalling.

5. Studying PPIs and altered signalling pathways in cancer

We are particularly interested in non-small cell lung cancer (NSCLC) and Pancreatic Ductal Adenocarcinoma (PDAC), which are the first and fourth leading causes of cancer death in Canada, respectively. In both types of cancer, the majority of cases are due to mutations in particular RTKs - EGFR in NSCLC and K-RAS in PDAC. A second mutation is commonly observed in oncogenic EGFR in NSCLC patients who initially respond to therapy that treats the EGFR mutation. The second mutation, which occurs in ~50% of the responsive patients, renders the oncogene resistant to treatment. At this point, there is no available therapy remaining. Through use of MaMTH, we hope to delineate the interactomes of the oncogenes, and to identify and characterize potential therapeutic targets.

SELECT PUBLICATIONS

• A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae. Lam MH, Snider J, Rehal M, Wong V, Aboualizadeh F, Drecun L, Wong O, Jubran B, Li M, Ali M, Jessulat M, Deineko V, Miller R, Lee Me, Park HO, Davidson A, Babu M, Stagljar I. J Mol Biol. 2015 Jun 5;427(11):2088-103.
• The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells. Petschnigg J, Groisman B, Kotlyar M, Taipale M, Zheng Y, Kurat CF, Sayad A, Sierra JR, Mattiazzi Usaj M, Snider J, Nachman A, Krykbaeva I, Tsao MS, Moffat J, Pawson T, Lindquist S, Jurisica I, Stagljar I. Nat Methods. 2014 May;11(5):585-92.
• Mapping the functional yeast ABC transporter interactome. Snider J, Hanif A, Lee ME, Jin K, Yu AR, Graham C, Chuk M, Damjanovic D, Wierzbicka M, Tang P, Balderes D, Wong V, Jessulat M, Darowski KD, San Luis BJ, Shevelev I, Sturley SL, Boone C, Greenblatt JF, Zhang Z, Paumi CM, Babu M, Park HO, Michaelis S, Stagljar I. Nat Chem Biol. 2013 Sep;9(9):565-72.