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crystal Igor Stagljar Laboratory
Department of Biochemistry
gold_bar Department of Molecular Genetics
muffler University of Toronto corner_bottom
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Research

We have developed 2 powerful technologies to study integral membrane proteins:

  1. Membrane Yeast Two-Hybrid (MYTH) Studies
  2. Mammalian Membrane Two-Hybrid Studies (MaMTH)

We are using MaMTH technology to investigate two major areas:

  1. Functional interactions of mammalian ABC transporters
  2. Developing MaMTH as a drug screening platform

Unlocking the immense complexity of the cell is a major goal of modern biological science. To this end, scientists seek to acquire a detailed understanding of the many distinct molecular systems within the cell, and how these various systems function together as an integrative whole to generate unique cellular physiologies. One powerful method of obtaining such an understanding is through the identification and characterization of the protein interactions involved in these processes. In particular, large-scale proteomics studies involving the generation of complex ‘interactome’ maps of protein interconnectivity provide an unparalleled global view of the interplay between the different systems within the cell.

One major protein class of great biological importance is integral membrane proteins. Membrane proteins comprise nearly 30% of the proteome of most organisms, and have a diverse range of cellular functions, including roles as receptors, transporters, adhesion molecules, and enzymes, among others.

The association of integral membrane protein dysfunction with a range of different diseases, in addition to their accessibility and suitability for use as a target of therapeutic drugs, also makes them a protein class of significant biomedical relevance. However, the unique biochemistry and hydrophobicity of integral membrane proteins make them difficult to study using conventional biochemical approaches. Our lab is focused on the development and application of new technologies for use in the identification and characterization of the interactors of integral membrane proteins.

Currently our lab is focused on projects employing two powerful technologies we’ve developed for use in the study of integral membrane proteins:

  1. the original
    Membrane Yeast Two-Hybrid (MYTH)
  2. and
  3. the newly created
    Mammalian Membrane Two-Hybrid (MaMTH)

Our primary interests are in the identification and characterization of the interaction partners of integral membrane proteins associated with disease states, to obtain a better understanding of their molecular function, and in the identification of novel drugs and drug targets for use in therapeutic treatments.

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I. Membrane Yeast Two-Hybrid (MYTH) Studies

Previously, our lab developed the Membrane Yeast Two-Hybrid (MYTH), a powerful tool for use in the identification of membrane proteins in vivo, using the model organism Saccharomyces cerevisiae. An overview of the MYTH system can be seen in Figure 1.

Figure 1: Outline of the MYTH technology.

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.

Our lab has recently used the MYTH system to map the interactome of all non-mitochondrial ABC transporters found in Saccharomyces cereivisiae, and found that ABC transporters physically associate with a functionally diverse array of proteins, displaying a far greater integration with cellular processes than previously reported. The results of this work appear in the Sept 2013 issue of Nature Chemical Biology.

Currently, we are making use of the MYTH system to identify PPIs between all known human receptor tyrosine kinases and protein phosphatases. Receptor tyrosine Kinases (RTKs) are vital for many cellular functions such as cellular growth and differentiation. There are 58 known RTKs that fall into 20 subfamilies. Protein phosphatases (PPs) act on RTKs in many different ways including acting as negative regulators (rendering active RTKs inactive), helping maintain RTK inactivity, and in some cases acting as a positive regulators of RTK activity.

Interestingly, many studies have implicated RTKs and PPs in several cancers; for example, EGFR is mutated or amplified in 35% of glioblastomas and ~80% of head and neck cancer, and also correlates with poor prognosis and resistance to therapy. ErbB2 is mutated in ~30% of breast cancer, and alterations have also been found in pancreas, colon, endometrial, lung, and ovarian cancers. ErbB family receptors also play a role in non-cancer diseases, such as atherosclerosis and psoriasis. At least 30 different PPs have also been implicated in playing roles in cancer, including examples such as PTPN12, PTPN23, and DEP-1, which have recently been discovered to act as tumor suppressors.

To elucidate the RTK/PP interactome, we are employing our Membrane Yeast Two-Hybrid (MYTH) system, which is an ideal proteomic tool to study in vivo interactions of membrane proteins.  In this MYTH screen, 58 RTK baits are expressed as integral membrane proteins in yeast, mimicking their native states in mammalian cells and therefore maintaining their structure and function. These are then screened against a set of ~150 phosphatases similarly expressed in yeast.  Results have been obtained for all human ErbB RTK family members (EGFR, ErbB2, ErbB3, and ErbB4) and several novel, as well as interesting previously cited interactions, have been identified. These interactions have been confirmed in mammalian cells, and currently follow up experiments are being performed in order to analyze the effects they have on ErbB family signaling proteins. Screening of the remaining RTKS is currently underway, and nearing completion.

In this way, the MYTH system, coupled with downstream experiments performed in mammalian cells, offers a systems approach to identify interactors that may potentially lead to the discovery of novel drug targets, contribute to therapeutic research, and shed new light on the mechanism of transmembrane signaling.

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II. Mammalian Membrane Two-Hybrid Studies (MaMTH)

Building upon our earlier successes with MYTH, we have recently developed a powerful new technology, the Mammalian Membrane Two-Hybrid (MaMTH), for use in the in vivo analysis of the protein-protein interactions of full-length integral membrane proteins directly in the context of mammalian cell lines. Like MYTH, MaMTH is based upon the concept of split ubiquitin, but has been specifically re-engineered for use in the context of mammalian cells, thereby eliminating many of the complications associated with studying mammalian proteins in a foreign host.

Figure 2: Outline of the MaMTH technology.

A major advantage of this system is its sensitivity, which allows it to detect subtle dynamic changes in interaction patterns in response to environmental changes (e.g. drugs, ligands, phosphorylation state changes etc.). Additionally the system is highly transferable, allowing it to be carried out in virtually any cell line, and is perfectly suited for use in a high-throughput format. An overview of the MaMTH technology/pipeline is provided in Figure 2.

Using MaMTH as a targeted protein interaction screening assay, we identified a protein called CRKII as a novel interactor of oncogenic EGFR (L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer (NSCLC) cells, thus identifying CRK II as a novel potential biomarker for NSCLC (Petschnigg et al. (2014) Nature Methods). To the best of our knowledge, high-throughput dynamic interaction screens (i.e. performed under multiple conditions for comparison) with full-length membrane proteins in human cells have not been possible in the field of proteomics thus far.

We are currently making use of the MaMTH technology to investigate two major areas -

  1. Functional interactions of mammalian ABC transporters
  2. Developing MaMTH as a drug screening platform
  3. Assessing the functional role of CrkII protein in the onset of non-small cell lung cancer (NSCLC)
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A. Functional Interactions of Mammalian ABC Transporters

ABC Binding Cassette (ABC) proteins comprise one of the largest known protein superfamilies, using 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 great clinical interest because of the key roles they play in human health and disease. Dysfunction of ABC transporters is associated with a variety of human diseases, such as cystic fibrosis, pseudoxanthoma elasticum, adrenoleukodystrophy, Zellweger syndrome, familial hyperinsulinemic hypoglycemia of infancy, Dubin-Johnson syndrome, hepatic cholestasis, the retinal syndrome Stargardt’s dystrophy, and the cholesterol transport disorder Tangier disease. ABC transporters are also associated with multidrug resistance of cancer cells and pathogenic microorganisms, and can provide a serious barrier to effective drug therapy.

In an effort to gain a better understanding of the regulation and molecular function of these proteins we recently used the MYTH system to map the complete interactome of all non-mitochondrial ABC transporters found in the yeast Saccharomyces cerevisiae. Our results revealed that ABC transporters physically associate with a functionally diverse array of proteins and show far greater integration with cellular systems than previously known. We are currently seeking to expand upon this work by using our powerful new MaMTH technology to map the interactions of the complete complement of ABC transporters found in humans.

Figure 3: Inventory of Human ABC proteins. FifX- MaMTH development as drug screening platform.

The human genome carries 48 distinct genes encoding ABC proteins that can be arranged in seven subfamilies, designated A to G (Figure 3), of which 44 are integral membrane ABC transporters. We are currently in the process of systematically tagging each of these ABC transporters and screening them against a MaMTH prey library of ~ 13000 fully sequenced human ORFs to both identify novel interactions and validate previously reported interactions. Additionally, we are exploiting the high sensitivity of the MaMTH assay to map the dynamic interactomes of these transporters, specifically examining how their interaction partners change in response to various drugs and transport substrates. Our initial studies are focusing on the interactions of ABCC1 (MRP1), ABCB1 (MDR1) and ABCG2 (BCRP), which are associated with multidrug resistance in cancer cells, and the cystic fibrosis transmembrane receptor ABCC7 (CFTR). Preliminary results with CFTR have identified a number of novel interactions of significant medical interest, which we are currently in the process of functionally characterizing in greater detail.

Our work on human ABC transporters will build upon our earlier work in yeast and should greatly increase our understanding of the cellular role and regulation of this critically important class of integral membrane proteins, which in turn should help greatly in the development of novel therapeutics to better treat diseases associated with ABC transporter dysfunction.

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B. Developing MaMTH as a Drug Screening Platform

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 proteins analysis, which is of keen interest to the pharmaceutical market, as membrane proteins are the major class of drug targets.



Figure 4: Erlotinib-mediated inhibition of the EGFR phosphorylation detected by MaMTH assay.

Currently we are developing MaMTH as drug discovery platform, aiming to screen for small molecules (chemical compounds or antibody-derived peptides), which will affect disease-impaired protein networks in cells. To date, using the MaMTH approach we were able to detect, in a quantitative manner, very minor changes in protein-protein interactions in response to known drugs. One of our major areas of focus is on finding inhibitors of active versions of the Epidermal Growth Factor Receptor (EGFR), frequently mutated in human cancers. Using MaMTH we were able to measure inhibition of the activity of the most oncogenic EGFR variant found in lung cancer, in response to the clinically approved drug Erlotinib (Tarceva). The inhibitory effect can be measured as a decrease in EGFR phosphorylation state, which is reflected by reduced association with phosphorylation-dependent protein adaptors such as Shc1 (Figure 4).

Although Erlotinib is a potent drug, the majority of treated patients develop a secondary mutation conferring drug-resistance. At the current stage of our project we aim to screen for compounds inhibiting resistant EGFR variants, using various small molecule libraries available to us through collaborations. We believe that our 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.

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Last modified on 29 July, 2014 10:21 AM