Publications by authors named "Jamie Snider"

41 Publications

Receptor tyrosine kinases and cancer: oncogenic mechanisms and therapeutic approaches.

Oncogene 2021 Jun 2;40(24):4079-4093. Epub 2021 Jun 2.

Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease, notably cancer. Since their discovery, several mechanisms of RTK dysregulation have been identified, resulting in multiple cancer types displaying 'oncogenic addiction' to RTKs. As a result, RTKs have represented a major class for targeted therapeutics over the past two decades, with numerous small molecule-based tyrosine kinase inhibitor (TKI) therapeutics having been developed and clinically approved for several cancers. However, many of the current RTK inhibitor treatments eventually result in the rapid development of acquired resistance and subsequent tumor relapse. Recent technological advances and tools are being generated for the identification of novel RTK small molecule therapeutics. These newer technologies will be important for the identification of diverse types of RTK inhibitors, targeting both the receptors themselves as well as key cellular factors that play important roles in the RTK signaling cascade.
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http://dx.doi.org/10.1038/s41388-021-01841-2DOI Listing
June 2021

A homogeneous split-luciferase assay for rapid and sensitive detection of anti-SARS CoV-2 antibodies.

Nat Commun 2021 03 22;12(1):1806. Epub 2021 Mar 22.

Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Better diagnostic tools are needed to combat the ongoing COVID-19 pandemic. Here, to meet this urgent demand, we report a homogeneous immunoassay to detect IgG antibodies against SARS-CoV-2. This serological assay, called SATiN, is based on a tri-part Nanoluciferase (tNLuc) approach, in which the spike protein of SARS-CoV-2 and protein G, fused respectively to two different tNLuc tags, are used as antibody probes. Target engagement of the probes allows reconstitution of a functional luciferase in the presence of the third tNLuc component. The assay is performed directly in the liquid phase of patient sera and enables rapid, quantitative and low-cost detection. We show that SATiN has a similar sensitivity to ELISA, and its readouts are consistent with various neutralizing antibody assays. This proof-of-principle study suggests potential applications in diagnostics, as well as disease and vaccination management.
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http://dx.doi.org/10.1038/s41467-021-22102-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985487PMC
March 2021

Protein Interactions of the Mechanosensory Proteins Wsc2 and Wsc3 for Stress Resistance in .

G3 (Bethesda) 2020 09 2;10(9):3121-3135. Epub 2020 Sep 2.

Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, PO Box 365067, San Juan, PR 00936-067

Antifungal drug discovery and design is very challenging because of the considerable similarities in genetic features and metabolic pathways between fungi and humans. However, cell wall composition represents a notable point of divergence. Therefore, a research strategy was designed to improve our understanding of the mechanisms for maintaining fungal cell wall integrity, and to identify potential targets for new drugs that modulate the underlying protein-protein interactions in This study defines roles for and and their interacting protein partners in the cell wall integrity signaling and cell survival mechanisms that respond to treatments with fluconazole and hydrogen peroxide. By combined genetic and biochemical approaches, we report the discovery of 12 novel protein interactors of and Of these, interacting partners and , have opposing roles in the resistance and sensitivity to fluconazole treatments respectively. The interaction of with was confirmed by iMYTH and IP-MS approaches and is shown to play a dominant role in response to oxidative stress induced by hydrogen peroxide. Consistent with an earlier study, was also identified as an interacting partner of and cell wall integrity signaling proteins. Collectively, this study expands the interaction networks of the mechanosensory proteins of the Cell Wall Integrity pathway.
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http://dx.doi.org/10.1534/g3.120.401468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466973PMC
September 2020

Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition.

Nat Commun 2020 05 15;11(1):2440. Epub 2020 May 15.

Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Here, to overcome many limitations accompanying current available methods to detect protein-protein interactions (PPIs), we develop a live cell method called Split Intein-Mediated Protein Ligation (SIMPL). In this approach, bait and prey proteins are respectively fused to an intein N-terminal fragment (IN) and C-terminal fragment (IC) derived from a re-engineered split intein GP41-1. The bait/prey binding reconstitutes the intein, which splices the bait and prey peptides into a single intact protein that can be detected by regular protein detection methods such as Western blot analysis and ELISA, serving as readouts of PPIs. The method is robust and can be applied not only in mammalian cell lines but in animal models such as C. elegans. SIMPL demonstrates high sensitivity and specificity, and enables exploration of PPIs in different cellular compartments and tracking of kinetic interactions. Additionally, we establish a SIMPL ELISA platform that enables high-throughput screening of PPIs and their inhibitors.
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http://dx.doi.org/10.1038/s41467-020-16299-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229206PMC
May 2020

A drug discovery platform to identify compounds that inhibit EGFR triple mutants.

Nat Chem Biol 2020 05 24;16(5):577-586. Epub 2020 Feb 24.

Department for Lung Diseases Jordanovac, Clinical Hospital Centre Zagreb, University of Zagreb, Zagreb, Croatia.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.
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http://dx.doi.org/10.1038/s41589-020-0484-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123931PMC
May 2020

Highly Combinatorial Genetic Interaction Analysis Reveals a Multi-Drug Transporter Influence Network.

Cell Syst 2020 01 23;10(1):25-38.e10. Epub 2019 Oct 23.

Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Computer Science, University of Toronto, Toronto, ON M5T 3A1, Canada; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Many traits are complex, depending non-additively on variant combinations. Even in model systems, such as the yeast S. cerevisiae, carrying out the high-order variant-combination testing needed to dissect complex traits remains a daunting challenge. Here, we describe "X-gene" genetic analysis (XGA), a strategy for engineering and profiling highly combinatorial gene perturbations. We demonstrate XGA on yeast ABC transporters by engineering 5,353 strains, each deleted for a random subset of 16 transporters, and profiling each strain's resistance to 16 compounds. XGA yielded 85,648 genotype-to-resistance observations, revealing high-order genetic interactions for 13 of the 16 transporters studied. Neural networks yielded intuitive functional models and guided exploration of fluconazole resistance, which was influenced non-additively by five genes. Together, our results showed that highly combinatorial genetic perturbation can functionally dissect complex traits, supporting pursuit of analogous strategies in human cells and other model systems.
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http://dx.doi.org/10.1016/j.cels.2019.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989212PMC
January 2020

A Multireporter Bacterial 2-Hybrid Assay for the High-Throughput and Dynamic Assay of PDZ Domain-Peptide Interactions.

ACS Synth Biol 2019 05 18;8(5):918-928. Epub 2019 Apr 18.

Department of Biochemistry Molecular Pharmacology and Institute for Systems Genetics , NYU Langone Health , New York , New York 10016 , United States.

The accurate determination of protein-protein interactions has been an important focus of molecular biology toward which much progress has been made due to the continuous development of existing and new technologies. However, current methods can have limitations, including scale and restriction to high affinity interactions, limiting our understanding of a large subset of these interactions. Here, we describe a modified bacterial-hybrid assay that employs combined selectable and scalable reporters that enable the sensitive screening of large peptide libraries followed by the sorting of positive interactions by the level of reporter output. We have applied this tool to characterize a set of human and E. coli PDZ domains. Our results are consistent with prior characterization of these proteins, and the improved sensitivity increases our ability to predict known and novel in vivo binding partners. This approach allows for the recovery of a wide range of affinities with a high throughput method that does not sacrifice the scale of the screen.
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http://dx.doi.org/10.1021/acssynbio.8b00499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818413PMC
May 2019

Identification and Functional Testing of Novel Interacting Protein Partners for the Stress Sensors Wsc1p and Mid2p of .

G3 (Bethesda) 2019 04 9;9(4):1085-1102. Epub 2019 Apr 9.

Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, PO Box 365067, San Juan, PR 00936-5067

Wsc1p and Mid2p are transmembrane signaling proteins of cell wall stress in the budding yeast When an environmental stress compromises cell wall integrity, they activate a cell response through the Cell Wall Integrity (CWI) pathway. Studies have shown that the cytoplasmic domain of Wsc1p initiates the CWI signaling cascade by interacting with Rom2p, a Rho1-GDP-GTP exchange factor. Binding of Rom2p to the cytoplasmic tail of Wsc1p requires dephosphorylation of specific serine residues but the mechanism by which the sensor is dephosphorylated and how it subsequently interacts with Rom2p remains unclear. We hypothesize that Wsc1p and Mid2p must be physically associated with interacting proteins other than Rom2p that facilitate its interaction and regulate the activation of CWI pathway. To address this, a cDNA plasmid library of yeast proteins was expressed in bait strains bearing membrane yeast two-hybrid (MYTH) reporter modules of Wsc1p and Mid2p, and their interacting preys were recovered and sequenced. 14 previously unreported interactors were confirmed for Wsc1p and 29 for Mid2p The interactors' functionality were assessed by cell growth assays and CWI pathway activation by western blot analysis of Slt2p/Mpk1p phosphorylation in null mutants of each interactor under defined stress conditions. The susceptibility of these strains to different stresses were tested against antifungal agents and chemicals. This study reports important novel protein interactions of Wsc1p and Mid2p that are associated with the cellular response to oxidative stress induced by Hydrogen Peroxide and cell wall stress induced by Caspofungin.
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http://dx.doi.org/10.1534/g3.118.200985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469404PMC
April 2019

Systems analysis of the genetic interaction network of yeast molecular chaperones.

Mol Omics 2018 Apr;14(2):82-94

Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1M1, Canada. and Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada.

Molecular chaperones are typically promiscuous interacting proteins that function globally in the cell to maintain protein homeostasis. Recently, we had carried out experiments that elucidated a comprehensive interaction network for the core 67 chaperones and 15 cochaperones in the budding yeast Saccharomyces cerevisiae [Rizzolo et al., Cell Rep., 2017, 20, 2735-2748]. Here, the genetic (i.e. epistatic) interaction network obtained for chaperones was further analyzed, revealing that the global topological parameters of the resulting network have a more central role in mediating interactions in comparison to the rest of the proteins in the cell. Most notably, we observed Hsp10, Hsp70 Ssz1 chaperone, and Hsp90 cochaperone Cdc37 to be the main drivers of the network architecture. Systematic analysis on the physicochemical properties for all chaperone interactors further revealed the presence of preferential domains and folds that are highly interactive with chaperones such as the WD40 repeat domain. Further analysis with established cellular complexes revealed the involvement of R2TP chaperone in quaternary structure formation. Our results thus provide a global overview of the chaperone network properties in yeast, expanding our understanding of their functional diversity and their role in protein homeostasis.
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http://dx.doi.org/10.1039/C7MO00142HDOI Listing
April 2018

Recent Progress in CFTR Interactome Mapping and Its Importance for Cystic Fibrosis.

Front Pharmacol 2017 17;8:997. Epub 2018 Jan 17.

Department of Biochemistry, University of Toronto, Toronto, ON, Canada.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride channel found in secretory epithelia with a plethora of known interacting proteins. Mutations in the CFTR gene cause cystic fibrosis (CF), a disease that leads to progressive respiratory illness and other complications of phenotypic variance resulting from perturbations of this protein interaction network. Studying the collection of CFTR interacting proteins and the differences between the interactomes of mutant and wild type CFTR provides insight into the molecular machinery of the disease and highlights possible therapeutic targets. This mini review focuses on functional genomics and proteomics approaches used for systematic, high-throughput identification of CFTR-interacting proteins to provide comprehensive insight into CFTR regulation and function.
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http://dx.doi.org/10.3389/fphar.2017.00997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785726PMC
January 2018

Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping.

Cell Rep 2017 Sep;20(11):2735-2748

Department of Biochemistry, University of Toronto, Toronto, ON M5G 1M1, Canada; Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada. Electronic address:

A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.
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http://dx.doi.org/10.1016/j.celrep.2017.08.074DOI Listing
September 2017

Systematic protein-protein interaction mapping for clinically relevant human GPCRs.

Mol Syst Biol 2017 03 15;13(3):918. Epub 2017 Mar 15.

Donnelly Centre, University of Toronto, Toronto, ON, Canada

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371730PMC
http://dx.doi.org/10.15252/msb.20167430DOI Listing
March 2017

Detecting Membrane Protein-protein Interactions Using the Mammalian Membrane Two-hybrid (MaMTH) Assay.

Curr Protoc Chem Biol 2017 03 2;9(1):38-54. Epub 2017 Mar 2.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

Protein-protein interactions (PPIs) play an integral role in numerous cellular processes. Membrane protein interactions, in particular, are critical in cellular responses to stresses and stimuli, with dysfunction of these PPIs (e.g., due to aberrant expression and/or mutation of interaction partners) leading to a diverse array of pathological states. Exploration of the interaction space and dynamics of membrane proteins is difficult due to the limitations of current techniques used to study proteins in the biochemically complex environment of biological membranes. In the protocols below, we describe a newly developed membrane protein interaction assay called the Mammalian-Membrane Two-Hybrid (MaMTH), designed specifically for the detection of integral membrane PPIs in the context of living mammalian cells. Prior to using MaMTH, cell lines of interest are genetically modified to encode a reporter of choice. MaMTH "bait" and "prey" constructs of interest are also generated using Gateway cloning technology. The assay is then performed by co-transfection of baits and preys, with bait-prey interaction quantifiably assessed by way of a reporter signal (e.g., light (luciferase), fluorescence (GFP). © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpch.15DOI Listing
March 2017

A global genetic interaction network maps a wiring diagram of cellular function.

Science 2016 09;353(6306)

The Donnelly Centre, University of Toronto, 160 College Street, Toronto ON, Canada M5S 3E1.

We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing more than 23 million double mutants, identifying about 550,000 negative and about 350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell.
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http://dx.doi.org/10.1126/science.aaf1420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661885PMC
September 2016

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

Mol Syst Biol 2016 Apr 22;12(4):863. Epub 2016 Apr 22.

Joint IRB-BSC Program in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.

High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848762PMC
http://dx.doi.org/10.15252/msb.20156660DOI Listing
April 2016

Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen.

G3 (Bethesda) 2016 05 3;6(5):1469-74. Epub 2016 May 3.

Department of Biochemistry,University of Puerto Rico, San Juan, Puerto Rico 00936-5067

Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis.
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http://dx.doi.org/10.1534/g3.115.026609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856097PMC
May 2016

Membrane Yeast Two-Hybrid (MYTH) Mapping of Full-Length Membrane Protein Interactions.

Cold Spring Harb Protoc 2016 Jan 4;2016(1):pdb.top077560. Epub 2016 Jan 4.

Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Mapping of protein interaction networks is a major strategy for obtaining a global understanding of protein function in cells and represents one of the primary goals of proteomics research. Membrane proteins, which play key roles in human disease and as drug targets, are of considerable interest; however, because of their hydrophobic nature, mapping their interactions presents significant technical challenges and requires the use of special methodological approaches. One powerful approach is the membrane yeast two-hybrid (MYTH) assay, a split-ubiquitin-based system specifically suited to the study of full-length membrane protein interactions in vivo using the yeast Saccharomyces cerevisiae as a host. The system can be used in both low- and high-throughput formats to study proteins from a wide range of different organisms. There are two primary variants of MYTH: integrated (iMYTH), which involves endogenous expression and tagging of baits and is suitable for studying native yeast membrane proteins, and traditional (tMYTH), which involves ectopic plasmid-based expression of tagged baits and is suitable for studying membrane proteins from other organisms. Here we provide an introduction to the MYTH assay, including both the iMYTH and tMYTH variants. MYTH can be set up in almost any laboratory environment, with results typically obtainable within 4 to 6 wk.
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http://dx.doi.org/10.1101/pdb.top077560DOI Listing
January 2016

MYTH Screening: iMYTH and tMYTH Variants.

Cold Spring Harb Protoc 2016 Jan 4;2016(1):pdb.prot087825. Epub 2016 Jan 4.

Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Once a bait has been generated and validated for the membrane yeast two-hybrid (MYTH) assay, it can be used for either high-throughput screening to generate a detailed interaction map (interactome) or in low-throughput experiments to examine interactions with specific targets. Here we describe how to carry out high-throughput MYTH library screening of a validated bait generated using integrated or traditional MYTH. The principles herein can be easily adapted for use in a smaller-scale format if required. A typical MYTH library screen can be completed in ∼3-4 wk.
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http://dx.doi.org/10.1101/pdb.prot087825DOI Listing
January 2016

Generation and Validation of MYTH Baits: iMYTH and tMYTH Variants.

Cold Spring Harb Protoc 2016 Jan 4;2016(1):pdb.prot087817. Epub 2016 Jan 4.

Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Generation of baits for membrane yeast two-hybrid (MYTH) screening differs depending on the nature of the protein(s) being studied. When using native yeast proteins with cytoplasmic carboxyl termini, the integrated form of MYTH (iMYTH) is the method of choice. iMYTH involves endogenous carboxy-terminal tagging of the gene of interest within the yeast chromosome, leaving the gene under the control of its natural promoter. When studying proteins not native to yeast, or native yeast proteins with only cytoplasmic amino termini, traditional MYTH (tMYTH) must be used. In the tMYTH approach, amino- or carboxy-terminally tagged proteins are expressed ectopically from a plasmid. In this protocol, we describe the generation and validation of iMYTH and tMYTH baits. MYTH bait generation can typically be completed in ∼1-2 wk.
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http://dx.doi.org/10.1101/pdb.prot087817DOI Listing
January 2016

Fundamentals of protein interaction network mapping.

Mol Syst Biol 2015 Dec 17;11(12):848. Epub 2015 Dec 17.

Donnelly Centre, Department of Biochemistry, Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada

Studying protein interaction networks of all proteins in an organism ("interactomes") remains one of the major challenges in modern biomedicine. Such information is crucial to understanding cellular pathways and developing effective therapies for the treatment of human diseases. Over the past two decades, diverse biochemical, genetic, and cell biological methods have been developed to map interactomes. In this review, we highlight basic principles of interactome mapping. Specifically, we discuss the strengths and weaknesses of individual assays, how to select a method appropriate for the problem being studied, and provide general guidelines for carrying out the necessary follow-up analyses. In addition, we discuss computational methods to predict, map, and visualize interactomes, and provide a summary of some of the most important interactome resources. We hope that this review serves as both a useful overview of the field and a guide to help more scientists actively employ these powerful approaches in their research.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704491PMC
http://dx.doi.org/10.15252/msb.20156351DOI Listing
December 2015

ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

FASEB J 2015 Nov 28;29(11):4682-94. Epub 2015 Jul 28.

*Institute of Human Nutrition, Department of Neurology, **Department of Genetics and Development, and Department of Pediatrics, Columbia University Medical Center, New York, New York, USA; Department of Biological Sciences and Department of Chemistry, Columbia University, New York, New York, USA; Donnelly Center for Cellular and Biomolecular Research, Toronto, Ontario, Canada; Institute of Molecular Biosciences, BioTechMed Graz, University of Graz, Graz, Austria; Department of Biology, Providence College, Providence, Rhode Island, USA; and Marine Biological Laboratory, Woods Hole, Massachusetts, USA

A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.
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http://dx.doi.org/10.1096/fj.14-264796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608909PMC
November 2015

A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae.

J Mol Biol 2015 Jun 30;427(11):2088-103. Epub 2015 Jan 30.

Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada; Department of Biochemistry, University of Toronto, Medical Science Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Molecular Genetics, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Electronic address:

Sho1p, an integral membrane protein, plays a vital role in the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in the yeast Saccharomyces cerevisiae. Activated under conditions of high osmotic stress, it interacts with other HOG pathway proteins to mediate cell signaling events, ensuring that yeast cells can adapt and remain viable. In an attempt to further understand how the function of Sho1p is regulated through its protein-protein interactions (PPIs), we identified 49 unique Sho1p PPIs through the use of membrane yeast two-hybrid (MYTH), an assay specifically suited to identify PPIs of full-length integral membrane proteins in their native membrane environment. Secondary validation by literature search, or two complementary PPI assays, confirmed 80% of these interactions, resulting in a high-quality Sho1p interactome. This set of putative PPIs included both previously characterized interactors, along with a large subset of interactors that have not been previously identified as binding to Sho1p. The SH3 domain of Sho1p was found to be important for binding to many of these interactors. One particular novel interactor of interest is the glycerol transporter Fps1p, which was shown to require the SH3 domain of Sho1p for binding via its N-terminal soluble regulatory domain. Furthermore, we found that Fps1p is involved in the positive regulation of Sho1p function and plays a role in the phosphorylation of the downstream kinase Hog1p. This study represents the largest membrane interactome analysis of Sho1p to date and complements past studies on the HOG pathway by increasing our understanding of Sho1p regulation.
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http://dx.doi.org/10.1016/j.jmb.2015.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331858PMC
June 2015

Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes.

Mol Biol Cell 2015 Mar 21;26(6):1119-28. Epub 2015 Jan 21.

Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, Vancouver, BC V5Z 4H4, Canada

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain-containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.
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http://dx.doi.org/10.1091/mbc.E14-08-1281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357511PMC
March 2015

'More than skin-deep': biological essentialism in response to a distinctiveness threat in a stigmatized fan community.

Br J Soc Psychol 2015 Jun 12;54(2):359-70. Epub 2014 Aug 12.

Niagara County Community College, Sanborn, New York, USA.

We investigated how group distinctiveness threats affect essentialist beliefs about group membership in a stigmatized fan community. An experiment conducted on 817 members of the fan community revealed that highly identified fans who perceived significant stigmatization were the most likely to endorse essentialist beliefs about group membership when exposed to a distinctiveness threat via comparison to a highly similar (vs. dissimilar) outgroup. These results bridge essentialism research and research on distinctiveness threat by demonstrating the mutability of group essentialism beliefs as a defensive response to distinctiveness threats. Implications for future research are discussed.
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http://dx.doi.org/10.1111/bjso.12079DOI Listing
June 2015

The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells.

Nat Methods 2014 May 23;11(5):585-92. Epub 2014 Mar 23.

1] Donnelly Centre, University of Toronto, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. [3] Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.
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http://dx.doi.org/10.1038/nmeth.2895DOI Listing
May 2014

The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli.

PLoS One 2014 15;9(1):e85529. Epub 2014 Jan 15.

Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

MoxR ATPases are widespread throughout bacteria and archaea. The experimental evidence to date suggests that these proteins have chaperone-like roles in facilitating the maturation of dedicated protein complexes that are functionally diverse. In Escherichia coli, the MoxR ATPase RavA and its putative cofactor ViaA are found to exist in early stationary-phase cells at 37 °C at low levels of about 350 and 90 molecules per cell, respectively. Both proteins are predominantly localized to the cytoplasm, but ViaA was also unexpectedly found to localize to the cell membrane. Whole genome microarrays and synthetic lethality studies both indicated that RavA-ViaA are genetically linked to Fe-S cluster assembly and specific respiratory pathways. Systematic analysis of mutant strains of ravA and viaA indicated that RavA-ViaA sensitizes cells to sublethal concentrations of aminoglycosides. Furthermore, this effect was dependent on RavA's ATPase activity, and on the presence of specific subunits of NADH:ubiquinone oxidoreductase I (Nuo Complex, or Complex I). Importantly, both RavA and ViaA were found to physically interact with specific Nuo subunits. We propose that RavA-ViaA facilitate the maturation of the Nuo complex.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085529PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3893208PMC
December 2014

Mapping the functional yeast ABC transporter interactome.

Nat Chem Biol 2013 Sep 7;9(9):565-72. Epub 2013 Jul 7.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.
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http://dx.doi.org/10.1038/nchembio.1293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835492PMC
September 2013

Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae.

Nature 2012 Sep 2;489(7417):585-9. Epub 2012 Sep 2.

Banting and Best Department of Medical Research, Donnelly Centre, 160 College Street, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.
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http://dx.doi.org/10.1038/nature11354DOI Listing
September 2012

Investigation of membrane protein interactions using the split-ubiquitin membrane yeast two-hybrid system.

Methods Mol Biol 2012 ;812:225-44

Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.

Proteins are generally organized into molecular complexes, in which multiple interaction partners collaborate to carry out cellular processes. Thus, techniques to map protein-protein interactions have become pivotal for biological studies of as yet uncharacterized proteins. Investigation of interaction partners of membrane proteins is of special interest, as they play a major role in cellular processes and are often directly linked to human diseases. Owing to their hydrophobic nature, however, it has proven difficult to study their interaction partners. To circumvent this problem, a yeast-based genetic technology for the in vivo detection of membrane protein interactions, the split-ubiquitin membrane yeast two-hybrid (MYTH) system, has been developed. MYTH allows for detection of both stable and transient interactions and can be applied to large- and small-scale screens. It uses the split-ubiquitin approach, in which the reconstitution of two ubiquitin halves is mediated by a specific protein-protein interaction. Briefly, the bait membrane protein is fused to the C-terminal half of ubiquitin and an artificial transcription factor. The mutated N-terminal moiety of ubiquitin is fused to the prey protein. Upon interaction of bait and prey proteins, ubiquitin is reconstituted and further recognized by ubiquitin-specific proteases, which subsequently cleave off the transcription factor, thus resulting in reporter gene activation. To date, MYTH has been successfully applied to study interactions of membrane proteins from various organisms and has only recently been adapted for the identification of interaction partners of mammalian receptor tyrosine kinases.
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http://dx.doi.org/10.1007/978-1-61779-455-1_13DOI Listing
April 2012

The Rho1 GTPase acts together with a vacuolar glutathione S-conjugate transporter to protect yeast cells from oxidative stress.

Genetics 2011 Aug 30;188(4):859-70. Epub 2011 May 30.

Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210-1292, USA.

Maintenance of redox homeostasis is critical for the survival of all aerobic organisms. In the budding yeast Saccharomyces cerevisiae, as in other eukaryotes, reactive oxygen species (ROS) are generated during metabolism and upon exposure to environmental stresses. The abnormal production of ROS triggers defense mechanisms to avoid the deleterious consequence of ROS accumulation. Here, we show that the Rho1 GTPase is necessary to confer resistance to oxidants in budding yeast. Temperature-sensitive rho1 mutants (rho1(ts)) are hypersensitive to oxidants and exhibit high accumulation of ROS even at a semipermissive temperature. Rho1 associates with Ycf1, a vacuolar glutathione S-conjugate transporter, which is important for heavy metal detoxification in yeast. Rho1 and Ycf1 exhibit a two-hybrid interaction with each other and form a bimolecular fluorescent complex on the vacuolar membrane. A fluorescent-based complementation assay suggests that the GTP-bound Rho1 associates with Ycf1 and that their interaction is enhanced upon exposure to hydrogen peroxide. The rho1(ts) mutants also exhibit hypersensitivity to cadmium, while cells carrying a deletion of YCF1 or mutations in a component of the Pkc1-MAP kinase pathway exhibit little or minor sensitivity to oxidants. We thus propose that Rho1 protects yeast cells from oxidative stress by regulating multiple downstream targets including Ycf1. Since both Rho1 and Ycf1 belong to highly conserved families of proteins, similar mechanisms may exist in other eukaryotes.
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http://dx.doi.org/10.1534/genetics.111.130724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3176091PMC
August 2011