Publications by authors named "Igor Stagljar"

109 Publications

Toward the discovery of biological functions associated with the mechanosensor Mtl1p of Saccharomyces cerevisiae via integrative multi-OMICs analysis.

Sci Rep 2021 Apr 1;11(1):7411. Epub 2021 Apr 1.

Department of Biochemistry, Medical Sciences Campus, University of Puerto Rico, San Juan, PR, 00936-5067, USA.

Functional analysis of the Mtl1 protein in Saccharomyces cerevisiae has revealed that this transmembrane sensor endows yeast cells with resistance to oxidative stress through a signaling mechanism called the cell wall integrity pathway (CWI). We observed upregulation of multiple heat shock proteins (HSPs), proteins associated with the formation of stress granules, and the phosphatase subunit of trehalose 6-phosphate synthase which suggests that mtl1Δ strains undergo intrinsic activation of a non-lethal heat stress response. Furthermore, quantitative global proteomic analysis conducted on TMT-labeled proteins combined with metabolome analysis revealed that mtl1Δ strains exhibit decreased levels of metabolites of carboxylic acid metabolism, decreased expression of anabolic enzymes and increased expression of catabolic enzymes involved in the metabolism of amino acids, with enhanced expression of mitochondrial respirasome proteins. These observations support the idea that Mtl1 protein controls the suppression of a non-lethal heat stress response under normal conditions while it plays an important role in metabolic regulatory mechanisms linked to TORC1 signaling that are required to maintain cellular homeostasis and optimal mitochondrial function.
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http://dx.doi.org/10.1038/s41598-021-86671-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016984PMC
April 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

Distinct roles of UVRAG and EGFR signaling in skeletal muscle homeostasis.

Mol Metab 2021 May 6;47:101185. Epub 2021 Feb 6.

Toronto General Hospital Research Institute, University Health Network, Toronto, ON M5G 1L7, Canada; Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada; Division of Endocrinology and Metabolism, Department of Medicine, University Health Network and Sinai Health System, University of Toronto, Toronto, ON M5G 2C4, Canada. Electronic address:

Objective: Autophagy is a physiological self-eating process that can promote cell survival or activate cell death in eukaryotic cells. In skeletal muscle, it is important for maintaining muscle mass and function that is critical to sustain mobility and regulate metabolism. The UV radiation resistance-associated gene (UVRAG) regulates the early stages of autophagy and autophagosome maturation and plays a key role in endosomal trafficking. This study investigated the essential in vivo role of UVRAG in skeletal muscle biology.

Methods: To determine the role of UVRAG in skeletal muscle in vivo, we generated muscle-specific UVRAG knockout mice using the Cre-loxP system driven by Myf6 promoter that is exclusively expressed in skeletal muscle. Myf6-Cre UVRAG (M-UVRAG) mice were compared to littermate Myf6-Cre UVRAG (M-UVRAG) controls under basal conditions on a normal chow diet. Body composition, muscle function, and mitochondria morphology were assessed in muscles of the WT and KO mice at 24 weeks of age.

Results: M-UVRAG mice developed accelerated sarcopenia and impaired muscle function compared to M-UVRAG littermates at 24 weeks of age. Interestingly, these mice displayed improved glucose tolerance and increased energy expenditure likely related to upregulated Fgf21, a marker of muscle dysfunction. Skeletal muscle of the M-UVRAG mice showed altered mitochondrial morphology with increased mitochondrial fission and EGFR accumulation reflecting defects in endosomal trafficking. To determine whether increased EGFR signaling had a causal role in muscle dysfunction, the mice were treated with an EGFR inhibitor, gefitinib, which partially restored markers of muscle and mitochondrial deregulation. Conversely, constitutively active EGFR transgenic expression in UVRAG-deficient muscle led to further detrimental effects with non-overlapping distinct defects in muscle function, with EGFR activation affecting the muscle fiber type whereas UVRAG deficiency impaired mitochondrial homeostasis.

Conclusions: Our results show that both UVRAG and EGFR signaling are critical for maintaining muscle mass and function with distinct mechanisms in the differentiation pathway.
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http://dx.doi.org/10.1016/j.molmet.2021.101185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921879PMC
May 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.

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

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
May 2020

REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects.

Nat Commun 2020 02 19;11(1):965. Epub 2020 Feb 19.

Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, Toronto, ON, M5G1M1, Canada.

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.
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http://dx.doi.org/10.1038/s41467-019-14143-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031342PMC
February 2020

Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRAS.

Nat Commun 2020 01 24;11(1):499. Epub 2020 Jan 24.

Systems Biology Ireland, University College Dublin, Dublin, Ireland.

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRAS) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRAS (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.
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http://dx.doi.org/10.1038/s41467-019-14224-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981206PMC
January 2020

Highlights for the 60th anniversary of BBRC.

Authors:
Igor Stagljar

Biochem Biophys Res Commun 2019 12;520(4):699-700

Donnelly Centre, Department of Biochemistry, Department of Molecular Genetics, University of Toronto, Ontario, Canada; Mediterranean Institute for Life Sciences, Split, Croatia. Electronic address:

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http://dx.doi.org/10.1016/j.bbrc.2019.10.007DOI Listing
December 2019

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

Testing cancer inhibitors at scale.

Nat Biomed Eng 2018 04;2(4):203-204

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

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http://dx.doi.org/10.1038/s41551-018-0226-xDOI Listing
April 2018

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

SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue.

PLoS Pathog 2018 08 20;14(8):e1007269. Epub 2018 Aug 20.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.
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http://dx.doi.org/10.1371/journal.ppat.1007269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117100PMC
August 2018

Melatonin receptors limit dopamine reuptake by regulating dopamine transporter cell-surface exposure.

Cell Mol Life Sci 2018 Dec 24;75(23):4357-4370. Epub 2018 Jul 24.

Inserm, U1016, Institut Cochin, 22 Rue Mechain, 75014, Paris, France.

Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT and MT receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT or MT receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT and MT) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.
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http://dx.doi.org/10.1007/s00018-018-2876-yDOI Listing
December 2018

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

NDFIP allows NEDD4/NEDD4L-induced AQP2 ubiquitination and degradation.

PLoS One 2017 20;12(9):e0183774. Epub 2017 Sep 20.

Department of Physiology, Radboud University Medical Center, Nijmegen, The Netherlands.

Regulation of our water homeostasis is fine-tuned by dynamic translocation of Aquaporin-2 (AQP2)-bearing vesicles to and from the plasma membrane of renal principal cells. Whereas binding of vasopressin to its type-2 receptor initiates a cAMP-protein kinase A cascade and AQP2 translocation to the apical membrane, this is counteracted by protein kinase C-activating hormones, resulting in ubiquitination-dependent internalization of AQP2. The proteins targeting AQP2 for ubiquitin-mediated degradation are unknown. In collecting duct mpkCCD cells, siRNA knockdown of NEDD4 and NEDD4L E3 ligases yielded increased AQP2 abundance, but they did not bind AQP2. Membrane Yeast Two-Hybrid assays using full-length AQP2 as bait, identified NEDD4 family interacting protein 2 (NDFIP2) to bind AQP2. NDFIP2 and its homologue NDFIP1 have PY motifs by which they bind NEDD4 family members and bring them close to target proteins. In HEK293 cells, NDFIP1 and NDFIP2 bound AQP2 and were essential for NEDD4/NEDD4L-mediated ubiquitination and degradation of AQP2, an effect not observed with PY-lacking NDFIP1/2 proteins. In mpkCCD cells, downregulation of NDFIP1, NEDD4 and NEDD4L, but not NDFIP2, increased AQP2 abundance. In mouse kidney, Ndfip1 and Ndfip2 mRNA distribution was similar and high in proximal tubules and collecting ducts, which was also found for NDFIP1 proteins. Our results reveal that NEDD4/NEDD4L mediate ubiquitination and degradation of AQP2, but that NDFIP proteins are needed to connect NEDD4/NEDD4L to AQP2. As NDFIP1/2 bind many NEDD4 family E3 ligases, which are implicated in several cellular processes, NDFIP1/2 may be the missing link for AQP2 ubiquitination and degradation from different subcellular locations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183774PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5606929PMC
October 2017

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

The Parkinson's disease-associated GPR37 receptor interacts with striatal adenosine A receptor controlling its cell surface expression and function in vivo.

Sci Rep 2017 08 25;7(1):9452. Epub 2017 Aug 25.

Unitat de Farmacologia, Departament Patologia i Terapéutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.

G protein-coupled receptor 37 (GPR37) is an orphan receptor associated to Parkinson's disease (PD) neuropathology. Here, we identified GPR37 as an inhibitor of adenosine A receptor (AR) cell surface expression and function in vivo. In addition, we showed that GPR37 and AR do oligomerize in the striatum. Thus, a close proximity of GPR37 and AR at the postsynaptic level of striatal synapses was observed by double-labelling post-embedding immunogold detection. Indeed, the direct receptor-receptor interaction was further substantiated by proximity ligation in situ assay. Interestingly, GPR37 deletion promoted striatal AR cell surface expression that correlated well with an increased AR agonist-mediated cAMP accumulation, both in primary striatal neurons and nerve terminals. Furthermore, GPR37-/- mice showed enhanced AR agonist-induced catalepsy and an increased response to AR antagonist-mediated locomotor activity. Overall, these results revealed a key role for GPR37 controlling AR biology in the striatum, which may be relevant for PD management.
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http://dx.doi.org/10.1038/s41598-017-10147-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573386PMC
August 2017

Multiple functions of protein phosphatases in receptor tyrosine kinase signaling revealed by interactome analysis.

Mol Cell Oncol 2017 26;4(3):e1297101. Epub 2017 Feb 26.

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

To obtain a global picture of how protein phosphatases are involved in receptor tyrosine kinase (RTK) signaling, we mapped the RTK-phosphatase interactome. Analyses of selected interactions revealed detailed mechanisms of their actions. This study provides new knowledge to better understand cancer development and to identify novel therapeutic targets.
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http://dx.doi.org/10.1080/23723556.2017.1297101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462513PMC
February 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 Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome.

Mol Cell 2017 Jan 5;65(2):347-360. Epub 2017 Jan 5.

Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.
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http://dx.doi.org/10.1016/j.molcel.2016.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663465PMC
January 2017

Parkinson's disease-associated GPR37 receptor regulates cocaine-mediated synaptic depression in corticostriatal synapses.

Neurosci Lett 2017 01 19;638:162-166. Epub 2016 Dec 19.

Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain; Institut de Neurosciències, Universitat de Barcelona, Barcelona, Spain. Electronic address:

GPR37 is an orphan G protein-coupled receptor highly expressed in the brain. The precise function of GPR37 is still unknown, but a number of evidences indicate it modulates the dopaminergic system. Here, we aimed to determine the role of GPR37 on the control of cocaine-mediated electrophysiological effects (synaptic transmission and short-term plasticity) in corticostriatal synapses. Accordingly, we evaluated basal synaptic transmission and paired-pulse stimulation (PPS) in wild-type and GPR37KO mice slices. Regardless of the genotype, a low concentration of cocaine (2μM) did not modify basal synaptic transmission. Conversely, a higher dose of cocaine (30μM) decreased synaptic transmission in both genotypes, although with different intensities: approximately 30% in slices from wild-type mice and 45% in slices from GPR37-KO mice. On the other hand, no differences in PPS ratio were observed between wild-type and GPR37-KO cocaine-treated mice. Overall, our data suggest that GPR37 is involved in cocaine-induced modification of basal synaptic transmission without modifying cocaine effects in short-term plasticity.
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http://dx.doi.org/10.1016/j.neulet.2016.12.040DOI Listing
January 2017

Systematic Identification of Oncogenic EGFR Interaction Partners.

J Mol Biol 2017 01 9;429(2):280-294. Epub 2016 Dec 9.

MRC Laboratory for Molecular Cell Biology, University College London, London, WC1E 6BT, UK. Electronic address:

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (TK) that-once activated upon ligand binding-leads to receptor dimerization, recruitment of protein complexes, and activation of multiple signaling cascades. The EGFR is frequently overexpressed or mutated in various cancers leading to aberrant signaling and tumor growth. Hence, identification of interaction partners that bind to mutated EGFR can help identify novel targets for drug discovery. Here, we used a systematic approach to identify novel proteins that are involved in cancerous EGFR signaling. Using a combination of high-content imaging and a mammalian membrane two-hybrid protein-protein interaction method, we identified eight novel interaction partners of EGFR, of which half strongly interacted with oncogenic, hyperactive EGFR variants. One of these, transforming acidic coiled-coil proteins (TACC) 3, stabilizes EGFR on the cell surface, which results in an increase in downstream signaling via the mitogen-activated protein kinase and AKT pathway. Depletion of TACC3 from cells using small hairpin RNA (shRNA) knockdown or small-molecule targeting reduced mitogenic signaling in non-small cell lung cancer cell lines, suggesting that targeting TACC3 has potential as a new therapeutic approach for non-small cell lung cancer.
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http://dx.doi.org/10.1016/j.jmb.2016.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240790PMC
January 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. Department of Molecular Genetics, University of Toronto, 160 College Street, Toronto ON, Canada M5S 3E1. Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Sciences (CSRS), Saitama, Japan.

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

The power of OMICs.

Authors:
Igor Stagljar

Biochem Biophys Res Commun 2016 Oct 20;479(4):607-609. Epub 2016 Sep 20.

Donnelly Centre, University of Toronto, Ontario, Canada; Department of Biochemistry, University of Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Ontario, Canada. Electronic address:

Over the past two decades, the field of systems biology, which encompasses the numerous, widely popular "OMICs" approaches, has driven many significant advances in biomedical research, enabling researchers to generate huge datasets at multiple levels of biological organization. Despite such successes, some scientists still think that "OMICs"-based research introduces a lot of chaos into the biomedical field and claim that the resultant data are often not reproducible and do not reveal deep mechanistic aspects of biological processes. In this editorial, I argue the following: first, that "OMICs" technologies have improved significantly to yield much better datasets; and second, that follow-up studies on components identified in "OMICs" analyses have yielded many valuable biological insights.
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http://dx.doi.org/10.1016/j.bbrc.2016.09.095DOI Listing
October 2016

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

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

Donnelly Centre, University of Toronto, Toronto, ON, Canada Lunenfeld-Tanenbaum Research Institute Mt. Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Canadian Institute for Advanced Research, Toronto, ON, Canada Department of Computer Science, University of Toronto, Toronto, Ontario, Canada

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 regulation of equlibrative nucleoside transporter 1 (ENT1) by receptor-stimulated Ca2+-dependent calmodulin binding.

Am J Physiol Cell Physiol 2016 05 23;310(10):C808-20. Epub 2016 Mar 23.

Department of Biology, York University, Toronto, Canada; Department of Chemistry and Biology, Ryerson University, Toronto, Canada;

Equilibrative nucleoside transporters (ENTs) facilitate the flux of nucleosides, such as adenosine, and nucleoside analog (NA) drugs across cell membranes. A correlation between adenosine flux and calcium-dependent signaling has been previously reported; however, the mechanistic basis of these observations is not known. Here we report the identification of the calcium signaling transducer calmodulin (CaM) as an ENT1-interacting protein, via a conserved classic 1-5-10 motif in ENT1. Calcium-dependent human ENT1-CaM protein interactions were confirmed in human cell lines (HEK293, RT4, U-87 MG) using biochemical assays (HEK293) and the functional assays (HEK293, RT4), which confirmed modified nucleoside uptake that occurred in the presence of pharmacological manipulations of calcium levels and CaM function. Nucleoside and NA drug uptake was significantly decreased (∼12% and ∼39%, respectively) by chelating calcium (EGTA, 50 μM; BAPTA-AM, 25 μM), whereas increasing intracellular calcium (thapsigargin, 1.5 μM) led to increased nucleoside uptake (∼26%). Activation of N-methyl-d-aspartate (NMDA) receptors (in U-87 MG) by glutamate (1 mM) and glycine (100 μM) significantly increased nucleoside uptake (∼38%) except in the presence of the NMDA receptor antagonist, MK-801 (50 μM), or CaM antagonist, W7 (50 μM). These data support the existence of a previously unidentified novel receptor-dependent regulatory mechanism, whereby intracellular calcium modulates nucleoside and NA drug uptake via CaM-dependent interaction of ENT1. These findings suggest that ENT1 is regulated via receptor-dependent calcium-linked pathways resulting in an alteration of purine flux, which may modulate purinergic signaling and influence NA drug efficacy.
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http://dx.doi.org/10.1152/ajpcell.00243.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4895448PMC
May 2016