Publications by authors named "Stephen W Michnick"

86 Publications

Genetics' Piece of the PI: Inferring the Origin of Complex Traits and Diseases from Proteome-Wide Protein-Protein Interaction Dynamics.

Bioessays 2020 02 18;42(2):e1900169. Epub 2019 Dec 18.

Departement de Biochimie, Université de Montréal, 2900 Édouard-Montpetit, Montréal, Quebec, H3T 1J4, Canada.

How do common and rare genetic polymorphisms contribute to quantitative traits or disease risk and progression? Multiple human traits have been extensively characterized at the genomic level, revealing their complex genetic architecture. However, it is difficult to resolve the mechanisms by which specific variants contribute to a phenotype. Recently, analyses of variant effects on molecular traits have uncovered intermediate mechanisms that link sequence variation to phenotypic changes. Yet, these methods only capture a fraction of genetic contributions to phenotype. Here, in reviewing the field, it is proposed that complex traits can be understood by characterizing the dynamics of biochemical networks within living cells, and that the effects of genetic variation can be captured on these networks by using protein-protein interaction (PPI) methodologies. This synergy between PPI methodologies and the genetics of complex traits opens new avenues to investigate the molecular etiology of human diseases and to facilitate their prevention or treatment.
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http://dx.doi.org/10.1002/bies.201900169DOI Listing
February 2020

Induced and spontaneous colitis mouse models reveal complex interactions between IL-10 and IL-12/IL-23 pathways.

Cytokine 2019 09 31;121:154738. Epub 2019 May 31.

Department of Immunology-Oncology, Maisonneuve-Rosemont Hospital, Montreal, Quebec H1T 2M4, Canada; Département de microbiologie, infectiologie et immunologie, Université de Montréal, Montreal, Quebec H3C 3J7, Canada. Electronic address:

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD). These idiopathic and chronic diseases result from inflammation of the gastrointestinal tract and are mainly mediated by the immune system. Genome wide association studies link genes of the IL-12 and IL-23 biology to both CD and UC susceptibility. IL-12 and IL-23 cytokines share a functional subunit, p40, and their respective receptors also share a functional subunit, IL-12Rβ1. However, clinical trials targeting p40, and thus inhibiting both IL-12 and IL-23 pathways, provided mitigated effects on IBD, suggesting context dependent effects for each cytokine. In addition to IL-12 and IL-23, genetic deficiencies in IL-10 also result in severe IBD pathology. We generated various mouse models to determine how IL-12 or IL-23 interacts with IL-10 in IBD pathology. Whereas defects in both IL-10 and IL-12R do not impact the severity of the Dextran Sulfate Sodium (DSS)-induced colitis, combined deficiencies in both IL-10 and IL-23R aggravate the disease. In contrast to DSS-induced colitis, defects in IL-12R and IL-23R both protect from the spontaneous colitis observed in IL10 mice. Together, these studies exemplify the complexity of genetic and environmental interactions for identifying biological pathways predictive of pathological inflammatory processes.
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http://dx.doi.org/10.1016/j.cyto.2019.154738DOI Listing
September 2019

Probing Inhomogeneous Diffusion in the Microenvironments of Phase-Separated Polymers under Confinement.

J Am Chem Soc 2019 05 6;141(19):7751-7757. Epub 2019 May 6.

Department of Physics , McGill University , Montreal , Quebec H3A 2T8 , Canada.

Biomolecular condensates formed by liquid-liquid phase separation of proteins and nucleic acids have been recently discovered to be prevalent in biology. These dynamic condensates behave like biochemical reaction vessels, but little is known about their structural organization and biophysical properties, which are likely related to condensate size. Thus, it is critical that we study them on scales found in vivo. However, previous in vitro studies of condensate assembly and physical properties have involved condensates up to 1000 times larger than those found in vivo. Here, we apply confinement microscopy to visualize condensates and control their sizes by creating appropriate confinement length scales relevant to the cell environment. We observe anomalous diffusion of probe particles embedded within confined condensates, as well as heterogeneous dynamics in condensates formed from PEG/dextran and in ribonucleoprotein complexes of RNA and the RNA-binding protein Dhh1. We propose that the observed non-Gaussian dynamics indicate a hopping diffusion mechanism inside condensates. We also observe that, for dextran-rich condensates, but not for ribonucleo condensates, probe particle diffusion depends on condensate size.
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http://dx.doi.org/10.1021/jacs.8b13349DOI Listing
May 2019

Predicted amino acid motif repeats in proteins potentially encode extensive multivalent macromolecular assemblies in the human proteome.

Curr Opin Struct Biol 2019 02 9;54:171-178. Epub 2019 Apr 9.

Department of Biochemistry, Faculty of Medicine, University of Montreal, Canada. Electronic address:

There are emerging interests in understanding higher order assemblies of biopolymers within and between cells, such as protein-protein and protein-RNA biomolecular condensates. These biomolecular condensates are thought to assemble/disassemble via multivalent interactions, including those mediated particularly by unique repeated amino acid motifs (URM). We asked how common are proteins with such URMs, their incidence and abundance, by exhaustively enumerating repeating motifs of length 3-10 in the human proteome. We found that URMs are very common and widely distributed across the human proteome. Moreover, the number of repetitions and intervals between them do not correlate with their lengths, which suggests that the number of repeats among proteins in the proteome is independent of length, contrary to the notion that short motifs are more abundant then long motifs. Finally, we describe two examples of URMs in proteins known to form higher order biopolymer assemblies: multi-PDZ domain-containing proteins and the FUS family of RNA binding proteins. For the FUS family, we predicted a known sequence 'grammar', specific motifs and interval sequence compositions that are essential to phase separation and material properties of condensates formed by this family of proteins. In PDZ domain-containing proteins we found a novel repeated motif that was surprisingly both within and between individual PDZ domains. We speculate that these motifs could be binding sites for multivalent interactions, a residual result of the mechanism by which PDZ-domain duplications occurred or that the linker sequences between PDZ domains may encode cryptic PDZ domains.
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http://dx.doi.org/10.1016/j.sbi.2019.01.026DOI Listing
February 2019

Author Correction: Genome-wide SWAp-Tag yeast libraries for proteome exploration.

Nat Methods 2019 Feb;16(2):205

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.
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http://dx.doi.org/10.1038/s41592-018-0297-3DOI Listing
February 2019

Tuberculosis and impaired IL-23-dependent IFN-γ immunity in humans homozygous for a common missense variant.

Sci Immunol 2018 12;3(30)

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA.

Inherited IL-12Rβ1 and TYK2 deficiencies impair both IL-12- and IL-23-dependent IFN-γ immunity and are rare monogenic causes of tuberculosis, each found in less than 1/600,000 individuals. We show that homozygosity for the common P1104A allele, which is found in about 1/600 Europeans and between 1/1000 and 1/10,000 individuals in regions other than East Asia, is more frequent in a cohort of patients with tuberculosis from endemic areas than in ethnicity-adjusted controls ( = 8.37 × 10; odds ratio, 89.31; 95% CI, 14.7 to 1725). Moreover, the frequency of P1104A in Europeans has decreased, from about 9% to 4.2%, over the past 4000 years, consistent with purging of this variant by endemic tuberculosis. Surprisingly, we also show that TYK2 P1104A impairs cellular responses to IL-23, but not to IFN-α, IL-10, or even IL-12, which, like IL-23, induces IFN-γ via activation of TYK2 and JAK2. Moreover, TYK2 P1104A is properly docked on cytokine receptors and can be phosphorylated by the proximal JAK, but lacks catalytic activity. Last, we show that the catalytic activity of TYK2 is essential for IL-23, but not IL-12, responses in cells expressing wild-type JAK2. In contrast, the catalytic activity of JAK2 is redundant for both IL-12 and IL-23 responses, because the catalytically inactive P1057A JAK2, which is also docked and phosphorylated, rescues signaling in cells expressing wild-type TYK2. In conclusion, homozygosity for the catalytically inactive P1104A missense variant of selectively disrupts the induction of IFN-γ by IL-23 and is a common monogenic etiology of tuberculosis.
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http://dx.doi.org/10.1126/sciimmunol.aau8714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341984PMC
December 2018

Changes of Cell Biochemical States Are Revealed in Protein Homomeric Complex Dynamics.

Cell 2018 11 25;175(5):1418-1429.e9. Epub 2018 Oct 25.

Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, QC H3C 3J7, Canada; Centre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, QC H3C 3J7, Canada. Electronic address:

We report here a simple and global strategy to map out gene functions and target pathways of drugs, toxins, or other small molecules based on "homomer dynamics" protein-fragment complementation assays (hdPCA). hdPCA measures changes in self-association (homomerization) of over 3,500 yeast proteins in yeast grown under different conditions. hdPCA complements genetic interaction measurements while eliminating the confounding effects of gene ablation. We demonstrate that hdPCA accurately predicts the effects of two longevity and health span-affecting drugs, the immunosuppressant rapamycin and the type 2 diabetes drug metformin, on cellular pathways. We also discovered an unsuspected global cellular response to metformin that resembles iron deficiency and includes a change in protein-bound iron levels. This discovery opens a new avenue to investigate molecular mechanisms for the prevention or treatment of diabetes, cancers, and other chronic diseases of aging.
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http://dx.doi.org/10.1016/j.cell.2018.09.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242466PMC
November 2018

Genome-wide SWAp-Tag yeast libraries for proteome exploration.

Nat Methods 2018 08 9;15(8):617-622. Epub 2018 Jul 9.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.
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http://dx.doi.org/10.1038/s41592-018-0044-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6076999PMC
August 2018

Mechanics, Structure and Function of Biopolymer Condensates.

J Mol Biol 2018 11 18;430(23):4754-4761. Epub 2018 Jun 18.

Département de Biochimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, Canada H3C 3J7; Centre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, Canada H3C 3J7. Electronic address:

The spontaneous nature of biopolymer phase separation in cells entails that the resulting condensates can be thermodynamic machines, which, in the process of condensing, can take on distinct forms themselves and deform neighboring cellular structures. We introduce here general notions of material and mechanical properties of protein condensates with an emphasis on how molecular arrangements and intermolecular interaction within condensates determine their ability to do work on their surroundings. We further propose functional implications of these concepts to cellular and subcellular morphology and biogenesis.
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http://dx.doi.org/10.1016/j.jmb.2018.06.023DOI Listing
November 2018

Why does biopolymer condensation matter?

Nat Rev Mol Cell Biol 2018 10;19(10):613-614

Département de Biochimie, Université de Montréal, Montréal, Québec, Canada.

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http://dx.doi.org/10.1038/s41580-018-0023-0DOI Listing
October 2018

More than One Way to Skin a Catalyst.

Cell Chem Biol 2017 Oct;24(10):1196-1197

Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, QC H3C 3J7, Canada; Centre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, QC H3C 3J7, Canada. Electronic address:

In this issue of Cell Chemical Biology, Diaz et al. (2017) report a strategy to achieve temporal, spatial, and stoichiometric control over the protein kinase cAbl in living cells. They achieve this by splitting cAbl into two inactive fragments that form an active kinase upon small molecule addition, potentially providing a general way to probe the wiring of signal transduction networks.
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http://dx.doi.org/10.1016/j.chembiol.2017.10.004DOI Listing
October 2017

Exhaustive search of linear information encoding protein-peptide recognition.

PLoS Comput Biol 2017 04 20;13(4):e1005499. Epub 2017 Apr 20.

Department of Biochemistry and Molecular Medicine, University of Montreal, Montreal, Quebec, Canada.

High-throughput in vitro methods have been extensively applied to identify linear information that encodes peptide recognition. However, these methods are limited in number of peptides, sequence variation, and length of peptides that can be explored, and often produce solutions that are not found in the cell. Despite the large number of methods developed to attempt addressing these issues, the exhaustive search of linear information encoding protein-peptide recognition has been so far physically unfeasible. Here, we describe a strategy, called DALEL, for the exhaustive search of linear sequence information encoded in proteins that bind to a common partner. We applied DALEL to explore binding specificity of SH3 domains in the budding yeast Saccharomyces cerevisiae. Using only the polypeptide sequences of SH3 domain binding proteins, we succeeded in identifying the majority of known SH3 binding sites previously discovered either in vitro or in vivo. Moreover, we discovered a number of sites with both non-canonical sequences and distinct properties that may serve ancillary roles in peptide recognition. We compared DALEL to a variety of state-of-the-art algorithms in the blind identification of known binding sites of the human Grb2 SH3 domain. We also benchmarked DALEL on curated biological motifs derived from the ELM database to evaluate the effect of increasing/decreasing the enrichment of the motifs. Our strategy can be applied in conjunction with experimental data of proteins interacting with a common partner to identify binding sites among them. Yet, our strategy can also be applied to any group of proteins of interest to identify enriched linear motifs or to exhaustively explore the space of linear information encoded in a polypeptide sequence. Finally, we have developed a webserver located at http://michnick.bcm.umontreal.ca/dalel, offering user-friendly interface and providing different scenarios utilizing DALEL.
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http://dx.doi.org/10.1371/journal.pcbi.1005499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5417721PMC
April 2017

Delineating functional principles of the bow tie structure of a kinase-phosphatase network in the budding yeast.

BMC Syst Biol 2017 03 16;11(1):38. Epub 2017 Mar 16.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, H3C 3J7, Canada.

Background: Kinases and phosphatases (KP) form complex self-regulating networks essential for cellular signal processing. In spite of having a wealth of data about interactions among KPs and their substrates, we have very limited models of the structures of the directed networks they form and consequently our ability to formulate hypotheses about how their structure determines the flow of information in these networks is restricted.

Results: We assembled and studied the largest bona fide kinase-phosphatase network (KP-Net) known to date for the yeast Saccharomyces cerevisiae. Application of the vertex sort (VS) algorithm on the KP-Net allowed us to elucidate its hierarchical structure in which nodes are sorted into top, core and bottom layers, forming a bow tie structure with a strongly connected core layer. Surprisingly, phosphatases tend to sort into the top layer, implying they are less regulated by phosphorylation than kinases. Superposition of the widest range of KP biological properties over the KP-Net hierarchy shows that core layer KPs: (i), receive the largest number of inputs; (ii), form bottlenecks implicated in multiple pathways and in decision-making; (iii), and are among the most regulated KPs both temporally and spatially. Moreover, top layer KPs are more abundant and less noisy than those in the bottom layer. Finally, we showed that the VS algorithm depends on node degrees without biasing the biological results of the sorted network. The VS algorithm is available as an R package ( https://cran.r-project.org/web/packages/VertexSort/index.html ).

Conclusions: The KP-Net model we propose possesses a bow tie hierarchical structure in which the top layer appears to ensure highest fidelity and the core layer appears to mediate signal integration and cell state-dependent signal interpretation. Our model of the yeast KP-Net provides both functional insight into its organization as we understand today and a framework for future investigation of information processing in yeast and eukaryotes in general.
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http://dx.doi.org/10.1186/s12918-017-0418-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5353956PMC
March 2017

Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

Cold Spring Harb Protoc 2016 11 1;2016(11). Epub 2016 Nov 1.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

Protein-fragment complementation assays (PCAs) comprise a family of assays that can be used to study protein-protein interactions (PPIs), conformation changes, and protein complex dimensions. We developed PCAs to provide simple and direct methods for the study of PPIs in any living cell, subcellular compartments or membranes, multicellular organisms, or in vitro. Because they are complete assays, requiring no cell-specific components other than reporter fragments, they can be applied in any context. PCAs provide a general strategy for the detection of proteins expressed at endogenous levels within appropriate subcellular compartments and with normal posttranslational modifications, in virtually any cell type or organism under any conditions. Here we introduce a number of applications of PCAs in budding yeast, Saccharomyces cerevisiae These applications represent the full range of PPI characteristics that might be studied, from simple detection on a large scale to visualization of spatiotemporal dynamics.
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http://dx.doi.org/10.1101/pdb.top083543DOI Listing
November 2016

Real-Time Protein-Fragment Complementation Assays for Studying Temporal, Spatial, and Spatiotemporal Dynamics of Protein-Protein Interactions in Living Cells.

Cold Spring Harb Protoc 2016 11 1;2016(11). Epub 2016 Nov 1.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

Here, we present detailed protocols for direct, real-time protein-fragment complementation assays (PCAs) for studying the spatiotemporal dynamics of protein-protein interactions (PPIs). The assays require the use of two fluorescent reporter proteins-the "Venus" version of yellow fluorescent protein (vYFP), and the monomeric infrared fluorescent protein 1.4 (IFP 1.4)-or two luciferase reporter proteins-Renilla (Rluc) and Gaussia (Gluc). The luciferase PCAs can be used to study the temporal dynamics of PPIs in any cellular compartment and on membranes. The full reversibility of these PCAs assures accurate measurements of the kinetics of PPI assembly/disassembly for processes that occur anywhere in a living cell and over time frames of seconds to hours. vYFP PCA, and all PCAs based on green fluorescent protein and its variants, are irreversible and can be used to trap and visualize rare and transient complexes and follow dynamic relocalization of constitutive complexes. vYFP PCA is limited in that accurate measurements of temporal changes in PPIs are not possible owing to the slow maturation time of vYFP (minutes to hours) and the irreversibility of its PCA that traps the complexes, thereby preventing the dissociation of PPIs that, in some instances, might cause spurious mislocalization of protein complexes. The limitations of vYFP PCA are overcome with IFP PCA, which is fully reversible and thus can be used to study spatiotemporal dynamics of PPIs on the timescale of seconds. All of these PCAs are sensitive enough to detect interactions among proteins expressed at endogenous levels in vivo.
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http://dx.doi.org/10.1101/pdb.prot090068DOI Listing
November 2016

Dissecting the Contingent Interactions of Protein Complexes with the Optimized Yeast Cytosine Deaminase Protein-Fragment Complementation Assay.

Cold Spring Harb Protoc 2016 11 1;2016(11). Epub 2016 Nov 1.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

Here, we present a detailed protocol for studying in yeast cells the contingent interaction between a substrate and its multisubunit enzyme complex by using a death selection technique known as the optimized yeast cytosine deaminase protein-fragment complementation assay (OyCD PCA). In yeast, the enzyme cytosine deaminase (encoded by FCY1) is involved in pyrimidine metabolism. The PCA is based on an engineered form of yeast cytosine deaminase optimized by directed evolution for maximum activity (OyCD), which acts as a reporter converting the pro-drug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a toxic compound that kills the cell. Cells that have OyCD PCA activity convert 5-FC to 5-FU and die. Using this assay, it is possible to assess how regulatory subunits of an enzyme contribute to the overall interaction between the catalytic subunit and the potential substrates. Furthermore, OyCD PCA can be used to dissect different functions of mutant forms of a protein as a mutant can disrupt interaction with one partner, while retaining interaction with others. As it is scalable to a medium- or high-throughput format, OyCD PCA can be used to study hundreds to thousands of pairwise protein-protein interactions in different deletion strains. In addition, OyCD PCA vectors (pAG413GAL1-ccdB-OyCD-F[1] and pAG415GAL1-ccdB-OyCD-F[2]) have been designed to be compatible with the proprietary Gateway technology. It is therefore easy to generate fusion genes with the OyCD reporter fragments. As an example, we will focus on the yeast cyclin-dependent protein kinase 1 (Cdk1, encoded by CDC28), its regulatory cyclin subunits, and its substrates or binding partners.
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http://dx.doi.org/10.1101/pdb.prot090043DOI Listing
November 2016

The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein-Protein Interactions.

Cold Spring Harb Protoc 2016 11 1;2016(11). Epub 2016 Nov 1.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

Protein-fragment complementation assays (PCAs) can be used to study protein-protein interactions (PPIs) in any living cell, in vivo or in vitro, in any subcellular compartment or membranes. Here, we present a detailed protocol for performing and analyzing a high-throughput PCA screening to study PPIs in yeast, using dihydrofolate reductase (DHFR) as the reporter protein. The DHFR PCA is a simple survival-selection assay in which Saccharomyces cerevisiae DHFR (scDHFR) is inhibited by methotrexate, thus preventing nucleotide synthesis and causing arrest of cell division. Complementation of cells with a methotrexate-insensitive murine DHFR restores nucleotide synthesis, allowing cell proliferation. The methotrexate-resistant DHFR has two mutations (L22F and F31S) and is 10,000 times less sensitive to methotrexate than wild-type scDHFR, but retains full catalytic activity. The DHFR PCA is sensitive enough for PPIs to be detected for open reading frame (ORF)-PCA fragments expressed off of their endogenous promoters.
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http://dx.doi.org/10.1101/pdb.prot090027DOI Listing
November 2016

Unravelling the Mechanism of TrkA-Induced Cell Death by Macropinocytosis in Medulloblastoma Daoy Cells.

Mol Cell Biol 2016 10 26;36(20):2596-611. Epub 2016 Sep 26.

Department of Biochemistry, Western University, London, Ontario, Canada Graduate Program in Neuroscience, Western University, London, Ontario, Canada

Macropinocytosis is a normal cellular process by which cells internalize extracellular fluids and nutrients from their environment and is one strategy that Ras-transformed pancreatic cancer cells use to increase uptake of amino acids to meet the needs of rapid growth. Paradoxically, in non-Ras transformed medulloblastoma brain tumors, we have shown that expression and activation of the receptor tyrosine kinase TrkA overactivates macropinocytosis, resulting in the catastrophic disintegration of the cell membrane and in tumor cell death. The molecular basis of this uncontrolled form of macropinocytosis has not been previously understood. Here, we demonstrate that the overactivation of macropinocytosis is caused by the simultaneous activation of two TrkA-mediated pathways: (i) inhibition of RhoB via phosphorylation at Ser(185) by casein kinase 1, which relieves actin stress fibers, and (ii) FRS2-scaffolded Src and H-Ras activation of RhoA, which stimulate actin reorganization and the formation of lamellipodia. Since catastrophic macropinocytosis results in brain tumor cell death, improved understanding of the mechanisms involved will facilitate future efforts to reprogram tumors, even those resistant to apoptosis, to die.
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http://dx.doi.org/10.1128/MCB.00255-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038151PMC
October 2016

Evolution of domain-peptide interactions to coadapt specificity and affinity to functional diversity.

Proc Natl Acad Sci U S A 2016 07 17;113(27):E3862-71. Epub 2016 Jun 17.

Department of Biochemistry and Molecular Medicine, University of Montreal, Montreal, QC, Canada H3T 1J4

Evolution of complexity in eukaryotic proteomes has arisen, in part, through emergence of modular independently folded domains mediating protein interactions via binding to short linear peptides in proteins. Over 30 years, structural properties and sequence preferences of these peptides have been extensively characterized. Less successful, however, were efforts to establish relationships between physicochemical properties and functions of domain-peptide interactions. To our knowledge, we have devised the first strategy to exhaustively explore the binding specificity of protein domain-peptide interactions. We applied the strategy to SH3 domains to determine the properties of their binding peptides starting from various experimental data. The strategy identified the majority (∼70%) of experimentally determined SH3 binding sites. We discovered mutual relationships among binding specificity, binding affinity, and structural properties and evolution of linear peptides. Remarkably, we found that these properties are also related to functional diversity, defined by depth of proteins within hierarchies of gene ontologies. Our results revealed that linear peptides evolved to coadapt specificity and affinity to functional diversity of domain-peptide interactions. Thus, domain-peptide interactions follow human-constructed gene ontologies, which suggest that our understanding of biological process hierarchies reflect the way chemical and thermodynamic properties of linear peptides and their interaction networks, in general, have evolved.
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http://dx.doi.org/10.1073/pnas.1518469113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941456PMC
July 2016

Mechanisms and Consequences of Macromolecular Phase Separation.

Cell 2016 May;165(5):1067-1079

Département de Biochimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada; Centre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada. Electronic address:

Over a century ago, colloidal phase separation of matter into non-membranous bodies was recognized as a fundamental organizing principal of cell "protoplasm." Recent insights into the molecular properties of such phase-separated bodies present challenges to our understanding of cellular protein interaction networks, as well as opportunities for interpreting and understanding of native and pathological genetic and molecular interactions. Here, we briefly review examples of and discuss physical principles of phase-separated cellular bodies and then reflect on how knowledge of these principles may direct future research on their functions.
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http://dx.doi.org/10.1016/j.cell.2016.05.026DOI Listing
May 2016

IL23R (Interleukin 23 Receptor) Variants Protective against Inflammatory Bowel Diseases (IBD) Display Loss of Function due to Impaired Protein Stability and Intracellular Trafficking.

J Biol Chem 2016 Apr 17;291(16):8673-85. Epub 2016 Feb 17.

From the Department of Biochemistry, University of Montreal, Montreal, Quebec H3C 3J7, Canada,

Genome-wide association studies as well as murine models have shown that the interleukin 23 receptor (IL23R) pathway plays a pivotal role in chronic inflammatory diseases such as Crohn disease (CD), ulcerative colitis, psoriasis, and type 1 diabetes. Genome-wide association studies and targeted re-sequencing studies have revealed the presence of multiple potentially causal variants of the IL23R. Specifically the G149R, V362I, and R381Q IL23Rα chain variants are linked to protection against the development of Crohn disease and ulcerative colitis in humans. Moreover, the exact mechanism of action of these receptor variants has not been elucidated. We show that all three of these IL23Rα variants cause a reduction in IL23 receptor activation-mediated phosphorylation of the signal-transducing activator of transcription 3 (STAT3) and phosphorylation of signal transducing activator of transcription 4 (STAT4). The reduction in signaling is due to lower levels of cell surface receptor expression. For G149R, the receptor retention in the endoplasmic reticulum is due to an impairment of receptor maturation, whereas the R381Q and V362I variants have reduced protein stability. Finally, we demonstrate that the endogenous expression of IL23Rα protein from V362I and R381Q variants in human lymphoblastoid cell lines exhibited lower expression levels relative to susceptibility alleles. Our results suggest a convergent cause of IL23Rα variant protection against chronic inflammatory disease.
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http://dx.doi.org/10.1074/jbc.M116.715870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861437PMC
April 2016

Protein Kinase C ζ Interacts with a Novel Binding Region of Gαq to Act as a Functional Effector.

J Biol Chem 2016 Apr 17;291(18):9513-25. Epub 2016 Feb 17.

From the Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa," CSIC-UAM, Universidad Autónoma de Madrid, 28049-Madrid, Spain, Instituto de Investigación Sanitaria La Princesa, 29006-Madrid, Spain,

Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C ζ (PKCζ), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLCβ. We report herein that the activation-dependent Gαq/PKCζ complex involves the basic PB1-type II domain of PKCζ and a novel interaction module in Gαq different from the classical effector-binding site. Point mutations in this Gαq region completely abrogate ERK5 phosphorylation, indicating that Gαq/PKCζ association is required for the activation of the pathway. Indeed, PKCζ was demonstrated to directly bind ERK5 thus acting as a scaffold between Gαq and ERK5 upon GPCR activation. The inhibition of these protein complexes by G protein-coupled receptor kinase 2, a known Gαq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal that Gαq/PKCζ complexes link Gαq to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in Gαq and the effector PKCζ is a key event in Gαq signaling.
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http://dx.doi.org/10.1074/jbc.M115.684308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850291PMC
April 2016

Combining the Optimized Yeast Cytosine Deaminase Protein Fragment Complementation Assay and an In Vitro Cdk1 Targeting Assay to Study the Regulation of the γ-Tubulin Complex.

Methods Mol Biol 2016 ;1342:237-57

Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada, H3C 3J7.

Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, and associate these complexes to the appropriate cyclins using a cell-based protein-fragment complementation assay. Validation of the specific reliance of the OyCD interaction between Cdk1 and budding yeast γ-tubulin on the Clb3 cyclin, relative to the mitotic Clb2 cyclin, was performed by an in vitro kinase assay using the γ-tubulin complex as a substrate.
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http://dx.doi.org/10.1007/978-1-4939-2957-3_14DOI Listing
May 2016

In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs.

Sci Rep 2015 Jun 23;5:11133. Epub 2015 Jun 23.

Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.

Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.
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http://dx.doi.org/10.1038/srep11133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477410PMC
June 2015

Mapping biochemical networks with protein fragment complementation assays.

Methods Mol Biol 2015 ;1278:467-81

Département de Biochimie, Université de Montréal, C.P. 6128, succursale centre-ville, Montréal, QC, Canada, H3C 3J7.

Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. Although our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells. The experimental strategy is based on Protein fragment Complementation Assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, etc.). In this chapter we review some of the essential principles of PCA and provide details and protocols for applications of PCA, particularly in mammalian cells, based on three PCA reporters, dihydrofolate reductase, green fluorescent protein, and β-lactamase.
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http://dx.doi.org/10.1007/978-1-4939-2425-7_31DOI Listing
December 2015

A cell-signaling network temporally resolves specific versus promiscuous phosphorylation.

Cell Rep 2015 Feb 19;10(7):1202-14. Epub 2015 Feb 19.

Département de Biochimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, QC H3C 3J7, Canada; Centre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, QC H3C 3J7, Canada. Electronic address:

If specific and functional kinase- or phosphatase-substrate interactions are optimized for binding compared to promiscuous interactions, then changes in phosphorylation should occur faster on functional versus promiscuous substrates. To test this hypothesis, we designed a high temporal resolution global phosphoproteomics protocol to study the high-osmolarity glycerol (HOG) response in the budding yeast Saccharomyces cerevisiae. The method provides accurate, stimulus-specific measurement of phosphoproteome changes, quantitative analysis of phosphodynamics at sub-minute temporal resolution, and detection of more phosphosites. Rates of evolution of dynamic phosphosites were comparable to those of known functional phosphosites and significantly lower than static or longer-time-frame dynamic phosphosites. Kinetic profile analyses indicated that putatively functional kinase- or phosphatase-substrate interactions occur more rapidly, within 60 s, than promiscuous interactions. Finally, we report many changes in phosphorylation of proteins implicated in cytoskeletal and mitotic spindle dynamics that may underlie regulation of cell cycle and morphogenesis.
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http://dx.doi.org/10.1016/j.celrep.2015.01.052DOI Listing
February 2015

Fast and accurate discovery of degenerate linear motifs in protein sequences.

PLoS One 2014 10;9(9):e106081. Epub 2014 Sep 10.

Département de Biochimie and Centre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal, Succursale Centre-Ville, Montreal, Quebec, Canada.

Linear motifs mediate a wide variety of cellular functions, which makes their characterization in protein sequences crucial to understanding cellular systems. However, the short length and degenerate nature of linear motifs make their discovery a difficult problem. Here, we introduce MotifHound, an algorithm particularly suited for the discovery of small and degenerate linear motifs. MotifHound performs an exact and exhaustive enumeration of all motifs present in proteins of interest, including all of their degenerate forms, and scores the overrepresentation of each motif based on its occurrence in proteins of interest relative to a background (e.g., proteome) using the hypergeometric distribution. To assess MotifHound, we benchmarked it together with state-of-the-art algorithms. The benchmark consists of 11,880 sets of proteins from S. cerevisiae; in each set, we artificially spiked-in one motif varying in terms of three key parameters, (i) number of occurrences, (ii) length and (iii) the number of degenerate or "wildcard" positions. The benchmark enabled the evaluation of the impact of these three properties on the performance of the different algorithms. The results showed that MotifHound and SLiMFinder were the most accurate in detecting degenerate linear motifs. Interestingly, MotifHound was 15 to 20 times faster at comparable accuracy and performed best in the discovery of highly degenerate motifs. We complemented the benchmark by an analysis of proteins experimentally shown to bind the FUS1 SH3 domain from S. cerevisiae. Using the full-length protein partners as sole information, MotifHound recapitulated most experimentally determined motifs binding to the FUS1 SH3 domain. Moreover, these motifs exhibited properties typical of SH3 binding peptides, e.g., high intrinsic disorder and evolutionary conservation, despite the fact that none of these properties were used as prior information. MotifHound is available (http://michnick.bcm.umontreal.ca or http://tinyurl.com/motifhound) together with the benchmark that can be used as a reference to assess future developments in motif discovery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0106081PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160167PMC
May 2015

High-resolution mapping of protein concentration reveals principles of proteome architecture and adaptation.

Cell Rep 2014 May 10;7(4):1333-40. Epub 2014 May 10.

Département de Biochimie and Centre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montreal, QC H3C 3J7, Canada. Electronic address:

A single yeast cell contains a hundred million protein molecules. How these proteins are organized to orchestrate living processes is a central question in biology. To probe this organization in vivo, we measured the local concentration of proteins based on the strength of their nonspecific interactions with a neutral reporter protein. We first used a cytosolic reporter and measured local concentrations for ~2,000 proteins in S. cerevisiae, with accuracy comparable to that of mass spectrometry. Localizing the reporter to membranes specifically increased the local concentration measured for membrane proteins. Comparing the concentrations measured by both reporters revealed that encounter frequencies between proteins are primarily dictated by their abundances. However, to change these encounter frequencies and restructure the proteome, as in adaptation, we find that changes in localization have more impact than changes in abundance. These results highlight how protein abundance and localization contribute to proteome organization and reorganization.
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http://dx.doi.org/10.1016/j.celrep.2014.04.009DOI Listing
May 2014

An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions.

Nat Methods 2014 Jun 20;11(6):641-4. Epub 2014 Apr 20.

Département de Biochimie, Université de Montréal, Montréal, Québec, Canada.

We report a protein-fragment complementation assay (PCA) based on the engineered Deinococcus radiodurans infrared fluorescent protein IFP1.4. Unlike previous fluorescent protein PCAs, the IFP PCA is reversible, allowing analysis of spatiotemporal dynamics of hormone-induced signaling complexes in living yeast and mammalian cells at nanometer resolution. The inherently low background of infrared fluorescence permitted detection of subcellular reorganization of a signaling complex expressed at low abundance.
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http://dx.doi.org/10.1038/nmeth.2934DOI Listing
June 2014