Publications by authors named "Matthew Jessulat"

29 Publications

  • Page 1 of 1

The conserved Tpk1 regulates non-homologous end joining double-strand break repair by phosphorylation of Nej1, a homolog of the human XLF.

Nucleic Acids Res 2021 Jul 9. Epub 2021 Jul 9.

Department of Biochemistry, University of Regina, Regina, Saskatchewan S4S 0A2, Canada.

The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine-threonine kinase, encompassing three catalytic (Tpk1-3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.
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http://dx.doi.org/10.1093/nar/gkab585DOI Listing
July 2021

Deletion of yeast TPK1 reduces the efficiency of non-homologous end joining DNA repair.

Biochem Biophys Res Commun 2020 12 30;533(4):899-904. Epub 2020 Sep 30.

Department of Biology, Carleton University, Ottawa, Ontario, Canada; Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada. Electronic address:

Non-homologous end joining (NHEJ) is a highly conserved mechanism of DNA double-stranded break (DSB) repair. Here we utilize a computational protein-protein interaction method to identify human PRKACB as a potential candidate interacting with NHEJ proteins. We show that the deletion of its yeast homolog, TPK1 that codes for the protein kinase A catalytic subunit reduces the efficiency of NHEJ repair of breaks with overhangs and blunt ends in plasmid-based repair assays. Additionally, tpk1Δ mutants showed defects in the repair of chromosomal breaks induced by HO-site specific endonuclease. Our double deletion mutant analyses suggest that TPK1 and YKU80, a key player in NHEJ could function in parallel pathways. Altogether, here we report a novel involvement for TPK1 in NHEJ.
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http://dx.doi.org/10.1016/j.bbrc.2020.09.083DOI Listing
December 2020

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

Misconnecting the dots: altered mitochondrial protein-protein interactions and their role in neurodegenerative disorders.

Expert Rev Proteomics 2020 02 6;17(2):119-136. Epub 2020 Feb 6.

Department of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.

: Mitochondria (mt) are protein-protein interaction (PPI) hubs in the cell where mt-localized and associated proteins interact in a fashion critical for cell fitness. Altered mtPPIs are linked to neurodegenerative disorders (NDs) and drivers of pathological associations to mediate ND progression. Mapping altered mtPPIs will reveal how mt dysfunction is linked to NDs.: This review discusses how database sources reflect on the number of mt protein or interaction predictions, and serves as an update on mtPPIs in mt dynamics and homeostasis. Emphasis is given to mRNA expression profiles for mt proteins in human tissues, cellular models relevant to NDs, and altered mtPPIs in NDs such as Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD).: We highlight the scarcity of biomarkers to improve diagnostic accuracy and tracking of ND progression, obstacles in recapitulating NDs using human cellular models to underpin the pathophysiological mechanisms of disease, and the shortage of mt protein interactome reference database(s) of neuronal cells. These bottlenecks are addressed by improvements in induced pluripotent stem cell creation and culturing, patient-derived 3D brain organoids to recapitulate structural arrangements of the brain, and cell sorting to elucidate mt proteome disparities between cell types.
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http://dx.doi.org/10.1080/14789450.2020.1723419DOI Listing
February 2020

Rewiring of the Human Mitochondrial Interactome during Neuronal Reprogramming Reveals Regulators of the Respirasome and Neurogenesis.

iScience 2019 Sep 4;19:1114-1132. Epub 2019 Sep 4.

Department of Biochemistry, University of Regina, Regina, SK S4S 0A2, Canada. Electronic address:

Mitochondrial protein (MP) assemblies undergo alterations during neurogenesis, a complex process vital in brain homeostasis and disease. Yet which MP assemblies remodel during differentiation remains unclear. Here, using mass spectrometry-based co-fractionation profiles and phosphoproteomics, we generated mitochondrial interaction maps of human pluripotent embryonal carcinoma stem cells and differentiated neuronal-like cells, which presented as two discrete cell populations by single-cell RNA sequencing. The resulting networks, encompassing 6,442 high-quality associations among 600 MPs, revealed widespread changes in mitochondrial interactions and site-specific phosphorylation during neuronal differentiation. By leveraging the networks, we show the orphan C20orf24 as a respirasome assembly factor whose disruption markedly reduces respiratory chain activity in patients deficient in complex IV. We also find that a heme-containing neurotrophic factor, neuron-derived neurotrophic factor [NENF], couples with Parkinson disease-related proteins to promote neurotrophic activity. Our results provide insights into the dynamic reorganization of mitochondrial networks during neuronal differentiation and highlights mechanisms for MPs in respirasome, neuronal function, and mitochondrial diseases.
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http://dx.doi.org/10.1016/j.isci.2019.08.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831851PMC
September 2019

A Tag-Based Affinity Purification Mass Spectrometry Workflow for Systematic Isolation of the Human Mitochondrial Protein Complexes.

Adv Exp Med Biol 2019 ;1158:83-100

Department of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.

Mitochondria (mt) are double-membraned, dynamic organelles that play an essential role in a large number of cellular processes, and impairments in mt function have emerged as a causative factor for a growing number of human disorders. Given that most biological functions are driven by physical associations between proteins, the first step towards understanding mt dysfunction is to map its protein-protein interaction (PPI) network in a comprehensive and systematic fashion. While mass-spectrometry (MS) based approaches possess the high sensitivity ideal for such an endeavor, it also requires stringent biochemical purification of bait proteins to avoid detecting spurious, non-specific PPIs. Here, we outline a tagging-based affinity purification coupled with mass spectrometry (AP-MS) workflow for discovering new mt protein associations and providing novel insights into their role in mt biology and human physiology/pathology. Because AP-MS relies on the creation of proteins fused with affinity tags, we employ a versatile-affinity (VA) tag, consisting of 3× FLAG, 6 × His, and Strep III epitopes. For efficient delivery of affinity-tagged open reading frames (ORF) into mammalian cells, the VA-tag is cloned onto a specific ORF using Gateway recombinant cloning, and the resulting expression vector is stably introduced in target cells using lentiviral transduction. In this chapter, we show a functional workflow for mapping the mt interactome that includes tagging, stable transduction, selection and expansion of mammalian cell lines, mt extraction, identification of interacting protein partners by AP-MS, and lastly, computational assessment of protein complexes/PPI networks.
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http://dx.doi.org/10.1007/978-981-13-8367-0_6DOI Listing
September 2019

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

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

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

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

In Silico Engineering of Synthetic Binding Proteins from Random Amino Acid Sequences.

iScience 2019 Jan 4;11:375-387. Epub 2018 Dec 4.

Department of Biology, Carleton University, Ottawa, ON K1S5B6, Canada; Ottawa Institute of Systems Biology, Carleton University, Ottawa, ON K1S5B6, Canada; Institute of Biochemistry, Carleton University, Ottawa, ON K1S5B6, Canada. Electronic address:

Synthetic proteins with high affinity and selectivity for a protein target can be used as research tools, biomarkers, and pharmacological agents, but few methods exist to design such proteins de novo. To this end, the In-Silico Protein Synthesizer (InSiPS) was developed to design synthetic binding proteins (SBPs) that bind pre-determined targets while minimizing off-target interactions. InSiPS is a genetic algorithm that refines a pool of random sequences over hundreds of generations of mutation and selection to produce SBPs with pre-specified binding characteristics. As a proof of concept, we design SBPs against three yeast proteins and demonstrate binding and functional inhibition of two of three targets in vivo. Peptide SPOT arrays confirm binding sites, and a permutation array demonstrates target specificity. Our foundational approach will support the field of de novo design of small binding polypeptide motifs and has robust applicability while offering potential advantages over the limited number of techniques currently available.
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http://dx.doi.org/10.1016/j.isci.2018.11.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348295PMC
January 2019

A Map of Human Mitochondrial Protein Interactions Linked to Neurodegeneration Reveals New Mechanisms of Redox Homeostasis and NF-κB Signaling.

Cell Syst 2017 12 8;5(6):564-577.e12. Epub 2017 Nov 8.

Department of Biochemistry, University of Regina, Regina, SK S4S 0A2, Canada. Electronic address:

Mitochondrial protein (MP) dysfunction has been linked to neurodegenerative disorders (NDs); however, the discovery of the molecular mechanisms underlying NDs has been impeded by the limited characterization of interactions governing MP function. Here, using mass spectrometry (MS)-based analysis of 210 affinity-purified mitochondrial (mt) fractions isolated from 27 epitope-tagged human ND-linked MPs in HEK293 cells, we report a high-confidence MP network including 1,964 interactions among 772 proteins (>90% previously unreported). Nearly three-fourths of these interactions were confirmed in mouse brain and multiple human differentiated neuronal cell lines by primary antibody immunoprecipitation and MS, with many linked to NDs and autism. We show that the SOD1-PRDX5 interaction, critical for mt redox homeostasis, can be perturbed by amyotrophic lateral sclerosis-linked SOD1 allelic variants and establish a functional role for ND-linked factors coupled with IκBɛ in NF-κB activation. Our results identify mechanisms for ND-linked MPs and expand the human mt interaction landscape.
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http://dx.doi.org/10.1016/j.cels.2017.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746455PMC
December 2017

Uncharacterized ORF HUR1 influences the efficiency of non-homologous end-joining repair in Saccharomyces cerevisiae.

Gene 2018 Jan 5;639:128-136. Epub 2017 Oct 5.

Department of Biology, Carleton University, Ottawa, Ontario, Canada; Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada. Electronic address:

Non-Homologous End Joining (NHEJ) is a highly conserved pathway that repairs Double-Strand Breaks (DSBs) within DNA. Here we show that the deletion of yeast uncharacterized ORF HUR1, Hydroxyurea Resistance1 affects the efficiency of NHEJ. Our findings are supported by Protein-Protein Interaction (PPI), genetic interaction and drug sensitivity analyses. To assess the activity of HUR1 in DSB repair, we deleted its non-overlapping region with PMR1, referred to as HUR1-A. We observed that similar to deletion of TPK1 and NEJ1, and unlike YKU70 (important for NHEJ of DNA with overhang and not blunt end), deletion of HUR1-A reduced the efficiency of NHEJ in both overhang and blunt end plasmid repair assays. Similarly, a chromosomal repair assay showed a reduction for repair efficiency when HUR1-A was deleted. In agreement with a functional connection for Hur1p with Tpk1p and NEJ1p, double mutant strains Δhur1-A/Δtpk1, and Δhur1-A/Δnej1 showed the same reduction in the efficiency of plasmid repair, compared to both single deletion strains. Also, using a Homologous Recombination (HR) specific plasmid-based DSB repair assay we observed that deletion of HUR1-A influenced the efficiency of HR repair, suggesting that HUR1 might also play additional roles in other DNA repair pathways.
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http://dx.doi.org/10.1016/j.gene.2017.10.003DOI Listing
January 2018

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

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

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

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

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining.

Mol Cell Biol 2015 Jul 11;35(14):2448-63. Epub 2015 May 11.

Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina, Saskatchewan, Canada

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.
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http://dx.doi.org/10.1128/MCB.00007-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475915PMC
July 2015

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

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

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

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

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

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

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

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

Yeast mitochondrial protein-protein interactions reveal diverse complexes and disease-relevant functional relationships.

J Proteome Res 2015 Feb 20;14(2):1220-37. Epub 2015 Jan 20.

Terrence Donnelly Centre, University of Toronto , Toronto, Ontario M5S 3E1, Canada.

Although detailed, focused, and mechanistic analyses of associations among mitochondrial proteins (MPs) have identified their importance in varied biological processes, a systematic understanding of how MPs function in concert both with one another and with extra-mitochondrial proteins remains incomplete. Consequently, many questions regarding the role of mitochondrial dysfunction in the development of human disease remain unanswered. To address this, we compiled all existing mitochondrial physical interaction data for over 1200 experimentally defined yeast MPs and, through bioinformatic analysis, identified hundreds of heteromeric MP complexes having extensive associations both within and outside the mitochondria. We provide support for these complexes through structure prediction analysis, morphological comparisons of deletion strains, and protein co-immunoprecipitation. The integration of these MP complexes with reported genetic interaction data reveals substantial crosstalk between MPs and non-MPs and identifies novel factors in endoplasmic reticulum-mitochondrial organization, membrane structure, and mitochondrial lipid homeostasis. More than one-third of these MP complexes are conserved in humans, with many containing members linked to clinical pathologies, enabling us to identify genes with putative disease function through guilt-by-association. Although still remaining incomplete, existing mitochondrial interaction data suggests that the relevant molecular machinery is modular, yet highly integrated with non-mitochondrial processes.
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http://dx.doi.org/10.1021/pr501148qDOI Listing
February 2015

Mitochondrial targets for pharmacological intervention in human disease.

J Proteome Res 2015 Jan 12;14(1):5-21. Epub 2014 Dec 12.

Department of Biochemistry, Research and Innovation Centre, University of Regina , Regina, Saskatchewan S4S 0A2, Canada.

Over the past several years, mitochondrial dysfunction has been linked to an increasing number of human illnesses, making mitochondrial proteins (MPs) an ever more appealing target for therapeutic intervention. With 20% of the mitochondrial proteome (312 of an estimated 1500 MPs) having known interactions with small molecules, MPs appear to be highly targetable. Yet, despite these targeted proteins functioning in a range of biological processes (including induction of apoptosis, calcium homeostasis, and metabolism), very few of the compounds targeting MPs find clinical use. Recent work has greatly expanded the number of proteins known to localize to the mitochondria and has generated a considerable increase in MP 3D structures available in public databases, allowing experimental screening and in silico prediction of mitochondrial drug targets on an unprecedented scale. Here, we summarize the current literature on clinically active drugs that target MPs, with a focus on how existing drug targets are distributed across biochemical pathways and organelle substructures. Also, we examine current strategies for mitochondrial drug discovery, focusing on genetic, proteomic, and chemogenomic assays, and relevant model systems. As cell models and screening techniques improve, MPs appear poised to emerge as relevant targets for a wide range of complex human diseases, an eventuality that can be expedited through systematic analysis of MP function.
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http://dx.doi.org/10.1021/pr500813fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286170PMC
January 2015

A global investigation of gene deletion strains that affect premature stop codon bypass in yeast, Saccharomyces cerevisiae.

Mol Biosyst 2014 Apr 18;10(4):916-24. Epub 2014 Feb 18.

Department of Biology and Ottawa Institute of Systems Biology, Carleton University, Ottawa, Ontario, Canada.

Protein biosynthesis is an orderly process that requires a balance between rate and accuracy. To produce a functional product, the fidelity of this process has to be maintained from start to finish. In order to systematically identify genes that affect stop codon bypass, three expression plasmids, pUKC817, pUKC818 and pUKC819, were integrated into the yeast non-essential loss-of-function gene array (5000 strains). These plasmids contain three different premature stop codons (UAA, UGA and UAG, respectively) within the LacZ expression cassette. A fourth plasmid, pUKC815 that carries the native LacZ gene was used as a control. Transformed strains were subjected to large-scale β-galactosidase lift assay analysis to evaluate production of β-galactosidase for each gene deletion strain. In this way 84 potential candidate genes that affect stop codon bypass were identified. Three candidate genes, OLA1, BSC2, and YNL040W, were further investigated, and were found to be important for cytoplasmic protein biosynthesis.
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http://dx.doi.org/10.1039/c3mb70501cDOI Listing
April 2014

Phosphatase complex Pph3/Psy2 is involved in regulation of efficient non-homologous end-joining pathway in the yeast Saccharomyces cerevisiae.

PLoS One 2014 31;9(1):e87248. Epub 2014 Jan 31.

Department of Biology, Carleton University, Ottawa, Ontario, Canada ; Ottawa Institute of Systems Biology, Carleton University, Ottawa, Ontario, Canada.

One of the main mechanisms for double stranded DNA break (DSB) repair is through the non-homologous end-joining (NHEJ) pathway. Using plasmid and chromosomal repair assays, we showed that deletion mutant strains for interacting proteins Pph3p and Psy2p had reduced efficiencies in NHEJ. We further observed that this activity of Pph3p and Psy2p appeared linked to cell cycle Rad53p and Chk1p checkpoint proteins. Pph3/Psy2 is a phosphatase complex, which regulates recovery from the Rad53p DNA damage checkpoint. Overexpression of Chk1p checkpoint protein in a parallel pathway to Rad53p compensated for the deletion of PPH3 or PSY2 in a chromosomal repair assay. Double mutant strains Δpph3/Δchk1 and Δpsy2/Δchk1 showed additional reductions in the efficiency of plasmid repair, compared to both single deletions which is in agreement with the activity of Pph3p and Psy2p in a parallel pathway to Chk1p. Genetic interaction analyses also supported a role for Pph3p and Psy2p in DNA damage repair, the NHEJ pathway, as well as cell cycle progression. Collectively, we report that the activity of Pph3p and Psy2p further connects NHEJ repair to cell cycle progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087248PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909046PMC
October 2014

Mapping the functional yeast ABC transporter interactome.

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

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

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

Recent advances in protein-protein interaction prediction: experimental and computational methods.

Expert Opin Drug Discov 2011 Sep 29;6(9):921-35. Epub 2011 Jul 29.

Carleton University , Department of Biology , 209 Nesbitt Building, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6 , Canada

Introduction: Proteins within the cell act as part of complex networks, which allow pathways and processes to function. Therefore, understanding how proteins interact is a significant area of current research.

Areas Covered: This review aims to present an overview of key experimental techniques (yeast two-hybrid, tandem affinity purification and protein microarrays) used to discover protein-protein interactions (PPIs), as well as to briefly discuss certain computational methods for predicting protein interactions based on gene localization, phylogenetic information, 3D structural modeling or primary protein sequence data. Due to the large-scale applicability of primary sequence-based methods, the authors have chosen to focus on this strategy for our review. There is an emphasis on a recent algorithm called Protein Interaction Prediction Engine (PIPE) that can predict global PPIs. The readers will discover recent advances both in the practical determination of protein interaction and the strategies that are available to attempt to anticipate interactions without the time and costs of experimental work.

Expert Opinion: Global PPI maps can help understand the biology of complex diseases and facilitate the identification of novel drug target sites. This study describes different techniques used for PPI prediction that we believe will significantly impact the development of the field in a new future. We expect to see a growing number of similar techniques capable of large-scale PPI predictions.
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http://dx.doi.org/10.1517/17460441.2011.603722DOI Listing
September 2011

Short Co-occurring Polypeptide Regions Can Predict Global Protein Interaction Maps.

Sci Rep 2012 30;2:239. Epub 2012 Jan 30.

A goal of the post-genomics era has been to elucidate a detailed global map of protein-protein interactions (PPIs) within a cell. Here, we show that the presence of co-occurring short polypeptide sequences between interacting protein partners appears to be conserved across different organisms. We present an algorithm to automatically generate PPI prediction method parameters for various organisms and illustrate that global PPIs can be predicted from previously reported PPIs within the same or a different organism using protein primary sequences. The PPI prediction code is further accelerated through the use of parallel multi-core programming, which improves its usability for large scale or proteome-wide PPI prediction. We predict and analyze hundreds of novel human PPIs, experimentally confirm protein functions and importantly predict the first genome-wide PPI maps for S. pombe (∼9,000 PPIs) and C. elegans (∼37,500 PPIs).
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http://dx.doi.org/10.1038/srep00239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269044PMC
September 2012

Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

PLoS One 2011 Apr 27;6(4):e18510. Epub 2011 Apr 27.

Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.

Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018510PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083400PMC
April 2011

Chemical-genetic profile analysis of five inhibitory compounds in yeast.

BMC Chem Biol 2010 Aug 6;10. Epub 2010 Aug 6.

Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, K1 S 5B6, ON, Canada.

Background: Chemical-genetic profiling of inhibitory compounds can lead to identification of their modes of action. These profiles can help elucidate the complex interactions between small bioactive compounds and the cell machinery, and explain putative gene function(s).

Results: Colony size reduction was used to investigate the chemical-genetic profile of cycloheximide, 3-amino-1,2,4-triazole, paromomycin, streptomycin and neomycin in the yeast Saccharomyces cerevisiae. These compounds target the process of protein biosynthesis. More than 70,000 strains were analyzed from the array of gene deletion mutant yeast strains. As expected, the overall profiles of the tested compounds were similar, with deletions for genes involved in protein biosynthesis being the major category followed by metabolism. This implies that novel genes involved in protein biosynthesis could be identified from these profiles. Further investigations were carried out to assess the activity of three profiled genes in the process of protein biosynthesis using relative fitness of double mutants and other genetic assays.

Conclusion: Chemical-genetic profiles provide insight into the molecular mechanism(s) of the examined compounds by elucidating their potential primary and secondary cellular target sites. Our follow-up investigations into the activity of three profiled genes in the process of protein biosynthesis provided further evidence concerning the usefulness of chemical-genetic analyses for annotating gene functions. We termed these genes TAE2, TAE3 and TAE4 for translation associated elements 2-4.
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http://dx.doi.org/10.1186/1472-6769-10-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925817PMC
August 2010

Disruption of fungal cell wall by antifungal Echinacea extracts.

Med Mycol 2010 Nov;48(7):949-58

Biology Department, Carleton University, Ottawa, Ontario, Canada.

In addition to widespread use in reducing the symptoms of colds and flu, Echinacea is traditionally employed to treat fungal and bacterial infections. However, to date the mechanism of antimicrobial activity of Echinacea extracts remains unclear. We utilized a set of ∼4,600 viable gene deletion mutants of Saccharomyces cerevisiae to identify mutations that increase sensitivity to Echinacea. Thus, a set of chemical-genetic profiles for 16 different Echinacea treatments was generated, from which a consensus set of 23 Echinacea-sensitive mutants was identified. Of the 23 mutants, only 16 have a reported function. Ten of these 16 are involved in cell wall integrity/structure suggesting that a target for Echinacea is the fungal cell wall. Follow-up analyses revealed an increase in sonication-associated cell death in the yeasts S. cerevisiae and Cryptococcus neoformans after Echinacea extract treatments. Furthermore, fluorescence microscopy showed that Echinacea-treated S. cerevisiae was significantly more prone to cell wall damage than non-treated cells. This study further demonstrates the potential of gene deletion arrays to understand natural product antifungal mode of action and provides compelling evidence that the fungal cell wall is a target of Echinacea extracts and may thus explain the utility of this phytomedicine in treating mycoses.
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http://dx.doi.org/10.3109/13693781003767584DOI Listing
November 2010

In vivo investigation of protein-protein interactions for helicases using tandem affinity purification.

Methods Mol Biol 2010 ;587:99-111

Department of Biology and Ottawa Institute of Systems Biology, Carleton University, Ottawa, ON, Canada.

A key component in determining the functional role of any protein is the elucidation of its binding partners using protein-protein interaction (PPI) data. Here we examine the use of tandem affinity purification (TAP) tagging to study RNA/DNA helicase PPIs in Escherichia coli. The tag, which consists of a calmodulin-binding region, a TEV protease recognition sequence, and an IgG-binding domain, is introduced into E. coli using a lambdared recombination system. This method prevents the overproduction of the target protein, which could generate false interactions. The interacting proteins are then affinity purified using double affinity purification steps and are separated by SDS-PAGE followed by mass spectrometry identification. Each protein identified would represent a physical interaction in the cell. These interactions may potentially be mediated by an RNA/DNA template, for which the helicase would likely be needed to disrupt the secondary structures.
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http://dx.doi.org/10.1007/978-1-60327-355-8_7DOI Listing
May 2010

Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis.

BMC Genomics 2008 Dec 3;9:583. Epub 2008 Dec 3.

Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, K1S 5B6, Canada.

Background: Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s) for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, approximately 4700 strains) for increased sensitivity to paromomycin, which targets the process of protein synthesis.

Results: As expected, our analysis indicated that the majority of deletion strains (134) with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45), cellular component biogenesis and organization (28), DNA maintenance (21), transport (20), others (38) and unknown (39). These may represent minor cellular target sites (side-effects) for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis.

Conclusion: We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s).
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http://dx.doi.org/10.1186/1471-2164-9-583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613417PMC
December 2008

Interacting proteins Rtt109 and Vps75 affect the efficiency of non-homologous end-joining in Saccharomyces cerevisiae.

Arch Biochem Biophys 2008 Jan 9;469(2):157-64. Epub 2007 Nov 9.

Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., Canada K1S 5B6.

One of the key pathways for DNA double-stranded break (DSB) repair is the non-homologous end-joining (NHEJ) pathway, which directly re-ligates two broken ends of DNA. Using a plasmid repair assay screen, we identified that the deletion strain for RTT109 had a reduced efficiency for NHEJ in yeast. This deletion strain also had a reduced efficiency to repair induced chromosomal DSBs in vivo. Tandem-affinity purification of Rtt109 recovered Vps75 as a physical interacting protein. Deletion of VPS75 was also shown to have an effect on the efficiency of NHEJ in both the plasmid repair and the chromosomal repair assays. In addition, deletion mutants for both RTT109 and VPS75 showed hypersensitivity to different DNA damaging agents. Our genetic interaction analysis supports a role for RTT109 in DNA damage repair. We propose that one function of the Rtt109-Vps75 interacting protein pair is to affect the efficiency of NHEJ in yeast. Vps75 but not Rtt109 also seem to have an effect on the efficiency of DSB repair using homologous recombination.
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http://dx.doi.org/10.1016/j.abb.2007.11.001DOI Listing
January 2008

Antifungal and antioxidant activities of the phytomedicine pipsissewa, Chimaphila umbellata.

Phytochemistry 2008 Feb 22;69(3):738-46. Epub 2007 Oct 22.

Chemistry Department, Universidad Autónoma de Aguascalientes, Av. Universidad 940, Ags., CP 20100 México, Mexico.

Bioassay-guided fractionation of Chimaphila umbellata (L.) W. Bart (Pyrolaceae) ethanol extracts led to the identification of 2,7-dimethyl-1,4-naphthoquinone (chimaphilin) as the principal antifungal component. The structure of chimaphilin was confirmed by 1H and 13C NMR spectroscopy. The antifungal activity of chimaphilin was evaluated using the microdilution method with Saccharomyces cerevisiae (0.05mg/mL) and the dandruff-associated fungi Malassezia globosa (0.39mg/mL) and Malassezia restricta (0.55mg/mL). Pronounced antioxidant activity of C. umbellata crude extract was also identified using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, suggesting this phytomedicine has an antioxidant function in wound healing. A chemical-genetic profile was completed with chimaphilin using approximately 4700 S. cerevisiae gene deletion mutants. Cellular roles of deleted genes in the most susceptible mutants and secondary assays indicate that the targets for chimaphilin include pathways involved in cell wall biogenesis and transcription.
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http://dx.doi.org/10.1016/j.phytochem.2007.09.007DOI Listing
February 2008

Colony size measurement of the yeast gene deletion strains for functional genomics.

BMC Bioinformatics 2007 Apr 4;8:117. Epub 2007 Apr 4.

Department of Electrical and Computer Engineering, University of Toronto, Toronto, Canada.

Background: Numerous functional genomics approaches have been developed to study the model organism yeast, Saccharomyces cerevisiae, with the aim of systematically understanding the biology of the cell. Some of these techniques are based on yeast growth differences under different conditions, such as those generated by gene mutations, chemicals or both. Manual inspection of the yeast colonies that are grown under different conditions is often used as a method to detect such growth differences.

Results: Here, we developed a computerized image analysis system called Growth Detector (GD), to automatically acquire quantitative and comparative information for yeast colony growth. GD offers great convenience and accuracy over the currently used manual growth measurement method. It distinguishes true yeast colonies in a digital image and provides an accurate coordinate oriented map of the colony areas. Some post-processing calculations are also conducted. Using GD, we successfully detected a genetic linkage between the molecular activity of the plant-derived antifungal compound berberine and gene expression components, among other cellular processes. A novel association for the yeast mek1 gene with DNA damage repair was also identified by GD and confirmed by a plasmid repair assay. The results demonstrate the usefulness of GD for yeast functional genomics research.

Conclusion: GD offers significant improvement over the manual inspection method to detect relative yeast colony size differences. The speed and accuracy associated with GD makes it an ideal choice for large-scale functional genomics investigations.
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http://dx.doi.org/10.1186/1471-2105-8-117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1854909PMC
April 2007