Publications by authors named "Ashkan Golshani"

65 Publications

Propionic acid disrupts endocytosis, cell cycle, and cellular respiration in yeast.

BMC Res Notes 2021 Aug 28;14(1):335. Epub 2021 Aug 28.

Department of Biology, Carleton University, Ottawa, ON, Canada.

Objective: We previously identified propionic acid as a microbially-produced volatile organic compound with fungicidal activity against several pathogenic fungi. The purpose of this work is to better understand how propionic acid affects fungi by examining some of the effects of this compound on the yeast cell.

Results: We show that propionic acid causes a dramatic increase in the uptake of lucifer yellow in yeast cells, which is consistent with enhanced endocytosis. Additionally, using a propidium iodide assay, we show that propionic acid treatment causes a significant increase in the proportion of yeast cells in G and a significant decrease in the proportion of cells in G, suggesting that propionic acid causes a cell cycle arrest in yeast. Finally, we show that the reduction of MTT is attenuated in yeast cells treated with propionic acid, indicating that propionic acid disrupts cellular respiration. Understanding the effects of propionic acid on the yeast cell may aid in assessing the broader utility of this compound.
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http://dx.doi.org/10.1186/s13104-021-05752-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403364PMC
August 2021

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 Aug;49(14):8145-8160

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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373142PMC
August 2021

Identifying new variation at the J locus, previously identified as e6, in long juvenile 'Paranagoiana' soybean.

Theor Appl Genet 2021 Apr 2;134(4):1007-1014. Epub 2021 Jan 2.

Ottawa Research and Development Center, Agriculture and Agri-Food Canada, Ottawa, ON, Canada.

Key Message: A previously identified soybean maturity locus, E6, is discovered to be J, with the long juvenile allele in Paranagoiana now deemed j-x. Soybean grown at latitudes of ~20° or lower can produce lower grain yields due to the short days. This limitation can be overcome by using the long juvenile trait (LJ) which delays flowering under short day conditions. Two LJ loci have been mapped to the same location on Gm04, J and E6. The objective of this research was to investigate the e6 allele in 'Paranagoiana' and determine if E6 and J are the same locus or linked loci. KASP markers showed that e6 lines did not have the j-1 allele of LJ PI 159925. A population fixed for E1 but segregating for E6, with e6 introgressed from Paranagoiana, showed single gene control for flowering and maturity under short days. Sequencing Glyma.04G050200, the J gene, with long amplification Taq found that the e6 line 'Paranagoiana' contains a Ty1-copia retrotransposon of ~10,000 bp, inserted within exon 4. PCR amplification of the cDNA of Glyma.04G050200 also showed differences between the mRNA sequences (presence of insertion in j-x). Hence, we conclude that the loci E6 and J are one locus and deem this new variation found in Paranagoiana as j-x.
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http://dx.doi.org/10.1007/s00122-020-03746-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973924PMC
April 2021

Manganese-induced neurotoxicity in cerebellar granule neurons due to perturbation of cell network pathways with potential implications for neurodegenerative disorders.

Metallomics 2020 11 7;12(11):1656-1678. Epub 2020 Sep 7.

Laboratory of Bioinorganic and Environmental Toxicology - LABITA, Department of Exact and Earth Sciences, Federal University of São Paulo, Rua Prof. Artur Riedel, 275, CEP 09972-270, Diadema, SP, Brazil.

Manganese (Mn) is essential for living organisms, playing an important role in nervous system function. Nevertheless, chronic and/or acute exposure to this metal, especially during early life stages, can lead to neurotoxicity and dementia by unclear mechanisms. Thus, based on previous works of our group with yeast and zebrafish, we hypothesized that the mechanisms mediating manganese-induced neurotoxicity can be associated with the alteration of protein metabolism. These mechanisms may also depend on the chemical speciation of manganese. Therefore, the current study aimed at investigating the mechanisms mediating the toxic effects of manganese in primary cultures of cerebellar granule neurons (CGNs). By exposing cultured CGNs to different chemical species of manganese ([[2-[(dithiocarboxy)amino]ethyl]carbamodithioato]](2-)-kS,kS']manganese, named maneb (MB), and [[1,2-ethanediylbis[carbamodithioato]](2-)]manganese mixture with [[1,2-ethanediylbis[carbamodithioato]](2-)]zinc, named mancozeb (MZ), and manganese chloride (MnCl)), and using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, we observed that both MB and MZ induced similar cytotoxicity (LC∼ 7-9 μM), which was higher than that of MnCl (LC∼ 27 μM). Subsequently, we applied systems biology approaches, including metallomics, proteomics, gene expression and bioinformatics, and revealed that independent of chemical speciation, for non-cytotoxic concentrations (0.3-3 μM), Mn-induced neurotoxicity in CGNs is associated with metal dyshomeostasis and impaired protein metabolism. In this way, we verified that MB induced more post-translational alterations than MnCl, which can be a plausible explanation for cytotoxic differences between both chemical species. The metabolism of proteins is one of the most energy consuming cellular processes and its impairment appears to be a key event of some cellular stress processes reported separately in other studies such as cell cycle arrest, energy impairment, cell signaling, excitotoxicity, immune response, potential protein accumulation and apoptosis. Interestingly, we verified that Mn-induced neurotoxicity shares pathways associated with the development of Alzheimer's disease, Amyotrophic Lateral Sclerosis, Huntington's disease, and Parkinson's disease. This has been observed in baker's yeast and zebrafish suggesting that the mode of action of Mn may be evolutionarily conserved.
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http://dx.doi.org/10.1039/d0mt00085jDOI Listing
November 2020

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

Lithium Chloride Sensitivity in Yeast and Regulation of Translation.

Int J Mol Sci 2020 Aug 10;21(16). Epub 2020 Aug 10.

Department of Biology and Ottawa Institute of Systems Biology, Carleton University, Ottawa, ON K1S 5B6, Canada.

For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of , a phosphoglucomutase involved in sugar metabolism. Inhibition of leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, , , and , which increase yeast LiCl sensitivity when deleted. We further demonstrate that , , and influence translation and exert their activity through the 5'-Untranslated region (5'-UTR) of mRNA in yeast.
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http://dx.doi.org/10.3390/ijms21165730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461102PMC
August 2020

Sensitivity of yeast to lithium chloride connects the activity of YTA6 and YPR096C to translation of structured mRNAs.

PLoS One 2020 8;15(7):e0235033. Epub 2020 Jul 8.

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

Lithium Chloride (LiCl) toxicity, mode of action and cellular responses have been the subject of active investigations over the past decades. In yeast, LiCl treatment is reported to reduce the activity and alters the expression of PGM2, a gene that encodes a phosphoglucomutase involved in sugar metabolism. Reduced activity of phosphoglucomutase in the presence of galactose causes an accumulation of intermediate metabolites of galactose metabolism leading to a number of phenotypes including growth defect. In the current study, we identify two understudied yeast genes, YTA6 and YPR096C that when deleted, cell sensitivity to LiCl is increased when galactose is used as a carbon source. The 5'-UTR of PGM2 mRNA is structured. Using this region, we show that YTA6 and YPR096C influence the translation of PGM2 mRNA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235033PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343135PMC
September 2020

PIPE4: Fast PPI Predictor for Comprehensive Inter- and Cross-Species Interactomes.

Sci Rep 2020 Jan 29;10(1):1390. Epub 2020 Jan 29.

Department of Systems and Computer Engineering, Carleton University, Ottawa, Ontario, K1S 5B6, Canada.

The need for larger-scale and increasingly complex protein-protein interaction (PPI) prediction tasks demands that state-of-the-art predictors be highly efficient and adapted to inter- and cross-species predictions. Furthermore, the ability to generate comprehensive interactomes has enabled the appraisal of each PPI in the context of all predictions leading to further improvements in classification performance in the face of extreme class imbalance using the Reciprocal Perspective (RP) framework. We here describe the PIPE4 algorithm. Adaptation of the PIPE3/MP-PIPE sequence preprocessing step led to upwards of 50x speedup and the new Similarity Weighted Score appropriately normalizes for window frequency when applied to any inter- and cross-species prediction schemas. Comprehensive interactomes for three prediction schemas are generated: (1) cross-species predictions, where Arabidopsis thaliana is used as a proxy to predict the comprehensive Glycine max interactome, (2) inter-species predictions between Homo sapiens-HIV1, and (3) a combined schema involving both cross- and inter-species predictions, where both Arabidopsis thaliana and Caenorhabditis elegans are used as proxy species to predict the interactome between Glycine max (the soybean legume) and Heterodera glycines (the soybean cyst nematode). Comparing PIPE4 with the state-of-the-art resulted in improved performance, indicative that it should be the method of choice for complex PPI prediction schemas.
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http://dx.doi.org/10.1038/s41598-019-56895-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989690PMC
January 2020

Differential remodeling of the electron transport chain is required to support TLR3 and TLR4 signaling and cytokine production in macrophages.

Sci Rep 2019 12 11;9(1):18801. Epub 2019 Dec 11.

Department of Health Sciences, Carleton University, Ottawa, Ontario, Canada.

Increasing evidence suggests that mitochondria play a critical role in driving innate immune responses against bacteria and viruses. However, it is unclear if differential reprogramming of mitochondrial function contributes to the fine tuning of pathogen specific immune responses. Here, we found that TLR3 and TLR4 engagement on murine bone marrow derived macrophages was associated with differential remodeling of electron transport chain complex expression. This remodeling was associated with differential accumulation of mitochondrial and cytosolic ROS, which were required to support ligand specific inflammatory and antiviral cytokine production. We also found that the magnitude of TLR3, but not TLR4, responses were modulated by glucose availability. Under conditions of low glucose, TLR3 engagement was associated with increased ETC complex III expression, increased mitochondrial and cytosolic ROS and increased inflammatory and antiviral cytokine production. This amplification was selectively reversed by targeting superoxide production from the outer Q-binding site of the ETC complex III. These results suggest that ligand specific modulation of the ETC may act as a rheostat that fine tunes innate immune responses via mitochondrial ROS production. Modulation of these processes may represent a novel mechanism to modulate the nature as well as the magnitude of antiviral vs. inflammatory immune responses.
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http://dx.doi.org/10.1038/s41598-019-55295-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906364PMC
December 2019

Mode of action of nisin on .

Can J Microbiol 2020 Feb 19;66(2):161-168. Epub 2019 Nov 19.

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

Nisin is a class I polycyclic bacteriocin produced by the bacterium , which is used extensively as a food additive to inhibit the growth of foodborne Gram-positive bacteria. Nisin also inhibits growth of Gram-negative bacteria when combined with membrane-disrupting chelators such as citric acid. To gain insight into nisin's mode of action, we analyzed chemical-genetic interactions and identified nisin-sensitive strains in the Keio library of knockout mutants. The most sensitive mutants fell into two main groups. The first group accords with the previously proposed mode of action based on studies with Gram-positive bacteria, whereby nisin interacts with factors involved in cell wall, membrane, envelope biogenesis. We identified an additional, novel mode of action for nisin based on the second group of sensitive mutants that involves cell cycle and DNA replication, recombination, and repair. Further analyses supported these two distinct modes of action.
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http://dx.doi.org/10.1139/cjm-2019-0315DOI Listing
February 2020

Manganese-induced cellular disturbance in the baker's yeast, Saccharomyces cerevisiae with putative implications in neuronal dysfunction.

Sci Rep 2019 04 25;9(1):6563. Epub 2019 Apr 25.

Department of Biology and Ottawa Institute of Systems Biology, Carleton University, 1125 Colonel by Drive, Ottawa, ON, K1S 5B6, Canada.

Manganese (Mn) is an essential element, but in humans, chronic and/or acute exposure to this metal can lead to neurotoxicity and neurodegenerative disorders including Parkinsonism and Parkinson's Disease by unclear mechanisms. To better understand the effects that exposure to Mn exert on eukaryotic cell biology, we exposed a non-essential deletion library of the yeast Saccharomyces cerevisiae to a sub-inhibitory concentration of Mn followed by targeted functional analyses of the positive hits. This screen produced a set of 43 sensitive deletion mutants that were enriched for genes associated with protein biosynthesis. Our follow-up investigations demonstrated that Mn reduced total rRNA levels in a dose-dependent manner and decreased expression of a β-galactosidase reporter gene. This was subsequently supported by analysis of ribosome profiles that suggested Mn-induced toxicity was associated with a reduction in formation of active ribosomes on the mRNAs. Altogether, these findings contribute to the current understanding of the mechanism of Mn-triggered cytotoxicity. Lastly, using the Comparative Toxicogenomic Database, we revealed that Mn shared certain similarities in toxicological mechanisms with neurodegenerative disorders including amyotrophic lateral sclerosis, Alzheimer's, Parkinson's and Huntington's diseases.
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http://dx.doi.org/10.1038/s41598-019-42907-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484083PMC
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

Insights into the suitability of utilizing brown rats (Rattus norvegicus) as a model for healing spinal cord injury with epidermal growth factor and fibroblast growth factor-II by predicting protein-protein interactions.

Comput Biol Med 2019 01 30;104:220-226. Epub 2018 Nov 30.

Department of Biology, Carleton University, Ottawa, Canada; Department of Biochemistry, Carleton University, Ottawa, Canada. Electronic address:

The stimulation of the proliferation and differentiation of neural stem cells (NSCs) offers the possibility of a renewable source of replacement cells to treat numerous neurological diseases including spinal cord injury, traumatic brain injury and stroke. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) have been used to stimulate NSCs to renew, expand, and produce precursors for neural repair within an adult brown rat (Rattus norvegicus). To provide greater insight into the interspecies protein-protein interactions between human FGF-2 and EGF proteins and native R. norvegicus proteins, we have utilized the Massively Parallel Protein-Protein Interaction Prediction Engine (MP-PIPE) in an attempt to computationally shed light on the pathways potentially driving neurosphere proliferation. This study determined similar and differing protein interaction pathways between the two growth factors and the proteins in R. norvegicus compared with the proteins in H. sapiens. The protein-protein interactions predicted that EGF and FGF-2 may behave differently in rats than in humans. The identification and improved understanding of these differences may help to improve the clinical translation of NSC therapies from rats to humans.
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http://dx.doi.org/10.1016/j.compbiomed.2018.11.026DOI Listing
January 2019

Heavy metal sensitivities of gene deletion strains for ITT1 and RPS1A connect their activities to the expression of URE2, a key gene involved in metal detoxification in yeast.

PLoS One 2018 19;13(9):e0198704. Epub 2018 Sep 19.

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

Heavy metal and metalloid contaminations are among the most concerning types of pollutant in the environment. Consequently, it is important to investigate the molecular mechanisms of cellular responses and detoxification pathways for these compounds in living organisms. To date, a number of genes have been linked to the detoxification process. The expression of these genes can be controlled at both transcriptional and translational levels. In baker's yeast, Saccharomyces cerevisiae, resistance to a wide range of toxic metals is regulated by glutathione S-transferases. Yeast URE2 encodes for a protein that has glutathione peroxidase activity and is homologous to mammalian glutathione S-transferases. The URE2 expression is critical to cell survival under heavy metal stress. Here, we report on the finding of two genes, ITT1, an inhibitor of translation termination, and RPS1A, a small ribosomal protein, that when deleted yeast cells exhibit similar metal sensitivity phenotypes to gene deletion strain for URE2. Neither of these genes were previously linked to metal toxicity. Our gene expression analysis illustrates that these two genes affect URE2 mRNA expression at the level of translation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198704PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145592PMC
February 2019

Transcriptional Profiling Suggests Extensive Metabolic Rewiring of Human and Mouse Macrophages during Early Interferon Alpha Responses.

Mediators Inflamm 2018 25;2018:5906819. Epub 2018 Jul 25.

Department of Health Sciences, Carleton University, Ottawa, ON, Canada.

Emerging evidence suggests that cellular metabolism plays a critical role in regulating immune activation. Alterations in energy and lipid and amino acid metabolism have been shown to contribute to type I interferon (IFN) responses in macrophages, but the relationship between metabolic reprogramming and the establishment of early antiviral function remains poorly defined. Here, we used transcriptional profiling datasets to develop global metabolic signatures associated with early IFN- responses in two primary macrophage model systems: mouse bone marrow-derived macrophages (BMM) and human monocyte-derived macrophages (MDM). Short-term stimulation with IFN- (<4 hours) was associated with significant metabolic rewiring, with >500 metabolic genes altered in mouse and human macrophage models. Pathway and network analysis identified alterations in genes associated with cellular bioenergetics, cellular oxidant status, cAMP/AMP and cGMP/GMP ratios, branched chain amino acid catabolism, cell membrane composition, fatty acid synthesis, and -oxidation as key features of early IFN- responses. These changes may have important implications for initial establishment of antiviral function in these cells.
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http://dx.doi.org/10.1155/2018/5906819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6083555PMC
November 2018

A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis.

Cell Rep 2018 03;22(13):3427-3439

Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada; Ottawa Institute of Systems Biology, Ottawa, Ontario K1H 8M5, Canada. Electronic address:

Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.
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http://dx.doi.org/10.1016/j.celrep.2018.02.104DOI Listing
March 2018

Zinc oxide and silver nanoparticles toxicity in the baker's yeast, Saccharomyces cerevisiae.

PLoS One 2018 19;13(3):e0193111. Epub 2018 Mar 19.

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

Engineered nanomaterials (ENMs) are increasingly incorporated into a variety of commercial applications and consumer products; however, ENMs may possess cytotoxic properties due to their small size. This study assessed the effects of two commonly used ENMs, zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (AgNPs), in the model eukaryote Saccharomyces cerevisiae. A collection of ≈4600 S. cerevisiae deletion mutant strains was used to deduce the genes, whose absence makes S. cerevisiae more prone to the cytotoxic effects of ZnONPs or AgNPs. We demonstrate that S. cerevisiae strains that lack genes involved in transmembrane and membrane transport, cellular ion homeostasis, and cell wall organization or biogenesis exhibited the highest sensitivity to ZnONPs. In contrast, strains that lack genes involved in transcription and RNA processing, cellular respiration, and endocytosis and vesicular transport exhibited the highest sensitivity to AgNPs. Secondary assays confirmed that ZnONPs affected cell wall function and integrity, whereas AgNPs exposure decreased transcription, reduced endocytosis, and led to a dysfunctional electron transport system. This study supports the use of S. cerevisiae Gene Deletion Array as an effective high-throughput technique to determine cellular targets of ENM toxicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193111PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858749PMC
June 2018

Global landscape of cell envelope protein complexes in Escherichia coli.

Nat Biotechnol 2018 01 27;36(1):103-112. Epub 2017 Nov 27.

J. Craig Venter Institute, Rockville, Maryland, USA.

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the β-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.
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http://dx.doi.org/10.1038/nbt.4024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922438PMC
January 2018

The sensitivity of the yeast, , to acetic acid is influenced by and .

PeerJ 2017 14;5:e4037. Epub 2017 Nov 14.

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

The presence of acetic acid during industrial alcohol fermentation reduces the yield of fermentation by imposing additional stress on the yeast cells. The biology of cellular responses to stress has been a subject of vigorous investigations. Although much has been learned, details of some of these responses remain poorly understood. Members of heat shock chaperone HSP proteins have been linked to acetic acid and heat shock stress responses in yeast. Both acetic acid and heat shock have been identified to trigger different cellular responses including reduction of global protein synthesis and induction of programmed cell death. Yeast and code for two important heat shock proteins that together account for 1-2% of total cellular proteins. Both proteins have been linked to responses to acetic acid and heat shock. In contrast to the overall rate of protein synthesis which is reduced, the expression of and is induced in response to acetic acid stress. In the current study we identified two yeast genes and that are linked to acetic acid and heat shock sensitivity. We investigated the influence of these genes on the expression of HSP proteins. Our observations suggest that and influence translation in a CAP-independent manner.
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http://dx.doi.org/10.7717/peerj.4037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691786PMC
November 2017

Designing anti-Zika virus peptides derived from predicted human-Zika virus protein-protein interactions.

Comput Biol Chem 2017 Dec 2;71:180-187. Epub 2017 Nov 2.

Department of Biology and Institute of Biochemistry, and Ottawa Institute of Systems Biology, Carleton University, Ottawa, Ontario. K1S 5B6, Canada. Electronic address:

The production of anti-Zika virus (ZIKV) therapeutics has become increasingly important as the propagation of the devastating virus continues largely unchecked. Notably, a causal relationship between ZIKV infection and neurodevelopmental abnormalities has been widely reported, yet a specific mechanism underlying impaired neurological development has not been identified. Here, we report on the design of several synthetic competitive inhibitory peptides against key pathogenic ZIKV proteins through the prediction of protein-protein interactions (PPIs). Often, PPIs between host and viral proteins are crucial for infection and pathogenesis, making them attractive targets for therapeutics. Using two complementary sequence-based PPI prediction tools, we first produced a comprehensive map of predicted human-ZIKV PPIs (involving 209 human protein candidates). We then designed several peptides intended to disrupt the corresponding host-pathogen interactions thereby acting as anti-ZIKV therapeutics. The data generated in this study constitute a foundational resource to aid in the multi-disciplinary effort to combat ZIKV infection, including the design of additional synthetic proteins.
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http://dx.doi.org/10.1016/j.compbiolchem.2017.10.011DOI Listing
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

A Type-2 fuzzy data fusion approach for building reliable weighted protein interaction networks with application in protein complex detection.

Comput Biol Med 2017 09 23;88:18-31. Epub 2017 Jun 23.

Department of Biology, Carleton University, Ottawa, Canada. Electronic address:

Detecting the protein complexes is an important task in analyzing the protein interaction networks. Although many algorithms predict protein complexes in different ways, surveys on the interaction networks indicate that about 50% of detected interactions are false positives. Consequently, the accuracy of existing methods needs to be improved. In this paper we propose a novel algorithm to detect the protein complexes in 'noisy' protein interaction data. First, we integrate several biological data sources to determine the reliability of each interaction and determine more accurate weights for the interactions. A data fusion component is used for this step, based on the interval type-2 fuzzy voter that provides an efficient combination of the information sources. This fusion component detects the errors and diminishes their effect on the detection protein complexes. So in the first step, the reliability scores have been assigned for every interaction in the network. In the second step, we have proposed a general protein complex detection algorithm by exploiting and adopting the strong points of other algorithms and existing hypotheses regarding real complexes. Finally, the proposed method has been applied for the yeast interaction datasets for predicting the interactions. The results show that our framework has a better performance regarding precision and F-measure than the existing approaches.
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http://dx.doi.org/10.1016/j.compbiomed.2017.06.019DOI Listing
September 2017

Evolution of protein-protein interaction networks in yeast.

PLoS One 2017 1;12(3):e0171920. Epub 2017 Mar 1.

Department of Biology, Carleton University, Ottawa, Canada.

Interest in the evolution of protein-protein and genetic interaction networks has been rising in recent years, but the lack of large-scale high quality comparative datasets has acted as a barrier. Here, we carried out a comparative analysis of computationally predicted protein-protein interaction (PPI) networks from five closely related yeast species. We used the Protein-protein Interaction Prediction Engine (PIPE), which uses a database of known interactions to make sequence-based PPI predictions, to generate high quality predicted interactomes. Simulated proteomes and corresponding PPI networks were used to provide null expectations for the extent and nature of PPI network evolution. We found strong evidence for conservation of PPIs, with lower than expected levels of change in PPIs for about a quarter of the proteome. Furthermore, we found that changes in predicted PPI networks are poorly predicted by sequence divergence. Our analyses identified a number of functional classes experiencing fewer PPI changes than expected, suggestive of purifying selection on PPIs. Our results demonstrate the added benefit of considering predicted PPI networks when studying the evolution of closely related organisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171920PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382968PMC
August 2017

Elevated levels of ribosomal proteins eL36 and eL42 control expression of Hsp90 in rhabdomyosarcoma.

Translation (Austin) 2016 4;4(2):e1244395. Epub 2016 Oct 4.

Molecular Biomedicine Program, Children's Hospital of Eastern Ontario Research Institute , Ottawa, Ontario, Canada.

Mammalian 90 kDa heat shock protein (Hsp90) is a ubiquitous molecular chaperone whose expression is selectively upregulated during stress, although the precise control mechanism of this increase is yet to be fully elucidated. We used polysome profiling to show that Hsp90α mRNA is selectively translated, while global translation is inhibited during heat stress. Furthermore, we have identified 2 ribosomal proteins, eL36 and eL42 that modulate Hsp90α expression under both normal and heat shock conditions. Importantly, we noted that expression of eL36 and eL42 is elevated in a panel of human rhabdomyosarcomas where it drives high expression of Hsp90 and modulates sensitivity of these cells to an Hsp90 inhibitor 17-AAG.
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http://dx.doi.org/10.1080/21690731.2016.1244395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5173308PMC
October 2016

Mapping and identification of a potential candidate gene for a novel maturity locus, E10, in soybean.

Theor Appl Genet 2017 Feb 10;130(2):377-390. Epub 2016 Nov 10.

Agriculture and Agri-Food Canada, Ottawa Research and Development Centre, Ottawa, ON, K1A 0C6, Canada.

Key Message: E10 is a new maturity locus in soybean and FT4 is the predicted/potential functional gene underlying the locus. Flowering and maturity time traits play crucial roles in economic soybean production. Early maturity is critical for north and west expansion of soybean in Canada. To date, 11 genes/loci have been identified which control time to flowering and maturity; however, the molecular bases of almost half of them are not yet clear. We have identified a new maturity locus called "E10" located at the end of chromosome Gm08. The gene symbol E10e10 has been approved by the Soybean Genetics Committee. The e10e10 genotype results in 5-10 days earlier maturity than E10E10. A set of presumed E10E10 and e10e10 genotypes was used to identify contrasting SSR and SNP haplotypes. These haplotypes, and their association with maturity, were maintained through five backcross generations. A functional genomics approach using a predicted protein-protein interaction (PPI) approach (Protein-protein Interaction Prediction Engine, PIPE) was used to investigate approximately 75 genes located in the genomic region that SSR and SNP analyses identified as the location of the E10 locus. The PPI analysis identified FT4 as the most likely candidate gene underlying the E10 locus. Sequence analysis of the two FT4 alleles identified three SNPs, in the 5'UTR, 3'UTR and fourth exon in the coding region, which result in differential mRNA structures. Allele-specific markers were developed for this locus and are available for soybean breeders to efficiently develop earlier maturing cultivars using molecular marker assisted breeding.
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http://dx.doi.org/10.1007/s00122-016-2819-7DOI Listing
February 2017

Systematic Genetic Screens Reveal the Dynamic Global Functional Organization of the Bacterial Translation Machinery.

Cell Rep 2016 10;17(3):904-916

Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada. Electronic address:

Bacterial protein synthesis is an essential, conserved, and environmentally responsive process. Yet, many of its components and dependencies remain unidentified. To address this gap, we used quantitative synthetic genetic arrays to map functional relationships among >48,000 gene pairs in Escherichia coli under four culture conditions differing in temperature and nutrient availability. The resulting data provide global functional insights into the roles and associations of genes, pathways, and processes important for efficient translation, growth, and environmental adaptation. We predict and independently verify the requirement of unannotated genes for normal translation, including a previously unappreciated role of YhbY in 30S biogenesis. Dynamic changes in the patterns of genetic dependencies across the four growth conditions and data projections onto other species reveal overarching functional and evolutionary pressures impacting the translation system and bacterial fitness, underscoring the utility of systematic screens for investigating protein synthesis, adaptation, and evolution.
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http://dx.doi.org/10.1016/j.celrep.2016.09.040DOI Listing
October 2016

Conditional Epistatic Interaction Maps Reveal Global Functional Rewiring of Genome Integrity Pathways in Escherichia coli.

Cell Rep 2016 Jan 8;14(3):648-661. Epub 2016 Jan 8.

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

As antibiotic resistance is increasingly becoming a public health concern, an improved understanding of the bacterial DNA damage response (DDR), which is commonly targeted by antibiotics, could be of tremendous therapeutic value. Although the genetic components of the bacterial DDR have been studied extensively in isolation, how the underlying biological pathways interact functionally remains unclear. Here, we address this by performing systematic, unbiased, quantitative synthetic genetic interaction (GI) screens and uncover widespread changes in the GI network of the entire genomic integrity apparatus of Escherichia coli under standard and DNA-damaging growth conditions. The GI patterns of untreated cultures implicated two previously uncharacterized proteins (YhbQ and YqgF) as nucleases, whereas reorganization of the GI network after DNA damage revealed DDR roles for both annotated and uncharacterized genes. Analyses of pan-bacterial conservation patterns suggest that DDR mechanisms and functional relationships are near universal, highlighting a modular and highly adaptive genomic stress response.
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http://dx.doi.org/10.1016/j.celrep.2015.12.060DOI Listing
January 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

Advances and challenges in mucosal adjuvant technology.

Vaccine 2015 May 10;33(21):2399-405. Epub 2015 Apr 10.

Department of Pathology and Laboratory Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, ON, Canada; Department of Biotechnology, University of Ontario Institute of Technology, Toronto, ON, Canada. Electronic address:

Adjuvants play attractive roles in enhancement of immune response during vaccination; however, due to several challenges, only a limited number of adjuvants are licensed by health authorities. The lack of an effective mucosal adjuvant is even more significant as none of the licensed adjuvants revealed a strong enhancement in immune system after mucosal administration. Over the past two decades, several mucosal adjuvants have been developed to deliver antigens to the target cells in the mucosal immune system and increase specific immune responses. However, the safety and efficacy of these adjuvants for testing in human trials is still an important issue, requiring further study. In this article, we briefly review the challenges associated with most common mucosal adjuvants and discuss potential strategies for targeting the mucosal immune system.
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http://dx.doi.org/10.1016/j.vaccine.2015.03.096DOI Listing
May 2015

Efficient prediction of human protein-protein interactions at a global scale.

BMC Bioinformatics 2014 Dec 10;15:383. Epub 2014 Dec 10.

Department of Biology, Carleton University, Ottawa, Canada.

Background: Our knowledge of global protein-protein interaction (PPI) networks in complex organisms such as humans is hindered by technical limitations of current methods.

Results: On the basis of short co-occurring polypeptide regions, we developed a tool called MP-PIPE capable of predicting a global human PPI network within 3 months. With a recall of 23% at a precision of 82.1%, we predicted 172,132 putative PPIs. We demonstrate the usefulness of these predictions through a range of experiments.

Conclusions: The speed and accuracy associated with MP-PIPE can make this a potential tool to study individual human PPI networks (from genomic sequences alone) for personalized medicine.
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http://dx.doi.org/10.1186/s12859-014-0383-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272565PMC
December 2014
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