Publications by authors named "Sadhna Phanse"

51 Publications

Assembly principles of the human R2TP chaperone complex reveal the presence of R2T and R2P complexes.

Structure 2021 Sep 3. Epub 2021 Sep 3.

Department of Biochemistry, University of Toronto, 661 University Avenue, MaRS Centre, West Tower, Room 1612, Toronto, ON M5G 1M1, Canada; Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada. Electronic address:

R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2-based complexes are identified: R2TP, RUVBL1/2-RPAP3 (R2T), and RUVBL1/2-PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N-terminal domain mediating interactions with substrates whose sequences are primarily enriched for Armadillo repeat domains and other helical-type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.
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http://dx.doi.org/10.1016/j.str.2021.08.002DOI Listing
September 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

ZapG (YhcB/DUF1043), a novel cell division protein in gamma-proteobacteria linking the Z-ring to septal peptidoglycan synthesis.

J Biol Chem 2021 Jan-Jun;296:100700. Epub 2021 Apr 23.

Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia, USA. Electronic address:

YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall-acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.
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http://dx.doi.org/10.1016/j.jbc.2021.100700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163987PMC
August 2021

From fuzziness to precision medicine: on the rapidly evolving proteomics with implications in mitochondrial connectivity to rare human disease.

iScience 2021 Feb 6;24(2):102030. Epub 2021 Jan 6.

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

Mitochondrial (mt) dysfunction is linked to rare diseases (RDs) such as respiratory chain complex (RCC) deficiency, MELAS, and ARSACS. Yet, how altered mt protein networks contribute to these ailments remains understudied. In this perspective article, we identified 21 mt proteins from public repositories that associate with RCC deficiency, MELAS, or ARSACS, engaging in a relatively small number of protein-protein interactions (PPIs), underscoring the need for advanced proteomic and interactomic platforms to uncover the complete scope of mt connectivity to RDs. Accordingly, we discuss innovative untargeted label-free proteomics in identifying RD-specific mt or other macromolecular assemblies and mapping of protein networks in complex tissue, organoid, and stem cell-differentiated neurons. Furthermore, tag- and label-based proteomics, genealogical proteomics, and combinatorial affinity purification-mass spectrometry, along with advancements in detecting and integrating transient PPIs with single-cell proteomics and transcriptomics, collectively offer seminal follow-ups to enrich for RD-relevant networks, with implications in RD precision medicine.
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http://dx.doi.org/10.1016/j.isci.2020.102030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820543PMC
February 2021

Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex.

Nat Commun 2021 01 12;12(1):281. Epub 2021 Jan 12.

Department of Biochemistry, University of Toronto, Toronto, ON, M5G 1M1, Canada.

A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone-protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.
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http://dx.doi.org/10.1038/s41467-020-20553-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804408PMC
January 2021

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Mol Cell 2020 12 19;80(6):1104-1122.e9. Epub 2020 Nov 19.

Center for Network Systems Biology, Boston University, Boston, MA, USA; Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.
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http://dx.doi.org/10.1016/j.molcel.2020.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674017PMC
December 2020

Mitochondria under the spotlight: On the implications of mitochondrial dysfunction and its connectivity to neuropsychiatric disorders.

Comput Struct Biotechnol J 2020 14;18:2535-2546. Epub 2020 Sep 14.

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

Neuropsychiatric disorders (NPDs) such as bipolar disorder (BD), schizophrenia (SZ) and mood disorder (MD) are hard to manage due to overlapping symptoms and lack of biomarkers. Risk alleles of BD/SZ/MD are emerging, with evidence suggesting mitochondrial (mt) dysfunction as a critical factor for disease onset and progression. Mood stabilizing treatments for these disorders are scarce, revealing the need for biomarker discovery and artificial intelligence approaches to design synthetically accessible novel therapeutics. Here, we show mt involvement in NPDs by associating 245 mt proteins to BD/SZ/MD, with 7 common players in these disease categories. Analysis of over 650 publications suggests that 245 NPD-linked mt proteins are associated with 800 other mt proteins, with mt impairment likely to rewire these interactions. High dosage of mood stabilizers is known to alleviate manic episodes, but which compounds target mt pathways is another gap in the field that we address through mood stabilizer-gene interaction analysis of 37 prescriptions and over-the-counter psychotropic treatments, which we have refined to 15 mood-stabilizing agents. We show 26 of the 245 NPD-linked mt proteins are uniquely or commonly targeted by one or more of these mood stabilizers. Further, induced pluripotent stem cell-derived patient neurons and three-dimensional human brain organoids as reliable BD/SZ/MD models are outlined, along with multiomics methods and machine learning-based decision making tools for biomarker discovery, which remains a bottleneck for precision psychiatry medicine.
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http://dx.doi.org/10.1016/j.csbj.2020.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522539PMC
September 2020

Exploring the Impact of Mutations on the Total and Mitochondrial Proteome of Human Skin Fibroblasts.

Front Cell Dev Biol 2020 11;8:423. Epub 2020 Jun 11.

Department of Science and High Technology, Center of Neuroscience, University of Insubria, Busto Arsizio, Italy.

Mutations in gene are the most frequent cause of familial forms of Parkinson's disease (PD). This gene encodes Parkin, an E3 ubiquitin ligase involved in several cellular mechanisms, including mitophagy. Parkin loss-of-function is responsible for the cellular accumulation of damaged mitochondria, which in turn determines an increment of reactive oxygen species (ROS) levels, lower ATP production, and apoptosis activation. Given the importance of mitochondrial dysfunction and mitophagy impairment in PD pathogenesis, the aim of the present study was to investigate both total and mitochondrial proteome alterations in human skin fibroblasts of -mutated patients. To this end, both total and mitochondria-enriched protein fractions from fibroblasts of five -mutated patients and five control subjects were analyzed by quantitative shotgun proteomics to identify proteins specifically altered by Parkin mutations (mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD015880). Both the network-based and gene set enrichment analyses pointed out pathways in which Rab GTPase proteins are involved. To have a more comprehensive view of the mitochondrial alterations due to mutations, we investigated the impact of Parkin loss on mitochondrial function and network morphology. We unveiled that the mitochondrial membrane potential was reduced in -mutated patients, without inducing PINK1 accumulation, even when triggered with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Lastly, the analysis of the mitochondrial network morphology did not reveal any significant alterations in -mutated patients compared to control subjects. Thus, our results suggested that the network morphology was not influenced by the mitochondrial depolarization and by the lack of Parkin, revealing a possible impairment of fission and, more in general, of mitochondrial dynamics. In conclusion, the present work highlighted new molecular factors and pathways altered by mutations, which will unravel possible biochemical pathways altered in the sporadic form of PD.
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http://dx.doi.org/10.3389/fcell.2020.00423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300190PMC
June 2020

BraInMap Elucidates the Macromolecular Connectivity Landscape of Mammalian Brain.

Cell Syst 2020 04;10(4):333-350.e14

Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Broad Institute of Massachusetts Institute of Technology and Harvard University, Boston, MA, USA.

Connectivity webs mediate the unique biology of the mammalian brain. Yet, while cell circuit maps are increasingly available, knowledge of their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.
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http://dx.doi.org/10.1016/j.cels.2020.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938770PMC
April 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

Chemical-genetic profiling reveals limited cross-resistance between antimicrobial peptides with different modes of action.

Nat Commun 2019 12 16;10(1):5731. Epub 2019 Dec 16.

Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary.

Antimicrobial peptides (AMPs) are key effectors of the innate immune system and promising therapeutic agents. Yet, knowledge on how to design AMPs with minimal cross-resistance to human host-defense peptides remains limited. Here, we systematically assess the resistance determinants of Escherichia coli against 15 different AMPs using chemical-genetics and compare to the cross-resistance spectra of laboratory-evolved AMP-resistant strains. Although generalizations about AMP resistance are common in the literature, we find that AMPs with different physicochemical properties and cellular targets vary considerably in their resistance determinants. As a consequence, cross-resistance is prevalent only between AMPs with similar modes of action. Finally, our screen reveals several genes that shape susceptibility to membrane- and intracellular-targeting AMPs in an antagonistic manner. We anticipate that chemical-genetic approaches could inform future efforts to minimize cross-resistance between therapeutic and human host AMPs.
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http://dx.doi.org/10.1038/s41467-019-13618-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915728PMC
December 2019

ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores.

Commun Biol 2019 13;2:410. Epub 2019 Nov 13.

1Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1M1 Canada.

Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in and ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.
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http://dx.doi.org/10.1038/s42003-019-0656-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853987PMC
May 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 Gaussian process-based definition reveals new and bona fide genetic interactions compared to a multiplicative model in the Gram-negative Escherichia coli.

Bioinformatics 2020 02;36(3):880-889

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

Motivation: A digenic genetic interaction (GI) is observed when mutations in two genes within the same organism yield a phenotype that is different from the expected, given each mutation's individual effects. While multiplicative scoring is widely applied to define GIs, revealing underlying gene functions, it remains unclear if it is the most suitable choice for scoring GIs in Escherichia coli. Here, we assess many different definitions, including the multiplicative model, for mapping functional links between genes and pathways in E.coli.

Results: Using our published E.coli GI datasets, we show computationally that a machine learning Gaussian process (GP)-based definition better identifies functional associations among genes than a multiplicative model, which we have experimentally confirmed on a set of gene pairs. Overall, the GP definition improves the detection of GIs, biological reasoning of epistatic connectivity, as well as the quality of GI maps in E.coli, and, potentially, other microbes.

Availability And Implementation: The source code and parameters used to generate the machine learning models in WEKA software were provided in the Supplementary information.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz673DOI Listing
February 2020

EPIC: software toolkit for elution profile-based inference of protein complexes.

Nat Methods 2019 08 15;16(8):737-742. Epub 2019 Jul 15.

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

Protein complexes are key macromolecular machines of the cell, but their description remains incomplete. We and others previously reported an experimental strategy for global characterization of native protein assemblies based on chromatographic fractionation of biological extracts coupled to precision mass spectrometry analysis (chromatographic fractionation-mass spectrometry, CF-MS), but the resulting data are challenging to process and interpret. Here, we describe EPIC (elution profile-based inference of complexes), a software toolkit for automated scoring of large-scale CF-MS data to define high-confidence multi-component macromolecules from diverse biological specimens. As a case study, we used EPIC to map the global interactome of Caenorhabditis elegans, defining 612 putative worm protein complexes linked to diverse biological processes. These included novel subunits and assemblies unique to nematodes that we validated using orthogonal methods. The open source EPIC software is freely available as a Jupyter notebook packaged in a Docker container (https://hub.docker.com/r/baderlab/bio-epic/).
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http://dx.doi.org/10.1038/s41592-019-0461-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995176PMC
August 2019

Acyldepsipeptide Analogs Dysregulate Human Mitochondrial ClpP Protease Activity and Cause Apoptotic Cell Death.

Cell Chem Biol 2018 08 28;25(8):1017-1030.e9. Epub 2018 Jun 28.

Department of Biochemistry, University of Toronto, 661 University Avenue, MaRS Centre, West Tower, Room 1612, Toronto, ON M5G 1M1, Canada; Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada. Electronic address:

Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment.
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http://dx.doi.org/10.1016/j.chembiol.2018.05.014DOI Listing
August 2018

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

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

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

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

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

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

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

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

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

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

Insights from protein-protein interaction studies on bacterial pathogenesis.

Expert Rev Proteomics 2017 09 8;14(9):779-797. Epub 2017 Sep 8.

b Department of Biochemistry , University of Regina , Regina , SK , Canada.

Introduction: The threat bacterial pathogens pose to human health is increasing with the number and distribution of antibiotic-resistant bacteria, while the rate of discovery of new antimicrobials dwindles. Proteomics is playing key roles in understanding the molecular mechanisms of bacterial pathogenesis, and in identifying disease outcome determinants. The physical associations identified by proteomics can provide the means to develop pathogen-specific treatment methods that reduce the spread of antibiotic resistance and alleviate the negative effects of broad-spectrum antibiotics on beneficial bacteria. Areas covered: This review discusses recent trends in proteomics and introduces new and developing approaches that can be applied to the study of protein-protein interactions (PPIs) underlying bacterial pathogenesis. The approaches examined encompass options for mapping proteomes as well as stable and transient interactions in vivo and in vitro. We also explored the coverage of bacterial and human-bacterial PPIs, knowledge gaps in this area, and how they can be filled. Expert commentary: Identifying potential antimicrobial candidates is confounded by the complex molecular biology of bacterial pathogenesis and the lack of knowledge about PPIs underlying this process. Proteomics approaches can offer new perspectives for mechanistic insights and identify essential targets for guiding the discovery of next generation antimicrobials.
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http://dx.doi.org/10.1080/14789450.2017.1365603DOI Listing
September 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

Identification of Human Neuronal Protein Complexes Reveals Biochemical Activities and Convergent Mechanisms of Action in Autism Spectrum Disorders.

Cell Syst 2015 Nov;1(5):361-374

Department of Genetics, Stanford Center for Genomics and Personalized Medicine, Stanford, California, 94305 USA.

The prevalence of autism spectrum disorders (ASDs) is rapidly growing, yet its molecular basis is poorly understood. We used a systems approach in which ASD candidate genes were mapped onto the ubiquitous human protein complexes and the resulting complexes were characterized. The studies revealed the role of histone deacetylases (HDAC1/2) in regulating the expression of ASD orthologs in the embryonic mouse brain. Proteome-wide screens for the co-complexed subunits with HDAC1 and six other key ASD proteins in neuronal cells revealed a protein interaction network, which displayed preferential expression in fetal brain development, exhibited increased deleterious mutations in ASD cases, and were strongly regulated by FMRP and MECP2 causal for Fragile X and Rett syndromes, respectively. Overall, our study reveals molecular components in ASD, suggests a shared mechanism between the syndromic and idiopathic forms of ASDs, and provides a systems framework for analyzing complex human diseases.
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http://dx.doi.org/10.1016/j.cels.2015.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776331PMC
November 2015

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

Proteome-wide dataset supporting the study of ancient metazoan macromolecular complexes.

Data Brief 2016 Mar 12;6:715-21. Epub 2015 Dec 12.

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

Our analysis examines the conservation of multiprotein complexes among metazoa through use of high resolution biochemical fractionation and precision mass spectrometry applied to soluble cell extracts from 5 representative model organisms Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Strongylocentrotus purpuratus, and Homo sapiens. The interaction network obtained from the data was validated globally in 4 distant species (Xenopus laevis, Nematostella vectensis, Dictyostelium discoideum, Saccharomyces cerevisiae) and locally by targeted affinity-purification experiments. Here we provide details of our massive set of supporting biochemical fractionation data available via ProteomeXchange (PXD002319-PXD002328), PPIs via BioGRID (185267); and interaction network projections via (http://metazoa.med.utoronto.ca) made fully accessible to allow further exploration. The datasets here are related to the research article on metazoan macromolecular complexes in Nature [1].
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http://dx.doi.org/10.1016/j.dib.2015.11.062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4738005PMC
March 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

Panorama of ancient metazoan macromolecular complexes.

Nature 2015 Sep 7;525(7569):339-44. Epub 2015 Sep 7.

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, here we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we generated a draft conservation map consisting of more than one million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering reveals a spectrum of conservation, ranging from ancient eukaryotic assemblies that have probably served cellular housekeeping roles for at least one billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, affinity purification and functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic importance and adaptive value to animal cell systems.
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http://dx.doi.org/10.1038/nature14877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036527PMC
September 2015
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