Publications by authors named "Anne-Claude Gingras"

223 Publications

Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane.

Nat Commun 2021 Oct 18;12(1):6064. Epub 2021 Oct 18.

Department of Biology, Stanford University, Stanford, CA, USA.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.
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http://dx.doi.org/10.1038/s41467-021-26326-4DOI Listing
October 2021

Resistance of SARS-CoV-2 Beta and Gamma variants to plasma collected from Canadian blood donors during the Spring of 2020.

Transfusion 2021 Oct 18. Epub 2021 Oct 18.

Epidemiology and Surveillance, Canadian Blood Services, Ottawa, Canada.

Background: This pilot study assesses the ability of plasma collected from Canadian blood donors in the first wave of the SARS-CoV-2 pandemic to neutralize later SARS-CoV-2 variants of concern (VOCs).

Study Design And Methods: A repeated cross-sectional design was employed, and a random cross-sectional sample of all available Canadian Blood Services retention samples (n=1500/month) was drawn monthly for April and May of 2020. Qualitative IgG analysis was performed on aliquots of specimens using anti-spike, anti-receptor binding domain and anti-nucleocapsid protein enzyme-linked immunosorbent assays as well as the Abbott Architect SARS CoV-2 IgG assay (Abbott Laboratories, USA) against anti-nucleocapsid protein. Selected plasma specimens were then assessed for neutralization against VOCs using pseudotyped lentivirus inhibition assays as well as plaque reduction neutralization test 50% (PRNT ).

Results: Six specimens with a high neutralizing titer against wild-type SARS-CoV-2 and three specimens with a low neutralizing titer against wild-type SARS-CoV-2 were chosen for further analysis against VOCs. Four of six high neutralizing titer specimens had a reduced neutralizing capacity against Beta VOCs by both neutralization methods. Three of six high neutralizing titer specimens had reduced neutralization capacity against Gamma VOCs.

Conclusions: This preliminary data can be used as a justification for limiting the use of first wave plasma products in upcoming clinical trials but cannot be used to speculate on general trends in the immunity of Canadian blood donors to SARS-CoV-2. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1111/trf.16713DOI Listing
October 2021

Implementation of serological and molecular tools to inform COVID-19 patient management: protocol for the GENCOV prospective cohort study.

BMJ Open 2021 09 30;11(9):e052842. Epub 2021 Sep 30.

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Introduction: There is considerable variability in symptoms and severity of COVID-19 among patients infected by the SARS-CoV-2 virus. Linking host and virus genome sequence information to antibody response and biological information may identify patient or viral characteristics associated with poor and favourable outcomes. This study aims to (1) identify characteristics of the antibody response that result in maintained immune response and better outcomes, (2) determine the impact of genetic differences on infection severity and immune response, (3) determine the impact of viral lineage on antibody response and patient outcomes and (4) evaluate patient-reported outcomes of receiving host genome, antibody and viral lineage results.

Methods And Analysis: A prospective, observational cohort study is being conducted among adult patients with COVID-19 in the Greater Toronto Area. Blood samples are collected at baseline (during infection) and 1, 6 and 12 months after diagnosis. Serial antibody titres, isotype, antigen target and viral neutralisation will be assessed. Clinical data will be collected from chart reviews and patient surveys. Host genomes and T-cell and B-cell receptors will be sequenced. Viral genomes will be sequenced to identify viral lineage. Regression models will be used to test associations between antibody response, physiological response, genetic markers and patient outcomes. Pathogenic genomic variants related to disease severity, or negative outcomes will be identified and genome wide association will be conducted. Immune repertoire diversity during infection will be correlated with severity of COVID-19 symptoms and human leucocyte antigen-type associated with SARS-CoV-2 infection. Participants can learn their genome sequencing, antibody and viral sequencing results; patient-reported outcomes of receiving this information will be assessed through surveys and qualitative interviews.

Ethics And Dissemination: This study was approved by Clinical Trials Ontario Streamlined Ethics Review System (CTO Project ID: 3302) and the research ethics boards at participating hospitals. Study findings will be disseminated through peer-reviewed publications, conference presentations and end-users.
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http://dx.doi.org/10.1136/bmjopen-2021-052842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8487020PMC
September 2021

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence: Navigating the absence of a gold standard.

PLoS One 2021 23;16(9):e0257743. Epub 2021 Sep 23.

Microbiology Department, Canadian Blood Services, Ottawa, Canada.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence studies bridge the gap left from case detection, to estimate the true burden of the COVID-19 pandemic. While multiple anti-SARS-CoV-2 immunoassays are available, no gold standard exists.

Methods: This serial cross-sectional study was conducted using plasma samples from 8999 healthy blood donors between April-September 2020. Each sample was tested by four assays: Abbott SARS-Cov-2 IgG assay, targeting nucleocapsid (Abbott-NP) and three in-house IgG ELISA assays (targeting spike glycoprotein, receptor binding domain, and nucleocapsid). Seroprevalence rates were compared using multiple composite reference standards and by a series of Bayesian Latent Class Models.

Result: We found 13 unique diagnostic phenotypes; only 32 samples (0.4%) were positive by all assays. None of the individual assays resulted in seroprevalence increasing monotonically over time. In contrast, by using the results from all assays, the Bayesian Latent Class Model with informative priors predicted seroprevalence increased from 0.7% (95% credible interval (95% CrI); 0.4, 1.0%) in April/May to 0.7% (95% CrI 0.5, 1.1%) in June/July to 0.9% (95% CrI 0.5, 1.3) in August/September. Assay characteristics varied over time. Overall Spike had the highest sensitivity (93.5% (95% CrI 88.7, 97.3%), while the sensitivity of the Abbott-NP assay waned from 77.3% (95% CrI 58.7, 92.5%) in April/May to 64.4% (95% CrI 45.6, 83.0) by August/September.

Discussion: Our results confirmed very low seroprevalence after the first wave in Canada. Given the dynamic nature of this pandemic, Bayesian Latent Class Models can be used to correct for imperfect test characteristics and waning IgG antibody signals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0257743PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8459951PMC
October 2021

Subcellular proteomics.

Nat Rev Methods Primers 2021 29;1. Epub 2021 Apr 29.

Department of Biochemistry, University of Cambridge, Cambridge, UK.

The eukaryotic cell is compartmentalized into subcellular niches, including membrane-bound and membrane-less organelles. Proteins localize to these niches to fulfil their function, enabling discreet biological processes to occur in synchrony. Dynamic movement of proteins between niches is essential for cellular processes such as signalling, growth, proliferation, motility and programmed cell death, and mutations causing aberrant protein localization are associated with a wide range of diseases. Determining the location of proteins in different cell states and cell types and how proteins relocalize following perturbation is important for understanding their functions, related cellular processes and pathologies associated with their mislocalization. In this Primer, we cover the major spatial proteomics methods for determining the location, distribution and abundance of proteins within subcellular structures. These technologies include fluorescent imaging, protein proximity labelling, organelle purification and cell-wide biochemical fractionation. We describe their workflows, data outputs and applications in exploring different cell biological scenarios, and discuss their main limitations. Finally, we describe emerging technologies and identify areas that require technological innovation to allow better characterization of the spatial proteome.
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http://dx.doi.org/10.1038/s43586-021-00029-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8451152PMC
April 2021

Clionamines stimulate autophagy, inhibit Mycobacterium tuberculosis survival in macrophages, and target Pik1.

Cell Chem Biol 2021 Sep 11. Epub 2021 Sep 11.

Departments of Chemistry and Earth Ocean & Atmospheric Sciences, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada. Electronic address:

The pathogen Mycobacterium tuberculosis (Mtb) evades the innate immune system by interfering with autophagy and phagosomal maturation in macrophages, and, as a result, small molecule stimulation of autophagy represents a host-directed therapeutics (HDTs) approach for treatment of tuberculosis (TB). Here we show the marine natural product clionamines activate autophagy and inhibit Mtb survival in macrophages. A yeast chemical-genetics approach identified Pik1 as target protein of the clionamines. Biotinylated clionamine B pulled down Pik1 from yeast cell lysates and a clionamine analog inhibited phosphatidyl 4-phosphate (PI4P) production in yeast Golgi membranes. Chemical-genetic profiles of clionamines and cationic amphiphilic drugs (CADs) are closely related, linking the clionamine mode of action to co-localization with PI4P in a vesicular compartment. Small interfering RNA (siRNA) knockdown of PI4KB, a human homolog of Pik1, inhibited the survival of Mtb in macrophages, identifying PI4KB as an unexploited molecular target for efforts to develop HDT drugs for treatment of TB.
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http://dx.doi.org/10.1016/j.chembiol.2021.07.017DOI Listing
September 2021

Evaluation of the SARS-CoV-2 Antibody Response to the BNT162b2 Vaccine in Patients Undergoing Hemodialysis.

JAMA Netw Open 2021 09 1;4(9):e2123622. Epub 2021 Sep 1.

Division of Nephrology, Department of Medicine, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada.

Importance: Patients undergoing hemodialysis have a high mortality rate associated with COVID-19, and this patient population often has a poor response to vaccinations. Randomized clinical trials for COVID-19 vaccines included few patients with kidney disease; therefore, vaccine immunogenicity is uncertain in this population.

Objective: To evaluate the SARS-CoV-2 antibody response in patients undergoing chronic hemodialysis following 1 vs 2 doses of BNT162b2 COVID-19 vaccination compared with health care workers serving as controls and convalescent serum.

Design, Setting, And Participants: A prospective, single-center cohort study was conducted between February 2 and April 17, 2021, in Toronto, Ontario, Canada. Participants included 142 patients receiving in-center hemodialysis and 35 health care worker controls.

Exposures: BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine.

Main Outcomes And Measures: SARS-CoV-2 IgG antibodies to the spike protein (anti-spike), receptor binding domain (anti-RBD), and nucleocapsid protein (anti-NP).

Results: Among the 142 participants undergoing maintenance hemodialysis, 94 (66%) were men; median age was 72 (interquartile range, 62-79) years. SARS-CoV-2 IgG antibodies were measured in 66 patients receiving 1 vaccine dose following a public health policy change, 76 patients receiving 2 vaccine doses, and 35 health care workers receiving 2 vaccine doses. Detectable anti-NP suggestive of natural SARS-CoV-2 infection was detected in 15 of 142 (11%) patients at baseline, and only 3 patients had prior COVID-19 confirmed by reverse transcriptase polymerase chain reaction testing. Two additional patients contracted COVID-19 after receiving 2 doses of vaccine. In 66 patients receiving a single BNT162b2 dose, seroconversion occurred in 53 (80%) for anti-spike and 36 (55%) for anti-RBD by 28 days postdose, but a robust response, defined by reaching the median levels of antibodies in convalescent serum from COVID-19 survivors, was noted in only 15 patients (23%) for anti-spike and 4 (6%) for anti-RBD in convalescent serum from COVID-19 survivors. In patients receiving 2 doses of BNT162b2 vaccine, seroconversion occurred in 69 of 72 (96%) for anti-spike and 63 of 72 (88%) for anti-RBD by 2 weeks following the second dose and median convalescent serum levels were reached in 52 of 72 patients (72%) for anti-spike and 43 of 72 (60%) for anti-RBD. In contrast, all 35 health care workers exceeded the median level of anti-spike and anti-RBD found in convalescent serum 2 to 4 weeks after the second dose.

Conclusions And Relevance: This study suggests poor immunogenicity 28 days following a single dose of BNT162b2 vaccine in the hemodialysis population, supporting adherence to recommended vaccination schedules and avoiding delay of the second dose in these at-risk individuals.
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http://dx.doi.org/10.1001/jamanetworkopen.2021.23622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8414193PMC
September 2021

The macrophage-derived protein PTMA induces filamentation of the human fungal pathogen Candida albicans.

Cell Rep 2021 Aug;36(8):109584

Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Evasion of killing by immune cells is crucial for fungal survival in the host. For the human fungal pathogen Candida albicans, internalization by macrophages induces a transition from yeast to filaments that promotes macrophage death and fungal escape. Nutrient deprivation, alkaline pH, and oxidative stress have been implicated as triggers of intraphagosomal filamentation; however, the impact of other host-derived factors remained unknown. Here, we show that lysates prepared from macrophage-like cell lines and primary macrophages robustly induce C. albicans filamentation. Enzymatic treatment of lysate implicates a phosphorylated protein, and bioactivity-guided fractionation coupled to mass spectrometry identifies the immunomodulatory phosphoprotein PTMA as a candidate trigger of C. albicans filamentation. Immunoneutralization of PTMA within lysate abolishes its activity, strongly supporting PTMA as a filament-inducing component of macrophage lysate. Adding to the known repertoire of physical factors, this work implicates a host protein in the induction of C. albicans filamentation within immune cells.
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http://dx.doi.org/10.1016/j.celrep.2021.109584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8454912PMC
August 2021

Early warning and rapid public health response to prevent COVID-19 outbreaks in long-term care facilities (LTCF) by monitoring SARS-CoV-2 RNA in LTCF site-specific sewage samples and assessment of antibodies response in this population: prospective study protocol.

BMJ Open 2021 08 20;11(8):e052282. Epub 2021 Aug 20.

Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.

Introduction: The COVID-19 pandemic has an excessive impact on residents in long-term care facilities (LTCF), causing high morbidity and mortality. Early detection of presymptomatic and asymptomatic COVID-19 cases supports the timely implementation of effective outbreak control measures but repetitive screening of residents and staff incurs costs and discomfort. Administration of vaccines is key to controlling the pandemic but the robustness and longevity of the antibody response, correlation of neutralising antibodies with commercial antibody assays, and the efficacy of current vaccines for emerging COVID-19 variants require further study. We propose to monitor SARS-CoV-2 in site-specific sewage as an early warning system for COVID-19 in LTCF and to study the immune response of the staff and residents in LTCF to COVID-19 vaccines.

Methods And Analysis: The study includes two parts: (1) detection and quantification of SARS-CoV-2 in LTCF site-specific sewage samples using a molecular assay followed by notification of Public Health within 24 hours as an early warning system for appropriate outbreak investigation and control measures and cost-benefit analyses of the system and (2) testing for SARS-CoV-2 antibodies among staff and residents in LTCF at various time points before and after COVID-19 vaccination using commercial assays and neutralising antibody testing performed at a reference laboratory.

Ethics And Dissemination: Ethics approval was obtained from the University of Alberta Health Research Ethics Board with considerations to minimise risk and discomforts for the participants. Early recognition of a COVID-19 case in an LTCF might prevent further transmission in residents and staff. There was no direct benefit identified to the participants of the immunity study. Anticipated dissemination of information includes a summary report to the immunity study participants, sharing of study data with the scientific community through the Canadian COVID-19 Immunity Task Force, and prompt dissemination of study results in meeting abstracts and manuscripts in peer-reviewed journals.
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http://dx.doi.org/10.1136/bmjopen-2021-052282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8382669PMC
August 2021

A Toolbox for Efficient Proximity-Dependent Biotinylation in Zebrafish Embryos.

Mol Cell Proteomics 2021 Jul 29;20:100128. Epub 2021 Jul 29.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, Sinai Health, Toronto, Ontario, Canada. Electronic address:

Understanding how proteins are organized in compartments is essential to elucidating their function. While proximity-dependent approaches such as BioID have enabled a massive increase in information about organelles, protein complexes, and other structures in cell culture, to date there have been only a few studies on living vertebrates. Here, we adapted proximity labeling for protein discovery in vivo in the vertebrate model organism, zebrafish. Using lamin A (LMNA) as bait and green fluorescent protein (GFP) as a negative control, we developed, optimized, and benchmarked in vivo TurboID and miniTurbo labeling in early zebrafish embryos. We developed both an mRNA injection protocol and a transgenic system in which transgene expression is controlled by a heat shock promoter. In both cases, biotin is provided directly in the egg water, and we demonstrate that 12 h of labeling are sufficient for biotinylation of prey proteins, which should permit time-resolved analysis of development. After statistical scoring, we found that the proximal partners of LMNA detected in each system were enriched for nuclear envelope and nuclear membrane proteins and included many orthologs of human proteins identified as proximity partners of lamin A in mammalian cell culture. The tools and protocols developed here will allow zebrafish researchers to complement genetic tools with powerful proteomics approaches.
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http://dx.doi.org/10.1016/j.mcpro.2021.100128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8383115PMC
July 2021

A proximity-dependent biotinylation map of a human cell.

Nature 2021 07 2;595(7865):120-124. Epub 2021 Jun 2.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health System, Toronto, Ontario, Canada.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy and biochemical fractionation coupled with mass spectrometry have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.
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http://dx.doi.org/10.1038/s41586-021-03592-2DOI Listing
July 2021

Comprehensive interactome profiling of the human Hsp70 network highlights functional differentiation of J domains.

Mol Cell 2021 06 5;81(12):2549-2565.e8. Epub 2021 May 5.

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:

Hsp70s comprise a deeply conserved chaperone family that has a central role in maintaining protein homeostasis. In humans, Hsp70 client specificity is provided by 49 different co-factors known as J domain proteins (JDPs). However, the cellular function and client specificity of JDPs have largely remained elusive. We have combined affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to characterize the interactome of all human JDPs and Hsp70s. The resulting network suggests specific functions for many uncharacterized JDPs, and we establish a role of conserved JDPs DNAJC9 and DNAJC27 in histone chaperoning and ciliogenesis, respectively. Unexpectedly, we find that the J domain of DNAJC27 but not of other JDPs can fully replace the function of endogenous DNAJC27, suggesting a previously unappreciated role for J domains themselves in JDP specificity. More broadly, our work expands the role of the Hsp70-regulated proteostasis network and provides a platform for further discovery of JDP-dependent functions.
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http://dx.doi.org/10.1016/j.molcel.2021.04.012DOI Listing
June 2021

TAZ-CAMTA1 and YAP-TFE3 alter the TAZ/YAP transcriptome by recruiting the ATAC histone acetyltransferase complex.

Elife 2021 04 29;10. Epub 2021 Apr 29.

Department of Pathology, University of Iowa, Iowa City, United States.

Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma that metastasizes early in its clinical course and lacks an effective medical therapy. The TAZ-CAMTA1 and YAP-TFE3 fusion proteins are chimeric transcription factors and initiating oncogenic drivers of EHE. A combined proteomic/genetic screen in human cell lines identified YEATS2 and ZZZ3, components of the daontaining histone acetyltransferase (ATAC) complex, as key interactors of both fusion proteins despite the dissimilarity of the C terminal fusion partners CAMTA1 and TFE3. Integrative next-generation sequencing approaches in human and murine cell lines showed that the fusion proteins drive a unique transcriptome by simultaneously hyperactivating a TEAD-based transcriptional program and modulating the chromatin environment via interaction with the ATAC complex. Interaction of the ATAC complex with both fusion proteins indicates that it is a key oncogenic driver and unifying enzymatic therapeutic target for this sarcoma. This study presents an approach to mechanistically dissect how chimeric transcription factors drive the formation of human cancers.
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http://dx.doi.org/10.7554/eLife.62857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143797PMC
April 2021

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

Mol Ther 2021 06 10;29(6):1984-2000. Epub 2021 Feb 10.

Center for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1H 8M5, Canada. Electronic address:

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.
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http://dx.doi.org/10.1016/j.ymthe.2021.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872859PMC
June 2021

Rag GTPases and phosphatidylinositol 3-phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex.

J Cell Biol 2021 02;220(2)

Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.
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http://dx.doi.org/10.1083/jcb.202002075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814351PMC
February 2021

TRK-Fused Gene (TFG), a protein involved in protein secretion pathways, is an essential component of the antiviral innate immune response.

PLoS Pathog 2021 01 7;17(1):e1009111. Epub 2021 Jan 7.

Faculty of Pharmacy, Université de Montréal, Montréal, Canada.

Antiviral innate immune response to RNA virus infection is supported by Pattern-Recognition Receptors (PRR) including RIG-I-Like Receptors (RLR), which lead to type I interferons (IFNs) and IFN-stimulated genes (ISG) production. Upon sensing of viral RNA, the E3 ubiquitin ligase TNF Receptor-Associated Factor-3 (TRAF3) is recruited along with its substrate TANK-Binding Kinase (TBK1), to MAVS-containing subcellular compartments, including mitochondria, peroxisomes, and the mitochondria-associated endoplasmic reticulum membrane (MAM). However, the regulation of such events remains largely unresolved. Here, we identify TRK-Fused Gene (TFG), a protein involved in the transport of newly synthesized proteins to the endomembrane system via the Coat Protein complex II (COPII) transport vesicles, as a new TRAF3-interacting protein allowing the efficient recruitment of TRAF3 to MAVS and TBK1 following Sendai virus (SeV) infection. Using siRNA and shRNA approaches, we show that TFG is required for virus-induced TBK1 activation resulting in C-terminal IRF3 phosphorylation and dimerization. We further show that the ability of the TRAF3-TFG complex to engage mTOR following SeV infection allows TBK1 to phosphorylate mTOR on serine 2159, a post-translational modification shown to promote mTORC1 signaling. We demonstrate that the activation of mTORC1 signaling during SeV infection plays a positive role in the expression of Viperin, IRF7 and IFN-induced proteins with tetratricopeptide repeats (IFITs) proteins, and that depleting TFG resulted in a compromised antiviral state. Our study, therefore, identifies TFG as an essential component of the RLR-dependent type I IFN antiviral response.
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http://dx.doi.org/10.1371/journal.ppat.1009111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790228PMC
January 2021

The PHLPP1 N-Terminal Extension is a Mitotic Cdk1 Substrate and Controls an Interactome Switch.

Mol Cell Biol 2021 Jan 4. Epub 2021 Jan 4.

Department of Pharmacology, University of California, San Diego, California, USA.

PH domain Leucine-Rich Repeat Protein Phosphatase 1 (PHLPP1) is a tumor suppressor that directly dephosphorylates a wide array of substrates, most notably the pro-survival kinase Akt. However, little is known about the molecular mechanisms governing PHLPP1 itself. Here we report that PHLPP1 is dynamically regulated in a cell cycle-dependent manner, and deletion of PHLPP1 results in mitotic delays and increased rates of chromosomal segregation errors. We show that PHLPP1 is hyperphosphorylated during mitosis by Cdk1 in a functionally uncharacterized region known as the PHLPP1 N-terminal extension (NTE). A proximity-dependent biotin identification (BioID) interaction screen revealed that during mitosis PHLPP1 dissociates from plasma membrane scaffolds, such as Scribble, by a mechanism that depends on its NTE, and gains proximity with kinetochore and mitotic spindle proteins such as KNL1 and TPX2. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows accurate progression through mitosis. The finding that PHLPP1 binds mitotic proteins in a cell cycle- and phosphorylation-dependent manner may have relevance to its tumor suppressive function.
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http://dx.doi.org/10.1128/MCB.00333-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088274PMC
January 2021

Mitochondrial Threonyl-tRNA Synthetase TARS2 Is Required for Threonine-Sensitive mTORC1 Activation.

Mol Cell 2021 01 18;81(2):398-407.e4. Epub 2020 Dec 18.

Goodman Cancer Research Centre, McGill University, Montreal, QC H3A 1A3, Canada; Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada. Electronic address:

Mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and proliferation by sensing fluctuations in environmental cues such as nutrients, growth factors, and energy levels. The Rag GTPases (Rags) serve as a critical module that signals amino acid (AA) availability to modulate mTORC1 localization and activity. Recent studies have demonstrated how AAs regulate mTORC1 activity through Rags. Here, we uncover an unconventional pathway that activates mTORC1 in response to variations in threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. TARS2 interacts with inactive Rags, particularly GTP-RagC, leading to increased GTP loading of RagA. mTORC1 activity in cells lacking TARS2 is resistant to Thr repletion, showing that TARS2 is necessary for Thr-dependent mTORC1 activation. The requirement of TARS2, but not cytoplasmic threonyl-tRNA synthetase TARS, for this effect demonstrates an additional layer of complexity in the regulation of mTORC1 activity.
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http://dx.doi.org/10.1016/j.molcel.2020.11.036DOI Listing
January 2021

Systematic Examination of Antigen-Specific Recall T Cell Responses to SARS-CoV-2 versus Influenza Virus Reveals a Distinct Inflammatory Profile.

J Immunol 2021 01 18;206(1):37-50. Epub 2020 Nov 18.

Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

There is a pressing need for an in-depth understanding of immunity to SARS-CoV-2. In this study, we investigated human T cell recall responses to fully glycosylated spike trimer, recombinant N protein, as well as to S, N, M, and E peptide pools in the early convalescent phase and compared them with influenza-specific memory responses from the same donors. All subjects showed SARS-CoV-2-specific T cell responses to at least one Ag. Both SARS-CoV-2-specific and influenza-specific CD4 T cell responses were predominantly of the central memory phenotype; however SARS-CoV-2-specific CD4 T cells exhibited a lower IFN-γ to TNF ratio compared with influenza-specific memory responses from the same donors, independent of disease severity. SARS-CoV-2-specific T cells were less multifunctional than influenza-specific T cells, particularly in severe cases, potentially suggesting exhaustion. Most SARS-CoV-2-convalescent subjects also produced IFN-γ in response to seasonal OC43 S protein. We observed granzyme B/IFN-γ, CD4, and CD8 proliferative responses to peptide pools in most individuals, with CD4 T cell responses predominating over CD8 T cell responses. Peripheral T follicular helper (pTfh) responses to S or N strongly correlated with serum neutralization assays as well as receptor binding domain-specific IgA; however, the frequency of pTfh responses to SARS-CoV-2 was lower than the frequency of pTfh responses to influenza virus. Overall, T cell responses to SARS-CoV-2 are robust; however, CD4 Th1 responses predominate over CD8 T cell responses, have a more inflammatory profile, and have a weaker pTfh response than the response to influenza virus within the same donors, potentially contributing to COVID-19 disease.
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http://dx.doi.org/10.4049/jimmunol.2001067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7750861PMC
January 2021

The GATOR-Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids.

Science 2020 10;370(6514):351-356

Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada.

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.
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http://dx.doi.org/10.1126/science.aaz0863DOI Listing
October 2020

Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients.

Sci Immunol 2020 10;5(52)

Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital, Sinai Health System, Toronto, ON, Canada.

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the antibody response in saliva and its relationship to systemic antibody levels. Here, we profiled by enzyme-linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor-binding domain (RBD) in serum and saliva of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3-115 days post-symptom onset (PSO), compared to negative controls. Anti-SARS-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16-30 days PSO. Longitudinal analysis revealed that anti-SARS-CoV-2 IgA and IgM antibodies rapidly decayed, while IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that serum and saliva IgG antibodies to SARS-CoV-2 are maintained in the majority of COVID-19 patients for at least 3 months PSO. IgG responses in saliva may serve as a surrogate measure of systemic immunity to SARS-CoV-2 based on their correlation with serum IgG responses.
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http://dx.doi.org/10.1126/sciimmunol.abe5511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050884PMC
October 2020

The NEMP family supports metazoan fertility and nuclear envelope stiffness.

Sci Adv 2020 Aug 28;6(35):eabb4591. Epub 2020 Aug 28.

Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON M5G 1X5, Canada.

Human genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in () with early menopause; however, it is unclear whether NEMP1 has any role in fertility. We show that whole-animal loss of NEMP1 homologs in , , zebrafish, and mice leads to sterility or early loss of fertility. Loss of Nemp leads to nuclear shaping defects, most prominently in the germ line. Biochemical, biophysical, and genetic studies reveal that NEMP proteins support the mechanical stiffness of the germline nuclear envelope via formation of a NEMP-EMERIN complex. These data indicate that the germline nuclear envelope has specialized mechanical properties and that NEMP proteins play essential and conserved roles in fertility.
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http://dx.doi.org/10.1126/sciadv.abb4591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455189PMC
August 2020

IRX3/5 regulate mitotic chromatid segregation and limb bud shape.

Development 2020 10 5;147(19). Epub 2020 Oct 5.

Program in Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation.
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http://dx.doi.org/10.1242/dev.180042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561487PMC
October 2020

A High-Density Human Mitochondrial Proximity Interaction Network.

Cell Metab 2020 09;32(3):479-497.e9

Montreal Neurological Institute, McGill University, Montreal, QC, Canada; Department of Human Genetics, McGill University, Montreal, QC, Canada. Electronic address:

We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human mitochondrial proximity interaction network. We identified 1,465 proteins, producing 15,626 unique high-confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles.
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http://dx.doi.org/10.1016/j.cmet.2020.07.017DOI Listing
September 2020

A simple protein-based surrogate neutralization assay for SARS-CoV-2.

JCI Insight 2020 10 2;5(19). Epub 2020 Oct 2.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.
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http://dx.doi.org/10.1172/jci.insight.142362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566699PMC
October 2020

Functional characterization of a PROTAC directed against BRAF mutant V600E.

Nat Chem Biol 2020 11 10;16(11):1170-1178. Epub 2020 Aug 10.

Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Quebec, Montreal, Canada.

The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.
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http://dx.doi.org/10.1038/s41589-020-0609-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862923PMC
November 2020

A Comprehensive, Flexible Collection of SARS-CoV-2 Coding Regions.

G3 (Bethesda) 2020 09 2;10(9):3399-3402. Epub 2020 Sep 2.

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

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.
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http://dx.doi.org/10.1534/g3.120.401554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467003PMC
September 2020

Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism.

Nat Metab 2020 06 1;2(6):499-513. Epub 2020 Jun 1.

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

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases, including cancer. Because cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to the loss of de novo fatty acid biosynthesis. Here, we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in fatty acid synthase (FASN), whose product catalyses the formation of long-chain fatty acids. FASN-mutant cells show a strong dependence on lipid uptake that is reflected in negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking and protein glycosylation. Further support for these functional relationships is derived from additional GI screens in query cell lines deficient in other genes involved in lipid metabolism, including LDLR, SREBF1, SREBF2 and ACACA. Our GI profiles also identify a potential role for the previously uncharacterized gene C12orf49 (which we call LUR1) in regulation of exogenous lipid uptake through modulation of SREBF2 signalling in response to lipid starvation. Overall, our data highlight the genetic determinants underlying the cellular adaptation associated with loss of de novo fatty acid synthesis and demonstrate the power of systematic GI mapping for uncovering metabolic buffering mechanisms in human cells.
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http://dx.doi.org/10.1038/s42255-020-0211-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566881PMC
June 2020

AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion protein network.

Nat Commun 2020 07 17;11(1):3586. Epub 2020 Jul 17.

Montreal Clinical Research Institute (IRCM), Montréal, QC, H2W 1R7, Canada.

Aberrant expression of receptor tyrosine kinase AXL is linked to metastasis. AXL can be activated by its ligand GAS6 or by other kinases, but the signaling pathways conferring its metastatic activity are unknown. Here, we define the AXL-regulated phosphoproteome in breast cancer cells. We reveal that AXL stimulates the phosphorylation of a network of focal adhesion (FA) proteins, culminating in faster FA disassembly. Mechanistically, AXL phosphorylates NEDD9, leading to its binding to CRKII which in turn associates with and orchestrates the phosphorylation of the pseudo-kinase PEAK1. We find that PEAK1 is in complex with the tyrosine kinase CSK to mediate the phosphorylation of PAXILLIN. Uncoupling of PEAK1 from AXL signaling decreases metastasis in vivo, but not tumor growth. Our results uncover a contribution of AXL signaling to FA dynamics, reveal a long sought-after mechanism underlying AXL metastatic activity, and identify PEAK1 as a therapeutic target in AXL positive tumors.
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http://dx.doi.org/10.1038/s41467-020-17415-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368075PMC
July 2020

Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation.

Nucleic Acids Res 2020 08;48(14):7924-7943

Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA.

Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.
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http://dx.doi.org/10.1093/nar/gkaa592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430652PMC
August 2020
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