Biology subjects

Boisvert, F.-M.

Publications and source records attributed to Boisvert, F.-M..

13 recordsLinked to original sources

bioRxiv2026

Temporal proximity proteomics reveals ANP32A roles in replication fork progression and end joining

Acidic nuclear phosphoprotein 32 family member A (ANP32A) regulates chromatin and histone homeostasis, but its contribution to DNA replication and repair remains unclear. Time-resolved AirID proximity labeling with data-independent acquisition mass spectrometry revealed phased remodeling of the ANP32A proximal proteome during hydroxyurea-induced replication stress. Early stress enriched DNA replication and repair factors, including FEN1, XRCC5/Ku80, and XRCC6/Ku70, whereas prolonged exposure shifted the network towards checkpoint and cell-cycle regulators. Orthogonal proximity ligation assays revealed sustained ANP32A-FEN1 proximity and transiently increased ANP32A-XRCC5 proximity. ANP32A was dynamically recruited to {gamma}H2AX-marked lesions following laser microirradiation and showed increased proximity to {gamma}H2AX after hydroxyurea treatment. ANP32A loss impaired proliferation and G1/S progression and moderately reduced replication fork velocity. However, it did not promote nascent strand degradation at stalled forks or measurably alter homologous recombination reporter activity. In contrast, ANP32A loss reduced non-homologous end-joining reporter activity, consistent with its stress-induced proximity to Ku proteins. These findings identify ANP32A as a chromatin-associated factor supporting replication fork progression and end joining while dispensable for stalled-fork protection, linking ANP32A-dependent chromatin regulation to genome maintenance.

cell biology↗

bioRxiv2025

NLRC5 expression in tumor cells is critical to activate adaptive and innate antitumor immune responses

Tumors evade cytotoxic T lymphocyte (CTL)-mediated killing by downregulating MHC class-I, mainly resulting from the loss of its transcriptional activator NLRC5. Expressing full-length NLRC5 (NLRC5-FL) or a shorter NLRC5-CIITA fusion protein termed NLRC5 super-activator (NLRC5-SA) in cancer cells upregulates MHC-I expression and promotes antitumor immunity. To distinguish the role of NLRC5 expressed within tumor cells and antigen presenting cells, we studied B16-F10 melanoma expressing NLRC5-FL (B16-N-FL) or NLRC5-SA (B16-N-SA) in Nlrc5+/+ and Nlrc5-/- mice. Both tumors were efficiently controlled in both Nlrc5+/+ and Nlrc5-/- hosts with abundant immune cell infiltration, enriched for activated and differentiated CD8+ and CD4+ T cells, NK, NKT and iNKT cells. B16-N-FL and B16-N-SA tumors showed increased collagen deposition and vascularization, with upregulation of CCL4 and CXCL9 chemokine genes in B16-N-SA tumors. Depletion of either CD8+ T cells or NK1.1+ cells increased the growth of B16-N-FL and B16-N-SA tumors in Nlrc5+/+ mice, and that of B16-N-SA tumors in Nlrc5-/- hosts. Proteomes of B16-N-FL and B16-N-SA cells showed downmodulation of dominant tumor antigens and upregulation of ubiquitination and protein processing pathway proteins. Differentially expressed proteins shared between B16-N-FL and B16-N-SA cells showed enrichment in phagosome and autophagy pathways. We conclude that tumor cell-intrinsic NLRC5 expression is critical for the activation of adaptive and innate immune cells, and establishment of an immune-supportive tumor microenvironment to permit immune cell infiltration and their effector functions and achieve tumor control. NLRC5 expression in APCs is dispensable to mediate these effects. Delivering NLRC5-SA is a promising approach to restore antitumor immune responses in MHC-I-low immune evasive tumors. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/695247v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1bc4f51org.highwire.dtl.DTLVardef@1fb5b03org.highwire.dtl.DTLVardef@11448edorg.highwire.dtl.DTLVardef@bff26c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Essential role of NLRC5 in cancer immune surveillance and cancer immunoediting

A key mechanism of immune escape from CD8+ T cell-mediated tumor control occurs via downregulation of NLRC5, the IFN{gamma}-induced transcriptional activator of MHC class-I. As NLRC5 deficiency does not abrogate CD8+ T cell development, we investigated whether NLRC5-dependent antitumor immune mechanisms are required for immune surveillance. Development of 3-methylcholanthrene (MCA)-induced endogenous fibrosarcoma was studied in Nlrc5-/- mice with Nlrc5+/+ and Rag1-/- mice serving as controls. Nlrc5-/- and Rag1-/- mice showed increased propensity to develop MCA-induced tumors with elevated growth rate compared to Nlrc5+/+ mice, and displayed significantly reduced survival. Tumors from Nlrc5+/+ and Nlrc5-/-mice, but not from Rag1-/- mice, contained necrotic areas and displayed T cell infiltration. Tumor cell lines established from MCA- induced tumors were evaluated for their sensitivity to immune-mediated growth control following implantation into immunocompetent C57BL/6 and immunodeficient Rag1-/- hosts. Tumors formed by Nlrc5+/+ tumor cell lines progressed unhindered in C57BL/6 hosts that reflected their immunoedited status, whereas cell lines from Nlrc5-/- and Rag1-/-tumors were efficiently controlled, indicating their non-immunoedited status. Proteomic analysis by mass spectrometry followed by pathway analysis revealed enrichment of granzyme-mediated cytolytic pathway in Nlrc5+/+ tumors that were absent in Nlrc5-/- tumors, which showed enrichment of humoral and innate immune pathways. Overall, our findings show that NLRC5 is required for robust tumor immune surveillance and tumor immunoediting and that compensatory humoral and innate immune mechanisms activated by the loss of NLRC5 are insufficient for cancer immune surveillance and cancer immunoediting.

immunology↗

bioRxiv2024

A Proximity MAP of RAB GTPases

RAB GTPases are the most abundant family of small GTPases and regulate multiple aspects of membrane trafficking events, from cargo sorting to vesicle budding, transport, docking, and fusion. To regulate these processes, RABs are tightly regulated by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). Activated RABs recruit effector proteins that regulate trafficking. Identifying RAB-associated proteins has proven to be difficult because their association with interacting proteins is often transient. Recent advances in proximity labeling approaches that allow for the covalent labeling of neighbors of proteins of interest now permit the cataloging of proteins in the vicinity of RAB GTPases. Here, we report APEX2 proximity labeling of 23 human RABs and their neighboring proteomes. We have used bioinformatic analyses to map specific proximal proteins for an extensive array of RAB GTPases, and RAB localization can be inferred from their adjacent proteins. Focusing on specific examples, we identified a physical interaction between RAB25 and DENND6A, which affects cell migration. We also show functional relationships between RAB14 and the EARP complex, or between RAB14 and SHIP164 and its close ortholog UHRF1BP1. Our dataset provides an extensive resource to the community and helps define novel functional connections between RAB GTPases and their neighboring proteins.

cell biology↗

bioRxiv2024

The Pseudogene RPS27AP5 Reveals Novel Ubiquitin and Ribosomal Protein Variants Involved in Specialised Ribosomal Functions.

Pseudogenes, traditionally considered non-functional gene copies resulting from evolutionary mutations, have garnered attention due to recent transcriptomics and proteomics revealing their unexpected expressions and consequential cellular functions. Ubiquitin, transcribed from UBA52 and RPS27A genes, fused to ribosomal proteins eL40 and eS31, and polyubiquitin precursors encoded by UBB and UBC genes, has additional pseudogenes labeled as non-functional. However, recent evidence challenges this notion, demonstrating that these pseudogenes produce ubiquitin variants with minimal differences from the canonical sequence, suggesting a new regulatory dimension in ubiquitin-mediated cellular processes. To systematically catalogue possible Ubiquitin (Ub) and Ubiquitin-like (Ubl) variants from pseudogenes, expression data was compiled, identifying potential functional variants. Among these pseudogenes, RPS27AP5 expresses both Ubiquitin variant (UbP5) and ribosomal protein variant (S27aP5), with precursor proteins maturing through cleavage and exhibiting behavior similar to their counterparts post-translation. Notably, S27aP5 integrates into translating ribosomes, increasing the 80S monosomal ribosomal fraction and indirectly influencing p16INK4A transcriptional activation. The discovery of a functional S27a pseudogene supports the concept that a subset of ribosomes may incorporate diverse subunits for specific translational functions.

cell biology↗

bioRxiv2023

Exploring the interactome of PML nuclear subdomains during fatty acid stress using APEX2-mediated proximity labeling

When exposed to excess fatty acids, specific cell types produce nuclear lipid droplets (nLDs) that associate with promyelocytic leukemia (PML) protein to form Lipid Associated PML Structures (LAPS) that are enriched in lipid biosynthetic enzymes but deficient in canonical proteins associated with PML nuclear bodies (PML NBs). To identify the PML interactome during lipid stress, we employed proximity-dependent biotin identification (BioID) in U2OS cells expressing PMLI and PMLII fused to the ascorbate peroxidase APEX2 and cultured in the absence or presence of oleate to enhance lipid droplet formation. The resulting interactome included proteins enriched under oleate-treated conditions, such mitogen activated protein kinase-activated protein kinase 2 (MK2), ESCRT proteins and the COPII vesicle transport proteins Sec23B, Sec24A and USO1. COPII proteins co-localized with both PML-NBs and LAPS but were selectively enriched in PML-NBs following oleate treatment. The nuclear localization of USO1 was uniquely dependent on PML expression. Thus, the APEX2-PML proximity interactome implicates PML domains in the nuclear function of a non-canonical network of COPII vesicle trafficking proteins. SUMMARY STATEMENTThis is the first study to utilize APEX2 proximity labelling to identify the protein interactome of PML nuclear substructures and how interactions are modified under conditions of fatty acid-induced stress.

cell biology↗

bioRxiv2023

CRISPR/Cas9-mediated knockout of the ubiquitin variant UbKEKS reveals a role in regulating nucleolar structures and composition.

Ubiquitination is a post-translational modification responsible for one of the most complex multi-layered communication and regulation system in the cell. Over the past decades, new ubiquitin variants and ubiquitin-like proteins arose to further enrich this mechanism. Among them, the recently discovered ubiquitin variant UbKEKS can specifically target several proteins and yet, functional consequences of this new modification remain unknown. The absence of UbKEKS induces accumulation of lamin A in the nucleoli, highlighting the need for deeper investigations about protein composition and functions regulation of this highly dynamic and membrane-less compartment. By using data independent acquisition mass spectrometry and microscopy, we show here that despite not impacting protein stability, UbKEKS is required to maintain normal nucleolar organization. The absence of UbKEKS increases nucleolis size and accentuate their circularity while disrupting dense fibrillar component and fibrillar center structures. Moreover, depletion of UbKEKS leads to distinct changes in nucleolar composition. Notably, lack of UbKEKS favors nucleolar sequestration of known apoptotic regulators such as IFI16 or p14ARF, resulting in an increase of apoptosis in UbKEKS knockout cells observed by flow cytometry and real-time cellular growth monitoring. Overall, the results presented here identifies the first cellular functions of the UbKEKS variant and lay the foundation stone to establish UbKEKS as a new universal layer of regulation in the already complex ubiquitination system.

cell biology↗

bioRxiv2022

Antagonistic roles of canonical and alternative RPA in tandem CAG repeat diseases

Tandem CAG repeat expansion mutations cause >15 neurodegenerative diseases, where ongoing expansions in patients brains are thought to drive disease onset and progression. Repeat length mutations will involve single-stranded DNAs prone to form mutagenic DNA structures. However, the involvement of single-stranded DNA binding proteins (SSBs) in the prevention or formation of repeat instability is poorly understood. Here, we assessed the role of two SSBs, canonical RPA (RPA1-RPA2-RPA3) and the related Alternative-RPA (Alt-RPA, RPA1-RPA4-RPA3), where the primate-specific RPA4 replaces RPA2. RPA is essential for all forms of DNA metabolism, while Alt-RPA has undefined functions. RPA and Alt-RPA are upregulated 2- and 10-fold, respectively, in brains of Huntington disease (HD) and spinocerebellar ataxia type 1 (SCA1) patients. Correct repair of slipped-CAG DNA structures, intermediates of expansion mutations, is enhanced by RPA, but blocked by Alt-RPA. Slipped-DNAs are bound and melted more efficiently by RPA than by Alt-RPA. Removal of excess slipped-DNAs by FAN1 nuclease is enhanced by RPA, but blocked by Alt-RPA. Protein-protein interactomes (BioID) reveal unique and shared partners of RPA and Alt-RPA, including proteins involved in CAG instability and known modifiers of HD and SCA1 disease. RPA overexpression inhibits rampant CAG expansions in SCA1 mouse brains, coinciding with improved neuron morphology and rescued motor phenotypes. Thus, SSBs are involved in repeat length mutations, where Alt-RPA antagonistically blocks RPA from suppressing CAG expansions and hence pathogenesis. The processing of repeat length mutations is one example by which an Alt-RPA{leftrightarrow}RPA antagonistic interaction can affect outcomes, illuminating questions as to which of the many processes mediated by canonical RPA may also be modulated by Alt-RPA.

genetics↗

bioRxiv2022

Subcellular distribution of PP1 isoforms in holoenzyme complexes

Unlike its counterpart Ser/Thr kinases, the predominant Ser/Thr protein phosphatase 1 (PP1) is a promiscuous enzyme that gains its subcellular localization and substrate specificity from a large panel of regulatory proteins with which it associates in predominantly dimeric complexes. Inhibition of specific PP1-mediated dephosphorylation events relies on targeting the regulatory rather than the catalytic subunit, which in turn relies on a comprehensive understanding of the holoenzyme complexes that underlie its distribution throughout the cell. Proteomic, bioinformatic and biochemical screens have assembled lists of putative regulatory proteins, which have been studied to varying degrees. We took a non-biased approach to link steady-state localization to complexes, using a combination of fluorescence imaging, cellular fractionation and quantitative affinity purification/mass spectrometry (AP/MS) to map interactomes for PP1[a]/{beta}/{gamma} in 3 human cell lines. Comparing the distribution of each isoform between the pool of identified regulatory subunits highlighted key signaling pathways and identified c20orf27 as a novel PP1 regulatory protein. Steady-state association of a large fraction of PP1 with the evolutionarily conserved SDS22 was demonstrated, as was redistribution at the entry to mitosis. This is consistent with recent work suggesting that SDS22 acts as a PP1 sink from which it can be recruited as needed. Moving forward, this approach can be used to assess the redistribution of PP1 during other cellular processes or in response to perturbations or disease states, facilitating identification of the relevant complexes and the design of strategies to target them therapeutically.

cell biology↗

bioRxiv2022

Ketolysis is a metabolic driver of CD8+ T cell effector function through histone acetylation

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. However, the metabolic pathways critical for optimal T cell responses remain poorly understood. Here, we identify ketone bodies (KBs) - including {beta}-hydroxybutyrate ({beta}OHB) and acetoacetate (AcAc) - as essential fuels supporting CD8+ T cell metabolism and effector function. Ketolysis is an intrinsic feature of highly functional CD8+ T effector (Teff) cells and {beta}OHB directly increases CD8+ Teff cell IFN-{gamma} production and cytolytic activity. Using metabolic tracers, we establish that CD8+ Teff cells preferentially use KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boost the respiratory capacity of CD8+ T cells and TCA cycle-dependent metabolic pathways that fuel T cell growth. Mechanistically, we find that {beta}OHB is a major substrate for acetyl-CoA production in CD8+ T cells and regulates effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses. One Sentence summaryKetone bodies promote CD8+ T cell metabolism and effector function through regulation of epigenetic programming

immunology↗

bioRxiv2020

The early endosomal protein Rab21 is critical for enterocyte functions and intestinal homeostasis

Membrane trafficking is defined as the vesicular transport of molecules into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to genetic diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo model system, we investigated enterocyte-specific functions for the early endosomal trafficking machinery in gut homeostasis. We focused on the small GTPase Rab21, which regulates specific steps in early endosomal trafficking. Rab21-depleted guts showed severe abnormalities in intestinal morphology, with deregulated homeostasis associated with a gain in mitotic cells and increased cell death. Increases in both apoptosis and yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using a RNA interference screen, we identified specific regulators of autophagy and membrane trafficking that phenocopied Rab21 loss. We further showed that Rab21-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling, and identified improperly trafficked cargoes in Rab21-depleted enterocytes. Our data shed light on an important role for early endosomal trafficking, and particularly Rab21, in enterocyte-mediated intestinal homeostasis.

cell biology↗

bioRxiv2020

SILAC proteomics implicates the ubiquitin conjugating enzyme UBE2D in SOCS1-mediated downmodulation of the MET receptor in hepatocytes

Suppressor of Cytokine Signaling 1 (SOCS1) functions as a tumor suppressor in hepatocellular carcinoma (HCC) and many other types of cancers. SOCS1 mediates its functions by inhibiting tyrosine kinases, promoting ubiquitination and proteasomal degradation of signal transducing proteins, and by modulating transcription factors. Here, we studied the impact of SOCS1 on the hepatocyte proteome using Stable Isotopic Labelling of Amino acids in Cell culture (SILAC)-based mass spectrometry on the Hepa1-6 murine HCC cell line stably expressing wildtype SOCS1 or a mutant SOCS1 with impaired SH2 domain. As SOCS1 regulates the hepatocyte growth factor (HGF) receptor MET, the SILAC-labelled cells were stimulated or not with HGF. Following mass spectrometry analysis, differentially modulated proteins were identified, quantified and analyzed for pathway enrichment. Of the 3440 proteins identified in Hepa-SOCS1 cells at steady state, 181 proteins were significantly modulated compared to control cells. The SH2 domain mutation and HGF increased the number of differentially modulated proteins. Protein interaction network analysis revealed enrichment of SOCS1-modulated proteins within multiprotein complexes such as ubiquitin conjugating enzymes, proteasome, mRNA spliceosome, mRNA exosome and mitochondrial ribosome. These findings suggest that SOCS1, induced by cytokines, growth factors and diverse other stimuli, may dynamically modulate of large macromolecular regulatory complexes to help maintain cellular homeostasis. Notably, the expression of UBE2D ubiquitin conjugating enzyme, which is implicated in the control of growth factor receptor tyrosine kinase signaling, was found to be regulated by SOCS1.

molecular biology↗

bioRxiv2019

Human Hepatocyte Nuclear Factor 4-α encodes isoforms with distinct transcriptional functions

HNF4 is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. In order to characterize the transcriptional capacities of all 12 HNF4 isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The 1 and 2 isoforms were the most potent regulators of gene expression while the 3 isoform exhibited significantly reduced activity. The 4, 5 and 6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4 isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4 isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.

cell biology↗