Biology subjects

Boyce, M.

Publications and source records attributed to Boyce, M..

15 recordsLinked to original sources

bioRxiv2026

Divergent Pathogenic PR-DUB Complex Variants Converge Functionally Via PRC2 Displacement From Chromatin

The PR-DUB complex is responsible for erasing the repressive histone modification, H2AK119ub1. ASXL1-3 proteins are mutually exclusive catalytic partners of BAP1 in the PR-DUB complex. Somatic heterozygous ASXL1-3 variants are associated with cancer, including myeloid malignancies, while de novo germline variants cause neurodevelopmental disorders such as Bohring-Opitz syndrome. These pathogenic variants are almost exclusively nonsense and frameshift and have been proposed to act as gain-of-function. However, the precise catalytic impact and mechanism of variant ASXL1-3 remains elusive. Using an isogenic embryonic stem cell model we have discovered that ASXL1 BOS variants drive reductions - but not global ablations - in H2AK119ub1, consistent with gain-of-function. This catalytic change occurs through the production of a truncated ASXL1 protein with enhanced stability. Hyper-stabilised ASXL1 drives a stoichiometric shift in PR-DUB assembly away from ASXL2 complexes. The drop in H2AK119ub1 levels ultimately reduces PRC2 binding and H3K27me3 deposition. Surprisingly, this phenotype is shared across PR-DUB loss-of-function models and indeed is emerging as a common phenotype across genetically and mechanistically distinct Polycomb-related chromatinopathies.

genetics↗

bioRxiv2026

Discovery of cell-active small molecule inhibitors of UDP-galactose 4'-epimerase

Glycosylation depends on tightly regulated pools of nucleotide-sugars (NS), yet the mechanisms controlling mammalian NS homeostasis and their downstream effects on glycoprotein biosynthesis remain poorly understood. UDP-galactose 4'-epimerase (GALE) catalyzes the reversible interconversion of UDP-galactose/UDP-glucose and UDP-N-acetylgalactosamine/UDP-N-acetylglucosamine, making it a central regulator of glycan precursor pools and an excellent model enzyme for studying NS metabolism. Here, we report the discovery of a cell-active small molecule inhibitor of human GALE through a high-throughput chemical screening strategy. Using a coupled luminescence-based assay, we identified the FDA-approved drug disulfiram as a GALE inhibitor. Biochemical analyses demonstrated that disulfiram directly inhibits GALE through covalent modification of cysteine residues, including C153, likely via its reactive metabolite diethyldithiocarbamate. In cultured human cells, disulfiram treatment phenocopied genetic GALE deletion, reducing terminally sialylated glycans, mucin-type O-glycans, and properly glycosylated mucin-domain glycoproteins. These effects were rescued by galactose supplementation, consistent with a mechanism of on-target GALE inhibition. Similar phenotypes were observed in human lung adenocarcinoma cells, supporting a broader role for GALE in regulating glycosylation and mucin biosynthesis across tissue types. Together, these studies establish a platform for the discovery of pharmacological GALE inhibitors as new research tools, identify disulfiram as a cell-active chemical probe for studying NS regulation, and suggest that targeting GALE might modulate mucin hypersecretion in muco-obstructive diseases and mucinous cancers.

biochemistry↗

bioRxiv2025

Dynamic O-GlcNAcylation of Sec23-interacting protein regulates COPII function

About one-third of the eukaryotic proteome transits the secretory pathway to reach its correct cellular or extracellular destination. At the earliest stage, transport from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) or Golgi apparatus is mediated by coat protein complex II (COPII). COPII coats consist of inner and outer layers formed by Sec23-Sec24 heterodimers and Sec13-Sec31 heterotetramers, respectively, which initially assemble at ER exit sites (ERES) to form transport carriers. Sec23-interacting protein (Sec23IP) links the inner and outer coats through its interactions with both Sec23A and Sec31A, positioning it as a key potential regulator of COPII function. However, the mechanisms controlling Sec23IP activity remain poorly understood. Here, we investigate how physiological stimuli regulate COPII function through the dynamic modification of Sec23IP by O-linked {beta}-N-acetylglucosamine (O-GlcNAc), a reversible, intracellular form of glycosylation. We first validated Sec23IP as a bona fide O-GlcNAcylated protein. Rescue experiments in Sec23IP knockout cells with a nearly unglycosylatable mutant protein demonstrated the essential role of O-GlcNAcylation in the intrinsically disordered domain in protein transport and in recruiting Sec31A to ERES. Moreover, O-GlcNAcylation of Sec23IP increased during protein transport, coinciding with a reduction in its interaction with Sec31A. These results indicate that distinct site-specific O-GlcNAcylation of Sec23IP spatiotemporally modulates its association with Sec31A to fine-tune ERES recruitment and COPII assembly/disassembly. Our work provides new insight into Sec23IP regulation and suggests that O-GlcNAc on other COPII proteins may govern carrier formation, uncoating, and transport.

cell biology↗

bioRxiv2025

Dynamic regulation of the COPII interactome and collagen trafficking by site-specific glycosylation of Sec24D

Coat protein complex II (COPII) mediates anterograde trafficking from the endoplasmic reticulum (ER). While the core COPII machinery is well-characterized, how cells regulate COPII to accommodate large cargoes, including collagens, remains incompletely understood. Here, we show that the cargo-selecting COPII subunit Sec24D is modified by site-specific O-linked {beta}-N-acetylglucosamine (O-GlcNAc) in its N-terminal intrinsically disordered region upon induction of collagen transport. These glycosylations are required for collagen trafficking in human cells and developing zebrafish. Crosslinking proteomics demonstrated that each O-GlcNAcylation influences the Sec24D interactome in a distinct way, revealing novel mediators of COPII function. In particular, Sec24D glycosylation is required for its interaction with myoferlin, which unexpectedly facilitates fusion of ER exit sites (ERES) and the ER-Golgi intermediate compartment (ERGIC) to enable collagen transport. Our results establish Sec24D O-GlcNAcylation as a dynamic regulator of COPII protein-protein interactions and collagen trafficking and identify myoferlin as a novel mediator of this process.

cell biology↗

bioRxiv2025

Evidence for Functional Regulation of the KLHL3/WNK Pathway by O-GlcNAcylation

The 42-member Kelch-like (KLHL) protein family are adaptors for ubiquitin E3 ligase complexes, governing the stability of a wide range of substrates. KLHL proteins are critical for maintaining proteostasis in a variety of tissues and are mutated in human diseases, including cancer, neurodegeneration, and familial hyperkalemic hypertension. However, the regulation of KLHL proteins remains incompletely understood. Previously, we reported that two KLHL family members, KEAP1 and gigaxonin, are regulated by O-linked {beta}-N-acetylglucosamine (O-GlcNAc), an intracellular form of glycosylation. Interestingly, some ubiquitination targets of KEAP1 and gigaxonin are themselves also O-GlcNAcylated, suggesting that multi-level control by this post-translational modification may influence many KLHL pathways. To test this hypothesis, we examined KLHL3, which ubiquitinates with-no-lysine (WNK) kinases to modulate downstream ion channel activity. Our biochemical and glycoproteomic data demonstrate that human KLHL3 and all four WNK kinases (WNK1-4) are O-GlcNAcylated. Moreover, our results suggest that O-GlcNAcylation affects WNK4 function in both osmolarity control and ferroptosis, with potential implications ranging from blood pressure regulation to neuronal health and survival. This work demonstrates the functional regulation of the KLHL3/WNK axis by O-GlcNAcylation and supports a broader model of O-GlcNAc serving as a general regulator of KLHL signaling and proteostasis.

cell biology↗

bioRxiv2025

Dynamic regulation of Sec24C by phosphorylation and O-GlcNAcylation during cell cycle progression

During mitosis, eukaryotic cells cease anterograde trafficking from the endoplasmic reticulum (ER) towards the Golgi. This cessation corresponds with the dispersal of the COPII transport protein, Sec24C, from juxtanuclear ER exit sites (ERES) into a diffusely cytosolic pool. Redistribution of Sec24 paralogs and other core COPII proteins may underlie the mitotic pause in secretion and may be required for the equal inheritance of endomembrane organelles and machinery by both daughter cells. Therefore, it is important to understand the mechanisms governing the mitotic relocalization of COPII components. Here, we explore the role of post-translational modifications (PTMs) of the model COPII protein Sec24C in this phenotypic switch during mitosis. In interphase, Sec24C is modified by O-linked {beta}-N-acetylglucosamine (O-GlcNAc), and we show that this glycan is rapidly removed upon mitotic entry, influencing the timing of Sec24C dispersal. Additionally, we identify novel, cell cycle phase-enriched phosphorylation events on Sec24C, including phosphosites that regulate the stability and localization of the protein, providing the first systematic characterization of dynamic PTMs on any Sec24 protein. Together, our data support the hypothesis that phosphorylation and glycosylation of Sec24C act in concert to induce rapid dispersal upon mitotic entry and may promote equal partitioning of the endomembrane system to daughter cells after division.

biochemistry↗

bioRxiv2023

O-GlcNAcylation regulates neurofilament-light assembly and function and is perturbed by Charcot-Marie-Tooth disease mutations

The neurofilament (NF) cytoskeleton is critical for neuronal morphology and function. In particular, the neurofilament-light (NF-L) subunit is required for NF assembly in vivo and is mutated in subtypes of Charcot-Marie-Tooth (CMT) disease. NFs are highly dynamic, and the regulation of NF assembly state is incompletely understood. Here, we demonstrate that human NF-L is modified in a nutrient-sensitive manner by O-linked-{beta}-N-acetylglucosamine (O-GlcNAc), a ubiquitous form of intracellular glycosylation. We identify five NF-L O-GlcNAc sites and show that they regulate NF assembly state. Interestingly, NF-L engages in O-GlcNAc-mediated protein-protein interactions with itself and with the NF component -internexin, implying that O-GlcNAc is a general regulator of NF architecture. We further show that NF-L O-GlcNAcylation is required for normal organelle trafficking in primary neurons, underlining its functional significance. Finally, several CMT-causative NF-L mutants exhibit perturbed O-GlcNAc levels and resist the effects of O-GlcNAcylation on NF assembly state, indicating a potential link between dysregulated O-GlcNAcylation and pathological NF aggregation. Our results demonstrate that site-specific glycosylation regulates NF-L assembly and function, and aberrant NF O-GlcNAcylation may contribute to CMT and other neurodegenerative disorders.

cell biology↗

bioRxiv2022

Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX

Although picornaviruses are conventionally considered nonenveloped, members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX. Authors SummaryMechanisms underlying nonlytic release of canonical nonenveloped viruses from infected cells are poorly understood. We show here that release of hepatitis A virus from cells in exosome-like vesicles requires nonredundant activities of two distinct Bro1-domain proteins associated with host cell machinery (ESCRT) for endosomal sorting, HD-PTP and ALIX. We demonstrate both Bro1 domain proteins are recruited to the viral capsid by the pX segment of the 1D capsid protein, and that they act in a non-redundant manner to mediate virus release. Fusing pX to a self-assembling nanocage protein resulted in ESCRT-dependent release mediated by a short pX peptide sequence conserved in hepatoviruses from bats to humans. Mutations within the pX sequence ablate release and result in noncytolytic virus accumulating intracellularly. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows nonlytic release of quasi-enveloped virus is an ancient evolutionary trait of hepatoviruses.

microbiology↗

bioRxiv2022

Selective inhibition of protein secretion by abrogating receptor-coat interactions during ER export.

Protein secretion is an essential cellular process that permits cell growth, movement and communication. Traffic of proteins within the eukaryotic secretory pathway is mediated by transport intermediates that bud from one compartment, populated with appropriate cargo proteins, and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human PCSK9 is a major determinant of cholesterol metabolism, whose secretion is mediated by a specific cargo adaptor of the ER export machinery, SEC24A. We map a series of protein-protein interactions between PCSK9, its ER export receptor, SURF4, and SEC24A, that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-PBA, a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

cell biology↗

bioRxiv2020

Evidence for nutrient-dependent regulation of the COPII coat by O-GlcNAcylation

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) is a dynamic form of intracellular glycosylation common in animals, plants and other organisms. O-GlcNAcylation is essential in mammalian cells and is dysregulated in myriad human diseases, such as cancer, neurodegeneration and metabolic syndrome. Despite this pathophysiological significance, key aspects of O-GlcNAc signaling remain incompletely understood, including its impact on fundamental cell biological processes. Here, we investigate the role of O-GlcNAcylation in the coat protein II complex (COPII), a system universally conserved in eukaryotes that mediates anterograde vesicle trafficking from the endoplasmic reticulum. We identify new O-GlcNAcylation sites on Sec24C, Sec24D and Sec31A, core components of the COPII system, and provide evidence for potential nutrient-sensitive pathway regulation through site-specific glycosylation. Our work suggests a new connection between metabolism and trafficking through the conduit of COPII protein O-GlcNAcylation.

cell biology↗

bioRxiv2020

Assembly intermediates of orthoreovirus captured in the cell

Traditionally molecular assembly pathways for viruses have been inferred from high resolution structures of stable intermediates purified in vitro, and from low resolution images of cell sections as well as from genetic approaches including conditionally lethal mutants. Here, we directly visualise a previously unsuspected single shelled icosahedral intermediate for a mammalian orthoreovirus, in addition to the expected virions, in cryo-preserved infected cells by cryo-electron tomography of cellular lamellae1,2. Particle classification and averaging yielded structures at resolutions as high as 5.6 [A], sufficient to identify secondary structural elements and place known molecular structures, allowing us to produce an atomic model of the intermediate, comprising 120 copies of protein {lambda}1 and 120 copies of {sigma}2. This {lambda}1 shell is in a collapsed form compared to the mature virions, with the molecules pushed inwards at the icosahedral 5-folds by ~100 [A]. This grossly indented shell, although produced by a mammalian reovirus, is reminiscent of the first assembly intermediate of prokaryotic dsRNA viruses belonging to a different virus family3, adding weight to the supposition that these diverse viruses share a common ancestor, and suggesting mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds enormous promise for the dissection of the replication cycle of many viruses.

microbiology↗

bioRxiv2019

Combined Atomic Force Microscope and Volumetric Light Sheet System for Mechanobiology

The central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AFM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN - 100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per volume. To showcase the unique advantages of combining Line Bessel light sheet imaging with AFM, we measured the forces exerted by a macrophage during Fc{gamma}R-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces.

biophysics↗

bioRxiv2019

Scientific societies advancing STEM workforce diversity: Lessons and Outcomes from the Minorities Affairs Committee of the American Society for Cell Biology

Promoting diversity and inclusiveness in the STEM academic workforce remains a key challenge and national priority. Scientific societies can play a significant role in this process through the creation and implementation of programs to foster STEM academic workforce diversification, and by providing mentoring and skills development training that empower scientists from under-represented minority (URM) backgrounds to succeed in their communities of practice. In this article, we provide examples of challenges met by scientific societies in these areas and present data from the American Society for Cell Biology, highlighting the benefits received by trainees through long-term engagement with its programs. The success of these initiatives illustrates the impact of discipline-specific programming by scientific societies in supporting the development of URM scientists and an increasingly diverse and inclusive academic STEM community.

scientific communication and education↗

bioRxiv2019

The human UDP-galactose 4’-epimerase (GALE) is required for cell surface glycome structure and function

Glycan biosynthesis relies on nucleotide-sugars (NS), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease, but how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, we examined uridine diphosphate (UDP)-galactose 4-epimerase (GALE), which interconverts two pairs of essential NSs. GALE deletion in human cells triggered major imbalances in its substrate NSs and consequent dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the Fas death receptor. NS dysregulation also directly impacted cell signaling, as GALE-/- cells exhibit Fas hypoglycosylation and hypersensitivity to Fas ligand-induced apoptosis. Our results reveal a new role for GALE-mediated NS regulation in supporting death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

cell biology↗

bioRxiv2019

Glycosylation of gigaxonin regulates intermediate filaments: Novel molecular insights into giant axonal neuropathy

Gigaxonin (also known as KLHL16) is an E3 ligase adaptor protein that promotes the ubiquitination and degradation of intermediate filament (IF) proteins. Mutations in human gigaxonin cause the fatal neurodegenerative disease giant axonal neuropathy (GAN), in which IF proteins accumulate and aggregate in axons throughout the nervous system, impairing neuronal function and viability. Despite this pathophysiological significance, the upstream regulation and downstream effects of normal and aberrant gigaxonin function remain incompletely understood. Here, we report that gigaxonin is modified by O-linked-beta-N-acetylglucosamine (O-GlcNAc), a prevalent form of intracellular glycosylation, in a nutrient- and growth factor-dependent manner. Mass spectrometry analyses of human gigaxonin revealed nine candidate sites of O-GlcNAcylation, two of which - serine 272 and threonine 277 - are required for its ability to mediate IF turnover in novel gigaxonin-deficient human cell models that we created. Taken together, these results suggest that nutrient-responsive gigaxonin O-GlcNAcylation forms a regulatory link between metabolism and IF proteostasis. Our work may have significant implications for understanding the non-genetic modifiers of GAN phenotypes and for the optimization of gene therapy for this disease.

cell biology↗