Browsing by Subject "O-GlcNAc"
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Item Open Access Characterization of the O-GlcNAc Modification on the COPII Outer Coat(2020) Condon, Brett M.The coat protein complex II (COPII) traffics cargoes from the endoplasmic reticulum to the Golgi and is an essential step early in the secretory pathway. COPII consists of five proteins required for in vitro vesicle formation: SAR1, SEC23, SEC24, SEC13, and SEC31. Although the functions of COPII components are well characterized, the regulation of vesicle trafficking is less well understood. While transcriptional and translational control of COPII components have been reported, regulation of COPII trafficking by post-translational modifications (PTMs) has emerged as a rapid regulatory strategy, allowing the pathway to respond to acute cellular signals and environmental stressors. COPII vesicles are known to be regulated by phosphorylation and ubiquitination, and many COPII components are also decorated with PTMs of unknown function. In addition to phosphate and ubiquitin, our lab and others have discovered that SEC23, SEC24, and SEC31 are modified by O-linked β-N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a dynamic PTM that regulates numerous cellular pathways, responds to a variety of cellular stressors, and is dysregulated in a wide range of human diseases. To better understand the regulation of COPII vesicle trafficking, we investigated the role of O-GlcNAc on SEC31A, a key protein of the COPII outer coat. We hypothesized that O-GlcNAcylation of SEC31A at specific sites would regulate its function. Here we report the identification of five novel O-GlcNAcylated residues on SEC31A identified by MS. Consistent with a regulatory modification, we observed dynamic changes in endogenous SEC31A O-GlcNAcylation in mammalian cell lines. We also found that site-specific O-GlcNAcylation at S1202 is required for the binding of SEC31A with its outer coat partner SEC13. Importantly, we report SEC31A O-GlcNAcylation increases upon glucose reduction, suggesting a mechanism for outer coat regulation in response to dynamic changes in nutrient availability.
In addition to protein trafficking, we have also found evidence that O-GlcNAc regulates the cytoskeleton. Intermediate filaments (IFs) are a major component of the metazoan cytoskeleton and are essential for normal cell morphology, motility, and signal transduction. There are six categories of IFs, which participate in diverse cellular processes and are mutated in over 80 human disease. Although distinct in function, all IFs share common structural features, and homo- or heterodimerize via a conserved rod domain. IFs are ultimately assembled into nonpolar, mature filaments. This enables IFs to slide past each other within a filament and confers unique viscoelastic properties that protect the cell from compressive forces. While the structure and function of IFs are well characterized, the real-time effects of PTMs on IFs are not well understood. We recently reported that O-GlcNAcylation at S49 regulates the homotypic protein-protein interactions of the type III IF protein vimentin, controlling its assembly into mature filaments. Because the properties of IFs depend on their assembly, we hypothesized that wild type and S49A vimentin may have different viscoelastic properties, whether fully assembled into mature filaments or not. To test this, we have collaborated with the Superfine group at UNC to combine atomic force microscopy and light sheet fluorescence microscopy into a single tool for interrogating cellular mechanobiology. We demonstrated the ability to couple single-molecule force measurements with volumetric live cell imaging in real time. This novel approach will allow us to study the regulation of IFs, answering longstanding questions in the field of IF research.
Despite the regulatory role of O-GlcNAc in numerous cellular processes, the mechanisms by which O-GlcNAc controls these processes are largely unknown. Our work has characterized the functional effect of O-GlcNAc in both protein trafficking and the cytoskeleton and provides a model for studying O-GlcNAc in other contexts.
Item Open Access Increasing O-GlcNAcylation is neuroprotective in young and aged brains after ischemic stroke.(Experimental neurology, 2021-05) Wang, Zhuoran; Li, Xuan; Spasojevic, Ivan; Lu, Liping; Shen, Yuntian; Qu, Xingguang; Hoffmann, Ulrike; Warner, David S; Paschen, Wulf; Sheng, Huaxin; Yang, WeiSpliced X-box binding protein-1 (XBP1s) together with the hexosamine biosynthetic pathway (HBP) and O-GlcNAcylation forms the XBP1s/HBP/O-GlcNAc axis. Our previous studies have provided evidence that activation of this axis is neuroprotective after ischemic stroke and critically, ischemia-induced O-GlcNAcylation is impaired in the aged brain. However, the XBP1s' neuroprotective role and its link to O-GlcNAcylation in stroke, as well as the therapeutic potential of targeting this axis in stroke, have not been well established. Moreover, the mechanisms underlying this age-related impairment of O-GlcNAcylation induction after brain ischemia remain completely unknown. In this study, using transient ischemic stroke models, we first demonstrated that neuron-specific overexpression of Xbp1s improved outcome, and pharmacologically boosting O-GlcNAcylation with thiamet-G reversed worse outcome observed in neuron-specific Xbp1 knockout mice. We further showed that thiamet-G treatment improved long-term functional recovery in both young and aged animals after transient ischemic stroke. Mechanistically, using an analytic approach developed here, we discovered that availability of UDP-GlcNAc was compromised in the aged brain, which may constitute a novel mechanism responsible for the impaired O-GlcNAcylation activation in the aged brain after ischemia. Finally, based on this new mechanistic finding, we evaluated and confirmed the therapeutic effects of glucosamine treatment in young and aged animals using both transient and permanent stroke models. Our data together support that increasing O-GlcNAcylation is a promising strategy in stroke therapy.Item Open Access Metabolic Regulation of Kelch-like Proteins Through O-glycosylation(2018) Chen, Po-HanO-GlcNAcylation is a reversible post-translational modification that decorates an O-linked ß-N-acetylglucosamine (O-GlcNAc) moiety onto the serine/threonine residues of target proteins. In mammals, this modification is regulated by only two enzymes: O-GlcNAc transferase (OGT, the writer) and O-GlcNAcase (OGA, the eraser). Several studies have revealed that O-GlcNAcylation can be responsive to metabolic status or stress stimulation. However, the specific O-GlcNAc targets in response to various nutrient and stress signals are not well defined. We conducted a global transcriptome profiling in triple-negative breast cancer cells to search for signaling events that respond to O-GlcNAc fluctuation. Unexpectedly, we found that the NRF2-dependent stress response positively correlates with lower OGT activity in multiple human tumor gene expression datasets. NRF2, a major transcriptional regulator of redox balance, is usually activated by oxidative stress but degraded by proteasome under basal conditions via the KEAP1-CUL3 ubiquitin ligase-mediated polyubiquitination. Using azidosugar metabolic labeling, bioorthogonal chemistry and mass spectrometry, we determined that the NRF2 negative regulator KEAP1 is O-GlcNAcylated within its BTB and Kelch motifs. KEAP1 belongs to the Kelch-like (KLHL) adaptor protein family, which was known to regulate substrate proteostasis via CUL3-mediated ubiquitination. Of 11 candidate O-GlcNAc sites on KEAP1, serine 104 is responsible for regulating NRF2 activity by promoting the KEAP1-CUL3 interaction. Interestingly, we found that other KLHL protein, gigaxonin, is also O-GlcNAcylated on up to nine candidate sites. Mutation of gigaxonin is known to cause giant axonal neuropathy (GAN), a neurodegenerative disease that is characterized by the accumulation of intermediate filaments in axons. We found gigaxonin O-GlcNAcylation is required for its ability to facilitate the ubiquitination and proteolysis of intermediate filaments. Mutation of specific gigaxonin O-GlcNAcylation sites compromised its optimal interactions with intermediate filament proteins. This finding provides new molecular insight into GAN pathogenesis. The link between proteostasis and nutrient-sensing is fundamentally important yet incompletely understood. Together, my dissertation work has revealed new connections among nutrient-sensitive glycosylation, KLHL protein function, proteostasis and downstream signaling, with relevance for human diseases.
Item Open Access O-GlcNAc-Mediated Protein-Protein Interactions in Cell Signaling(2018) Tarbet, Heather JeanProtein modification by O-linked β-N-acetylglucosamine (O-GlcNAc) is an essential signaling mechanism that affects diverse processes such as cell cycle, metabolism, and death. Aberrant signaling has been implicated in numerous human diseases including cancer and neurodegeneration. However, major aspects of O-GlcNAc signaling are poorly understood, including how substrates are recognized, and the functional consequences of these modifications on proteins. Based on recent literature and our preliminary results, we propose one important function of O-GlcNAc is mediating protein-protein interactions. In previous work with the Kohler group, we developed a method of covalently capturing proteins that interact through O-GlcNAc using a GlcNAc analog containing a diazirine photocrosslinking moiety (GlcNDAz). We used this approach to identify proteins engaging in O-GlcNAc-mediated protein-protein interactions, including vimentin, an O-GlcNAcylated intermediate filament (IF) protein. Vimentin is important for the integrity of mesenchymal cells and has been implicated in cell movement and various metastatic cancers. The purpose of vimentin glycosylation remains unknown. However, we hypothesize that O-GlcNAc is important in regulating vimentin’s participation in cell motility and stiffness. We set up systems to characterize glycosylation site mutants of vimentin in an array of phenotypic assays, including cell migration, cell cycle progression, and IF dynamics. Here, we show that site-specific modification of the prototypical IF protein vimentin with O-GlcNAc mediates its homotypic protein-protein interactions and is required in human cells for IF morphology and cell migration. In addition, we show that the intracellular pathogen Chlamydia trachomatis, which remodels the host IF cytoskeleton during infection, requires specific vimentin glycosylation sites and O-GlcNAc transferase activity to maintain its replicative niche. Our results provide new insight into the biochemical and cell biological functions of vimentin O-GlcNAcylation, and may have broad implications for our understanding of the regulation of IF proteins in general.
Despite the extensive number of OGT and OGA substrates identified, including IFs, the substrate specificity of both of these enzymes remains an open question due to the lack of consensus sequence among O-GlcNAc modified proteins. A prevailing general model is that OGT binds to cofactor proteins, which confer specificity by recruiting it to particular substrates or subcellular sites. While there are some examples of cofactor proteins, new approaches are needed to understand the molecular mechanism of OGT regulation especially in response to pathophysiologically important signals. To identify new cofactor proteins, we developed a method to tag, purify, and identify endogenous OGT cofactor proteins from live cells in a stimulus-dependent manner. We fused OGT to the peroxidase APEX2 and expressed it in cultured human cells. When cells are treated with cell-permeable biotin-phenol and H2O2, APEX2 generates biotin-phenoxyl radicals in situ, which react rapidly with proteins or other biomolecules, covalently attaching biotin to them. Crucially, because the phenoxyl radical is so short-lived, it diffuses < 50 nm from APEX2 before quenching. Therefore, fusion of APEX2 to a protein of interest permits the “proximity ligation” of biotin to other proteins within a short radius, with negligible background from distant proteins. In a pilot experiment, we showed that OGT-APEX2-expressing cells exhibit specific biotin labeling, and that the pattern of biotinylated proteins changes in response to glucose deprivation. Moreover, we found that the endogenous transcriptional coactivator HCF1 was biotinylated specifically in glucose-replete cells, consistent with the prior demonstration of a glucose-dependent HCF1/OGT association in another system. We concluded that our proximity ligation assay can identify glucose-dependent OGT binding partners in living cells. Additionally, this system can be used to identify new cofactor proteins in a stimulus dependent manner which will help elucidate their role in regulating OGT’s localization and substrate specificity.
Item Embargo Regulation of Human Neurofilaments and WNK Kinases by O-linked-β-N-acetylglucosamine(2024) Huynh, Duc TanO-linked-β-N-acetylglucosamine (O-GlcNAc) is a dynamic intracellular glycosylation that modifies thousands of nucleocytoplasmic proteins. Regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), O-GlcNAcylation is ubiquitous in all mammalian tissues and important in fundamental cell biological processes such as epigenetics, transcription, temporal regulation of signaling, and protein degradation. Particularly abundant in the brain, this modification is crucial in various aspects of mammalian neurobiology, ranging from cerebellar development, synaptic transmission, to neuron survival and maintenance. For these reasons, targeting O-GlcNAcylation has shown therapeutic potential in various mouse models of neurodegeneration and OGA inhibitors have emerged as promising drug candidates in clinical trials. Despite the broad pathophysiological significance, how OGT and OGA regulate their native substrates at the molecular, cellular, and physiological level remains an outstanding knowledge gap. My dissertation aims to address this question by interrogating two classes of OGT substrates: Intermediate filaments and with-no-lysine (WNK) kinases. In Chapter 2, I explore how the human neuronal cytoskeleton, integral to axonal health, is functionally regulated. Particularly, my project focuses on neurofilaments (NFs), the neuronal intermediate filaments comprising light, medium, and heavy subunits that form the NF polymers. In vivo, the light (NF-L) subunit is required to provide axonal support, mediate organelle motility, and influence high-order brain functions. Mutating NF-L causes aggregations in two subtypes of Charcot-Marie-Tooth (CMT) disease, a hereditary neuromuscular disease, or chronic axonal dysregulation, such as protein aggregation, disrupts normal NF-L filament organization. More importantly, NF-L concentrations spike abnormally in the biofluids of patients with myriad neurological disorders, conferring it a promising biomarker in pre-clinical studies and clinical trials. Despite a broad pathophysiological importance, the biochemical regulation of NF assembly and pathological aggregation remains ambiguous to date. Early proteomic data in rodent brains indicated that rodent NFs are O-GlcNAcylated. However, whether O-GlcNAcylation occurs in the human homologs has yet been systematically tested. In my work, I used biochemical assays to demonstrate dynamic human NF-L O-GlcNAcylation in cultured cells and post-mortem human brain tissues. With CRISPR knockin with proteomics, I identified two novel glycosites of human NF-L that expand those mapped in rodent homologs. Next, combining biochemistry, chemical biology, and proteomics, I showed that site-specific NF-L O-GlcNAcylation mediates NF-L interactions with α-internexin (an in vivo binding partner), tunes NF-L assembly state, and controls its overall filamentous organization. Further collaborating with Dr. Chantell Evans, I discerned that NF-L O-GlcNAcylation is required for neuronal mitochondrial and lysosomal motility. Establishing the functional links between NF-L O-GlcNAcylation and its biology, I last examined whether dynamic human NF-L O-GlcNAcylation dysregulation has any pathological relevance. Among CMT disease-causing mutants, NF-L O-GlcNAc signals are widely perturbed. Notably, mutations that lie proximal to NF-L glycosites abolish NF-L O-GlcNAc signals and resist the functional effects of O-GlcNAcylation. Altogether, these findings propose a regulatory role of O-GlcNAcylation in the neuronal cytoskeleton, motivate future opportunities to determine NF-L glycoforms in diseases involving NF dysregulation, and explore their potential diagnostic benefit. In Chapter 3, I investigate the functional control of WNK kinases, a group of kinases with an atypical placement of the catalytic lysine. WNK kinases phosphorylate SPAK and OSR1, the upstream regulators of sodium-chloride cotransporters, the sodium-potassium-2-chloride cotransporters, and the potassium-coupled chloride cotransporters. Mutations in WNK kinases are found in human patients with familial hyperkalemic hypertension characterized by hyperkalemia and hyperchloremic metabolic acidosis. Modulation of these transporters by WNKs is essential for cell volume control, the homeostasis of intracellular chloride, and osmoregulation. However, how WNK kinases are regulated at the molecular level is currently unclear. We serendipitously discovered that WNK kinases are O-GlcNAcylated. By proteomics and biochemistry, we determined six glycosites of WNK4, a paralog abundant in the kidney, and then interrogated the effects of ion concentrations and osmotic stress on WNK4 O-GlcNAcylation. The results showed no changes in WNK4 O-GlcNAcylation in response to fluctuating ion concentrations, but an upregulation of WNK4 site-specific O-GlcNAcylation in correspondence to higher osmolarity. Intriguingly, my results also suggest a potential crosstalk between WNK4 O-GlcNAcylation and WNK4 phosphorylation in the hyperosmolarity condition. In conclusion, my dissertation has uncovered some of the biological roles of O-GlcNAc modification. In interrogating two O-GlcNAc classes, I have provided mechanistic insights on how O-GlcNAc influences mammalian neurobiology, from regulating the human cytoskeletal network and organelle motility via NF-L O-GlcNAcylation to potentially modulating osmosensing via WNK kinase O-GlcNAcylation. Future works would continue to broaden these lines of research and apply biochemical characterization to cellular and tissue physiology, by characterizing the roles of this modification in physiologically relevant organisms.
Item Open Access Regulation of Sec24 by O-GlcNAcylation(2020) Bisnett, BrittanyCoat protein complex II (COPII) mediates forward vesicle trafficking from the endoplasmic reticulum (ER). As an evolutionarily conserved system, COPII is essential for maintaining the lipid and protein composition of cellular membranes. COPII consists of five proteins essential for in vitro vesicle formation: Sar1, Sec23, Sec24, Sec13, and Sec31. Beautiful biochemical, genetic, and structural studies provided many insights into the function and geometry of the COPII coat. However, how COPII is regulated in response to environmental or cellular stimuli remains under-explored.
Like other proteins, COPII components are extensively post-translationally modified (PTM), which may function to modulate COPII activity. Of particular interest is O-linked β-N-acetylglucosamine (O-GlcNAc), a nutrient-sensitive modification of serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins. Recently, we and others have found extensive O-GlcNAcylation of COPII components, including Sec23A, Sec24C, Sec24D, and Sec31A. To investigate the role of O-GlcNAc on these proteins, we used mass spectrometry to unambiguously identify O-GlcNAc-modified sites. We then used CRISPR/Cas9 to delete Sec23A, Sec24C, Sec24D, and Sec31A from a variety of cell lines and re-expressed unglycosylatable Ser/ThrAla mutants. This work primarily focuses on Sec24C and Sec24D.
We found that Sec24C is dynamically O-GlcNAcylated on a timescale of minutes, suggesting that this modification plays a regulatory/signaling role, as opposed to a structural role. We also found that Sec24C O-GlcNAcylation may regulate protein-protein interactions. In particular, we discovered that unglycosylatable Sec24C mutants altered the interaction between Sec24C and the Ebola protein VP40, suggesting that Sec24C O-GlcNAcylation may influence Ebola virus replication.
We also investigated the effect of a variety of cellular stressors on Sec24C O-GlcNAcylation using immunoprecipitation and immunoblot. We found that Sec24C O-GlcNAcylation was increased with rapamycin, suggesting that autophagy may trigger Sec24C O-GlcNAcylation. However, autophagy induction via amino-acid starvation failed to produce the same effect, suggesting that Sec24C O-GlcNAcylation may be the result of other signaling by mTORC1/2.
Finally, we identified six novel O-GlcNAc sites on Sec24D, that may function in COPII trafficking. Because Sec24D is required for collagen trafficking, we are currently testing the functionality of unglycosylatable Sec24D mutants in both human cell lines and a vertebrate model of skeletogenesis.
Together, this work suggests that O-GlcNAcylation plays multifaced roles on Sec24C and Sec24D and may function to integrate COPII function with cellular metabolism. Future studies on site-specific O-GlcNAcylation of Sec24 proteins will aid in our understanding of COPII regulation and provide valuable insight into the complex web of protein and lipid trafficking.
Item Open Access Role of O-GlcNAc in the Vertebrate Secretory Pathway(2018) Cox, Nathan JamesO-linked β-N-acetylglucosamine (O-GlcNAc) exerts myriad effects on protein localization, activation, inhibition, stability, conformational changes, or degradation. However, the biochemical effects of O-GlcNAc on the vast majority of substrates is unknown. Recently, we and others have shown that several coat protein complex II (COPII) components including SEC23A, SEC24C, and SEC31A are O-GlcNAcylated. The COPII coat complex consists of protein coated carriers that mediate secretory trafficking from the endoplasmic reticulum. To determine the effects of O-GlcNAc on COPII we used a combination of chemical, biochemical, cellular and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. We demonstrate that individual O-GlcNAcylation sites of SEC23A are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of cranio-lenticulo-sutural dysplasia (CLSD).
Next, we developed a proteomic workflow to address the challenges of identifying and quantifying novel changes in substrate O-GlcNAcylation in response to a stimulus. Current methods of O-GlcNAcome enrichment suffer from issues with specificity, reproducibility, time-resolution, or require specialized hardware. We developed a novel, unbiased glycoproteomics workflow to survey global changes in O-GlcNAc in response to stimuli. Our approach utilizes both stable isotope labeling with amino acids in cell culture (SILAC) for quantitation and metabolic labeling of O-GlcNAc for enrichment. Using our glycoproteomics workflow we examined the effects of brefeldin A (BFA), a fungal metabolite that disrupts vesicle trafficking, and cytokine deprivation on a pro-B cell line. We identified changes in the O-GlcNAcylation of Coatomer subunit gamma-1 (COPG) a coat protein complex I (COPI) component in response to BFA. Interestingly, COPI mediates traffic from the Golgi to the ER, as well as within the Golgi, and is the specific target of BFA. O-GlcNAcylation of COPI components may have effects similar to O-GlcNAc on COPII, possibly altering membrane binding or the trafficking of specific cargo.
Finally, we identified a candidate O-GlcNAc-mediated binding part of SEC23A using a combination chemical biology tools and mass spectrometry (MS). We identified ankycorbin, a vertebrate specific protein with no known function, as the candidate SEC23A O-GlcNAc-mediated binding partner. However, our attempts to validate this interaction were inconclusive.
Overall, this work examines the role of O-GlcNAc in the vertebrate secretory pathway. We demonstrate the effects of O-GlcNAc on SEC23A in the COPII pathway and identify a potentially novel method of COPI protein trafficking regulation via the O-GlcNAcylation of COPG.