Browsing by Author "Boyce, Michael"

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.

Glycosylation is a universally conserved and ubiquitous posttranslational modification required for myriad cellular processes. Biosynthesis of these moieties, termed glycans, 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. In early work, we discovered that GALE knockdown sensitizes human cells to ER stress, suggesting that GALE is important for stress responses. While increased NS levels during ER stress have been reported previously, we found that GALE knockdown blunted this response.

GALE deletion via CRISPR/Cas9 in human cells triggered major imbalances in its substrate NSs along with changes in fatty acid levels, suggesting that GALE is crucial for whole-cell metabolism. The novel link between GALE and ostensibly unrelated metabolic pathways may prove important for understanding disorders of galactose metabolism. Altered NS levels led to dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the Fas death receptor. These changes in glycan biosynthesis directly impacted protein function, as GALE-/- cells exhibit hypersensitivity to Fas ligand (FasL)-induced apoptosis. Critically, monosaccharide supplementation reverses both Fas misglycosylation and FasL sensitivity, supporting a glycan-centric mechanism for this phenotype. 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.

To probe effects of integrin misglycosylation in GALE-/- cells, we leveraged a quantitative assay for cell adhesion, finding that GALE-/- cells adhered more robustly than controls on a variety of extracellular matrices. Subsequent experiments revealed technical limitations of this assay and the link between NS metabolism, integrin glycosylation, and cell adhesion remains inconclusive. We observed GALE-dependent changes in integrin stability or lifetime, suggesting that glycosylation has important functional consequences for integrin subunits.

In a model of B cell maturation, we found that GALE protein levels are increased by B cell stimulation. Since B cell stimulation triggers the transcription factor Xbp1s, of which GALE is a reported target, this suggested an exciting connection between B cell maturation and NS metabolism. However, subsequent studies using Xbp1-/- B cells and drugs preventing Xbp1 activation suggest that GALE upregulation is independent of Xbp1s in this context.

Intracellular glycosylation via O-linked N-acetylglucosamine (O-GlcNAc) is a rapidly reversible signaling moiety in animals. In response to ER stress, increased O-GlcNAc glycosylation promotes cell survival in multiple contexts, but specific substrate proteins modified during this response remain unknown. To discover ER stress-regulated glycoproteins and characterize the functional impact of their glycosylation, we employed a chemical biology glycoproteomics workflow combining an azide-functionalized hexosamine mimetic with SILAC proteomics. Our results demonstrate that a subset of glycoproteins is differentially glycosylated in response to the ER-stress inducing drug tunicamycin, suggesting a regulated rearrangement of the intracellular glycome.

Although the structural and enzymological characteristics of NS biosynthetic enzymes are well-studied, the consequences of altered NS metabolism in response to signals or disease states is understudied. In this work, we used both genetic models of NS imbalance and stimuli previously reported to affect NS metabolism to measure downstream glycosylation and consequences thereof. This work lays the foundation for a more comprehensive understanding of NS metabolic regulation in mammalian physiology.