Browsing by Subject "Vimentin"

Protein 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.