2021
DOI: 10.1093/glycob/cwab055
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Evidence for nutrient-dependent regulation of the COPII coat by O-GlcNAcylation

Abstract: O-linked β-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 i… Show more

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Cited by 12 publications
(12 citation statements)
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“…Multiple single cell–derived KO clones per each guide RNA were isolated and screened for gene disruption by Western blotting. SEC24C and SEC24D KO cell lines were described previously ( 38 ). Cells were validated by immunoblotting or quantitative RT-PCR ( SI Appendix, Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Multiple single cell–derived KO clones per each guide RNA were isolated and screened for gene disruption by Western blotting. SEC24C and SEC24D KO cell lines were described previously ( 38 ). Cells were validated by immunoblotting or quantitative RT-PCR ( SI Appendix, Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Previous reports identified several O-GlcNAcylation sites on rodent NF-L orthologs [47][48][49][50][51][52] , but directed studies of human NF glycosites were lacking. To discover human NF-L glycosites, we epitope-tagged the NEFL genomic locus of human cells via established CRISPR/Cas9 methods 79 (Figure s1D) and affinity-purified endogenous NF-L from cultures treated with Thiamet-G, a standard tactic to improve the technically challenging detection of O-GlcNAc moieties [80][81][82][83] . Mass spectrometry (MS) analysis identified two novel glycosites (S48, S431), complementing the previously reported glycosites from rodent NF-L 47,48 (T21, S27, S34 -human numbering of cognate rodent residues) (Figure 1F).…”
Section: Site-specific O-glcnacylation Of the Human Nf-l Head And Tai...mentioning
confidence: 99%
“…Liquid chromatography-tandem MS analysis. For gel band analysis, colloidal blue-stained SDS-PAGE bands (Invitrogen Unpaged 4-12% Bis-Tris) were manually excised and subjected to reduction, alkylation, and in-gel tryptic digestion as described 82,83 . For in-solution analysis, samples were supplemented with 5% SDS, reduced with 10 mM DTT for 15 min at 80 °C, alkylated with 20 mM iodoacetamide for 30 min at RT, then supplemented with a final concentration of 1.2% phosphoric acid and 609 µL of S-Trap (Protifi) binding buffer (90% MeOH/100 mM triethylammonium bicarbonate [TEAB]).…”
Section: Nf-l O-glcnacylationmentioning
confidence: 99%
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“…Several cell signaling pathways have been suggested to function in this context. Governed by nutrient availability 51 58 , various forms of cell stress 58 , and the presence of growth factors 59 , COPII subunits and multiple COPII regulatory factors undergo post-translational modifications, including phosphorylation 51 , 52 , 59 61 , glycosylation 62 , 63 , and ubiquitylation 64 , leading to alterations in their local concentrations at sites of transport intermediate formation, which in turn tunes the kinetics of cargo export. Additionally, their total cellular levels are further controlled by events that alter protein stability 53 , 57 or gene expression 58 , 65 .…”
Section: Introductionmentioning
confidence: 99%