O-GlcNAc transferase recognizes protein substrates using an asparagine ladder in the TPR superhelix
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PROVIDER: PRJNA421921 | ENA |
REPOSITORIES: ENA
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Project description:Abstract: The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we have developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously as-sess OGT activity across >6000 human proteins. We used this assay to examine the contribution of a conserved asparagine ladder within the lumen of OGT’s superhelical tetratri-copeptide repeat (TPR) domain to substrate selection. When these residues were mutated, OGT retained full activity against short peptides, but showed low to no activity against most of the OGT substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes a majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain. This series contains microarray data both comparing the new chemoenzymatic method to antibody-based detection as well as comparing arrays treated with wild-type OGT, 5N5A mutant OGT, or controls not treated with enzyme. Note: all CTD-stained arrays or control array raw files are contained in GSE107911_RAW.tar
2018-02-26 | GSE107911 | GEO
Project description:The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously assess OGT activity across >6000 human proteins. With this assay we examined the contribution to substrate selection of a conserved asparagine ladder within the lumen of OGT's superhelical tetratricopeptide repeat (TPR) domain. When five asparagines were mutated, OGT retained significant activity against short peptides, but showed limited limited glycosylation of protein substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes the majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain.
| S-EPMC5937710 | biostudies-literature
Project description:Abstract: O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.
2019-07-24 | GSE132205 | GEO
Project description:Nucleocytoplasmic O-linked N-acetylglucosamine (O-GlcNAc) is an essential post-translational modification that is installed to thousands of protein substrates by O-GlcNAc transferase (OGT). Substrate selection by OGT and its isoforms is primarily mediated by the tetratricopeptide repeat (TPR) domain, yet the impact of truncations to the TPR domain on substrate and glycosite selection remains unresolved. Here, we report the effects of TPR truncations on the substrate and glycosite selection of OGT against the model protein GFP-JunB and the surrounding O-GlcNAc proteome in U2OS cells. Truncation of the TPR domain of OGT maintains glycosyltransferase activity but alters subcellular localization in cells. Examination of the glycoproteome across the TPR truncations revealed the broadest substrate activity from the canonical nucleocytoplasmic OGT, with the greatest changes in O-GlcNAc occurring on proteins associated with mRNA splicing processes. Glycosite analysis revealed alterations to the O-GlcNAc consensus sequence globally and differential glycosite selectivity on GFP-JunB as a function of the OGT TPR isoform. This dataset provides a foundation to analyze how perturbations to the TPR domain and expression of OGT isoforms affects the glycosylation of substrates, which will be critical for future protein engineering of OGT, the biology of OGT isoforms, and diseases associated with the TPR domain of OGT.
2021-11-01 | PXD028141 | Pride
Project description:BioID performed in HeLa cells for the TPR domain of The O-GlcNAc Transferase and eGFP (negative control). Used to determine TPR interactors and the TPR interactome in HeLa cells. Three biological replicates were performed, with each replicate having an experimental (TPR-BirA*) and negative (eGFP-BirA*) condition, and each condition was split into four fractions during in-gel digest for mass spectrometry.
2020-06-24 | MSV000085626 | MassIVE
Project description:O-Linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Yet, the study of post-translational modifications like O-GlcNAc has been limited by the lack of strategies to induce O-GlcNAcylation of a target protein in cells. Here, we report a generalizable genetic strategy to induce O-GlcNAc to specific target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT(4), a truncated form of OGT, yielded a nanobody-OGT(4) construct that selectively delivered O-GlcNAc to the target protein (e.g., JunB, cJun, Nup62) and reduced alteration of global O-GlcNAc levels in the cell. Chemical proteomics confirmed the selective increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics on the target protein revealed similar glycosite selectivity between nanobody-OGT(4) or global elevation of O-GlcNAc. Finally, we demonstrate the ability to selectively target endogenous ⍺-synuclein for glycosylation in HEK293T cells. Thus, the use of nanobodies to redirect OGT substrate selection is a versatile strategy to induce glycosylation to desired target proteins in cells that will facilitate discovery of O-GlcNAc functions and provide a mechanism to engineer O-GlcNAc signaling. The proximity-directed OGT approach for protein-selective O-GlcNAcylation is readily translated to additional protein targets and nanobodies, and may constitute a generalizable strategy to control post-translational modifications in cells.
2021-09-08 | PXD016041 | Pride
Project description:We used SLAM-seq approach to define a contribution of RNA synthesis into RNA abundance changes after Tpr loss. Basket nucleoporin Tpr was AID-tagged and depleted through Auxin Induced Degradation system. Loss of Tpr led to rapid changes in rates of RNA synthesis. The majority of transcripts were downregulated. Analysis of RNA-seq and SLAM-seq indicated that the changes in RNA abundance of most of Tpr-dependent up- and downregulated genes resulted from increased or decreased rates of RNA synthesis, respectively.
2020-06-15 | GSE149890 | GEO
Project description:O-linked ?-N-acetyl-D-glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis.
| S-EPMC5967971 | biostudies-literature
Project description:O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme, O-GlcNAc transferase (OGT), generates the entire cytosolic O-GlcNAc proteome, it is not understood how it recognizes its protein substrates, targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1, a key innate immunity-signalling O-GlcNAc protein, revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis, this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site.
| S-EPMC5493779 | biostudies-literature
Project description:O-GlcNAcylation is the modification of serine and threonine residues with beta-N-acetylglucosamine (O-GlcNAc) on intracellular proteins. To investigate the role of protein O-GlcNAcylation on intestinal homeostasis, we generated intestinal epithelial cell (IEC)-specific O-GlcNAc transferase (OGT) knockout in mice. The KO mice developed spontanous intestinal inflammation. To determine the underlying molecular mechanisms, we performed RNA sequencing of ileum and colon epithelial cells of wildtype and IEC-OGT KO mice.
2017-06-27 | GSE100473 | GEO