Dataset Information

Combined Antibody/Lectin-Enrichment Identifies Extensive Changes in the O-GlcNAc Subproteome Upon Oxidative Stress

ABSTRACT: O-GlcNAc is thought to regulate proteins in a mannaer analogous to other PTMs, modulating cellular functions including the cellular stress response. The aim of this study was to identify specific cellular networks and protein complexes that are differentially O-GlcNAcylated and/or expressed upon acute oxidative stress (H2O2 for 1 and 2 h). We achieved this by employing SILAC and a novel anti-O-GlcNAc G5 lectibody IP strategy that combind O-GlcNAc specific antibodies and the lectin WGA. We identified 990 proteins among all three treatments including 202 non-redundant O-GlcNAc modified peptides. Differentially expressed proteins clustered into canonical 14-3-3/PI3K signaling pathways including the 14-3-3 complex, chaperonins, RNA Pol II Mediator and nuclear pore complexes.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Fibroblast

DISEASE(S): Ischemia

SUBMITTER: Albert Lee

LAB HEAD: Natasha E Zachara

PROVIDER: PXD000849 | Pride | 2016-09-29

REPOSITORIES: Pride

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Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress.

Lee Albert A Miller Devin D Henry Roger R Paruchuri Venkata D P VD O'Meally Robert N RN Boronina Tatiana T Cole Robert N RN Zachara Natasha E NE

Journal of proteome research 20161014 12

O-Linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies and regulates over 3000 nuclear, cytoplasmic, and mitochondrial proteins. Upon exposure to stress and injury, cells and tissues increase the O-GlcNAc modification, or O-GlcNAcylation, of numerous proteins promoting the cellular stress response and thus survival. The aim of this study was to identify proteins that are differentially O-GlcNAcylated upon acute oxidative stress (H<sub>2</sub>O<sub ...[more]

PMID: 27669760

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Project description:Thermally dimorphic human fungal pathogens undergo a reversible program of cellular differentiation in response to their environment that is essential for infectivity and pathogenicity. In the soil, these organisms grow as highly polarized, multicellular hyphal filaments that produce infectious particles. When inhaled by a mammalian host, these cells switch to a unicellular yeast form that causes disease even in healthy hosts. Temperature is considered to be the primary environmental cue that promotes reversible cellular differentiation; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. In a search for other signals that regulate morphogenesis, we considered the monosaccharide N-acetylglucosamine (GlcNAc), which is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc was a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift, indicating that fungal cells sense GlcNAc to promote filamentation. The synchronous morphologic transition stimulated by low temperature and GlcNAc allowed us to examine the temporal regulation of the transcriptome during morphogenesis to reveal candidate genes involved in establishing the filamentous growth program. Through this analysis, we identified two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes. For each time-course sample, cDNA was coupled to Cy5 and a reference cDNA pool was made by combining RNA from t = 0 and all late time course samples, which was coupled to Cy3. For end point microarray experiments (i.e., established yeast samples compared to established filamentous samples), G217B yeast cDNA was coupled to Cy5 and filament cDNA was coupled to Cy3.

			Action	DRS
	SILAC-1.raw	Raw
	SILAC-10.raw	Raw
	SILAC-11.raw	Raw
	SILAC-12.raw	Raw
	SILAC-13.raw	Raw