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Hédou J, Bastide B, Page A, Michalski JC, Morelle W. Mapping of O-linked beta-N-acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle. Proteomics 2009 9(8) 19322778
Abstract:
O-linked beta-N-acetylglucosamine (O-GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O-GlcNAc modified. Moreover, it was demonstrated that O-GlcNAc moieties involved in contractile protein interactions could modulate Ca(2+) activation parameters of contraction. In order to better understand how O-GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O-GlcNAc modification in purified contractile protein homogenates. Using an MS-based method that relies on mild beta-elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O-GlcNAc site in the subdomain four of actin and four O-GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O-GlcNAc sites might modulate the actin-tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein-protein interactions but could also modulate the contractile properties of skeletal muscle.
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Khidekel N, Ficarro SB, Clark PM, Bryan MC, Swaney DL, Rexach JE, Sun YE, Coon JJ, Peters EC, Hsieh-Wilson LC. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nature chemical biology 2007 3(6) 17496889
Abstract:
The addition of the monosaccharide beta-N-acetyl-D-glucosamine to proteins (O-GlcNAc glycosylation) is an intracellular, post-translational modification that shares features with phosphorylation. Understanding the cellular mechanisms and signaling pathways that regulate O-GlcNAc glycosylation has been challenging because of the difficulty of detecting and quantifying the modification. Here, we describe a new strategy for monitoring the dynamics of O-GlcNAc glycosylation using quantitative mass spectrometry-based proteomics. Our method, which we have termed quantitative isotopic and chemoenzymatic tagging (QUIC-Tag), combines selective, chemoenzymatic tagging of O-GlcNAc proteins with an efficient isotopic labeling strategy. Using the method, we detect changes in O-GlcNAc glycosylation on several proteins involved in the regulation of transcription and mRNA translocation. We also provide the first evidence that O-GlcNAc glycosylation is dynamically modulated by excitatory stimulation of the brain in vivo. Finally, we use electron-transfer dissociation mass spectrometry to identify exact sites of O-GlcNAc modification. Together, our studies suggest that O-GlcNAc glycosylation occurs reversibly in neurons and, akin to phosphorylation, may have important roles in mediating the communication between neurons.
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Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proceedings of the National Academy of Sciences of the United States of America 2004 101(36) 15340146
Abstract:
The covalent modification of intracellular proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as a crucial regulatory posttranslational modification akin to phosphorylation. Numerous studies point to the significance of O-GlcNAc in cellular processes such as nutrient sensing, protein degradation, and gene expression. Despite its importance, the breadth and functional roles of O-GlcNAc are only beginning to be elucidated. Advances in our understanding will require the development of new strategies for the detection and study of O-GlcNAc-modified proteins in vivo. Herein we report the direct, high-throughput analysis of O-GlcNAc-glycosylated proteins from the mammalian brain. The proteins were identified by using a chemoenzymatic approach that exploits an engineered galactosyltransferase enzyme to selectively label O-GlcNAc proteins with a ketone-biotin tag. The tag permits enrichment of low-abundance O-GlcNAc species from complex mixtures and localization of the modification to short amino acid sequences. Using this approach, we discovered 25 O-GlcNAc-glycosylated proteins from the brain, including regulatory proteins associated with gene expression, neuronal signaling, and synaptic plasticity. The functional diversity represented by this set of proteins suggests an expanded role for O-GlcNAc in regulating neuronal function. Moreover, the chemoenzymatic strategy described here should prove valuable for identifying O-GlcNAc-modified proteins in various tissues and facilitate studies of the physiological significance of O-GlcNAc across the proteome.
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