A 'molecular switchboard' - covalent modifications to proteins and their impact on transcription

Proteins undergo a remarkable variety of posttranslational modifications, with more than 200 distinct modifications identified to date. Increasing evidence suggests that many proteins bear multiple, distinct modifications, and the ability of one modification to antagonize or synergize the deposition of another can have significant biological consequences. Here, we illustrate the importance of posttranslational modifications within the context of transcriptional regulation, and we offer a perspective on the emerging role of combinatorial networks of modifications. Finally, we discuss the potential for chemical approaches to transform our understanding of the field

Parallel identification of O-GlcNAc-modified proteins from cell lysates

We report a new strategy for the parallel identification of O-GlcNAc-glycosylated proteins from cell lysates. The approach permits specific proteins of interest to be rapidly interrogated for the modification in any tissue or cell type and can be extended to peptides to facilitate the mapping of glycosylation sites. As an illustration of the approach, we identified four new O-GlcNAc-glycosylated proteins of low cellular abundance (c-Fos, c-Jun, ATF-1, and CBP) and two short regions of glycosylation in the enzyme O-GlcNAc transferase (OGT). The ability to target specific proteins across various tissue or cell types complements emerging proteomic technologies and should advance our understanding of this important posttranslational modification

A Chemoenzymatic Approach toward the Rapid and Sensitive Detection of O-GlcNAc Posttranslational Modifications

We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of β-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications

Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics

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

A Chemoenzymatic Strategy toward Understanding O-GlcNAc Glycosylation in the Brain

Posttranslational modification to proteins represents a fundamental mechanism by which protein function is extended and elaborated. In the brain, modifications such as phosphorylation play critical roles in mediating neuronal communication and development. Unique among carbohydrate modifications is the addition of a single monosaccharide, N-acetyl-D-glucosamine, to serine and threonine residues of proteins (O-GlcNAc glycosylation). The modification shares intriguing features with phosphorylation, including its intracellular and dynamic nature. The enzyme responsible for adding the modification to proteins is necessary for life at the single cell level and O-GlcNAc glycosylation has been linked to nutrient sensing, gene expression, and in the brain, to neurodegeneration. Despite tantalizing evidence for the modification’s importance, understanding O-GlcNAc glycosylation has been hampered by insufficient strategies to study it at single-protein level as well as across the proteome. Here we describe the development of a new, chemoenzymatic strategy to facilitate the discovery of O-GlcNAc proteins, as well as the first studies aimed at understanding O-GlcNAc proteome-wide, in the brain. Our approach capitalizes on an engineered enzyme and synthetic unnatural substrate to specifically 'tag' O-GlcNAc-modified proteins for rapid and sensitive detection. We applied the methodology to the discovery of low-abundance, endogenous O-GlcNAc proteins from cells. We also combined the approach with mass spectrometry for the isolation of O-GlcNAc peptides and the mapping of glycosylation sites, the first step toward functional analysis of the modification. Overall, our efforts led to the identification of nearly fifty new O-GlcNAc proteins, several of which serve as targets for mechanistic study. Many of the proteins function in the control of transcription and translation, highlighting the proposed role for O-GlcNAc in regulating gene expression. Additionally, we provide evidence that O-GlcNAc glycosylation is particularly prevalent on proteins at the nerve terminal, or synaptosome, where it may function to control vesicle cycling and neurotransmitter release. Finally, our work has also led to the first bioanalytical, quantitative assays for O-GlcNAc dynamics in both cells and tissue. Here, we have shown that O-GlcNAc is reversible in neuronal tissue and can respond rapidly and robustly to neuronal stimulation in vivo.</p