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 '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

Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation

The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition “click” reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)–(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation

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

Chemoenzymatic Probes for Detecting and Imaging Fucose-α(1-2)-galactose Glycan Biomarkers

The disaccharide motif fucose-α(1-2)-galactose (Fucα(1-2)Gal) is involved in many important physiological processes, such as learning and memory, inflammation, asthma, and tumorigenesis. However, the size and structural complexity of Fucα(1-2)Gal-containing glycans have posed a significant challenge to their detection. We report a new chemoenzymatic strategy for the rapid, sensitive detection of Fucα(1-2)Gal glycans. We demonstrate that the approach is highly selective for the Fucα(1-2)Gal motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of the motif on live cells, discriminating malignant from normal cells. This approach represents a new potential strategy for biomarker detection and expands the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease

Exploring Kinase Cosubstrate Promiscuity: Monitoring Kinase Activity through Dansylation

No AbstractPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61546/1/cbic_200800393_sm_miscellaneous_information.pd

Generalized quantum Fokker-Planck, diffusion and Smoluchowski equations with true probability distribution functions

Traditionally, the quantum Brownian motion is described by Fokker-Planck or diffusion equations in terms of quasi-probability distribution functions, e.g., Wigner functions. These often become singular or negative in the full quantum regime. In this paper a simple approach to non-Markovian theory of quantum Brownian motion using {\it true probability distribution functions} is presented. Based on an initial coherent state representation of the bath oscillators and an equilibrium canonical distribution of the quantum mechanical mean values of their co-ordinates and momenta we derive a generalized quantum Langevin equation in $c$-numbers and show that the latter is amenable to a theoretical analysis in terms of the classical theory of non-Markovian dynamics. The corresponding Fokker-Planck, diffusion and the Smoluchowski equations are the {\it exact} quantum analogues of their classical counterparts. The present work is {\it independent} of path integral techniques. The theory as developed here is a natural extension of its classical version and is valid for arbitrary temperature and friction (Smoluchowski equation being considered in the overdamped limit).Comment: RevTex, 16 pages, 7 figures, To appear in Physical Review E (minor revision

Analysis of transition state mimicry by tight binding aminothiazoline inhibitors provides insight into catalysis by human : O-GlcNAcase

The modification of nucleocytoplasmic proteins with O-linked N-Acetylglucosamine (O-GlcNAc) plays diverse roles in multicellular organisms. Inhibitors of O-GlcNAc hydrolase (OGA), the enzyme that removes O-GlcNAc from proteins, lead to increased O-GlcNAc levels in cells and are seeing widespread adoption in the field as a research tool used in cells and in vivo. Here we synthesize and study a series of tight binding carbohydrate-based inhibitors of human OGA (hOGA). The most potent of these 2′-Aminothiazolines binds with a sub-nanomolar Ki value to hOGA (510 ± 50 pM) and the most selective has greater than 1800000-fold selectivity for hOGA over mechanistically related human lysosomal β-hexosaminidase. Structural data of inhibitors in complex with an hOGA homologue reveals the basis for variation in binding among these compounds. Using linear free energy analyses, we show binding of these 2′-Aminothiazoline inhibitors depends on the pKa of the aminothiazoline ring system, revealing the protonation state of the inhibitor is a key driver of binding. Using series of inhibitors and synthetic substrates, we show that 2′-Aminothiazoline inhibitors are transition state analogues of hOGA that bind to the enzyme up to 1-million fold more tightly than the substrate. These collective data support an oxazoline, rather than a protonated oxazolinium ion, intermediate being formed along the reaction pathway. Inhibitors from this series will prove generally useful tools for the study of O-GlcNAc. The new insights gained here, into the catalytic mechanism of hOGA and the fundamental drivers of potency and selectivity of OGA inhibitors, should enable tuning of hOGA inhibitors with desirable properties