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Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

In eukaryotes, gene expression is performed by three RNA polymerases that are targeted to promoters by molecular complexes. A unique common factor, the TATA-box binding protein (TBP), is thought to serve as a platform to assemble pre-initiation complexes competent for transcription. Herein, we describe a novel molecular mechanism of nutrient regulation of gene transcription by dynamic O-GlcNAcylation of TBP. We show that O-GlcNAcylation at T114 of TBP blocks its interaction with BTAF1, hence the formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. Transcriptomic and metabolomic analyses of TBP(T114A) CRISPR/Cas9 edited cells showed that loss of O-GlcNAcylation at T114 increases TBP binding to BTAF1 and directly impacts expression of 408 genes. Lack of O-GlcNAcylation at T114 is associated with a striking reprogramming of cellular metabolism induced by a profound modification of the transcriptome, leading to gross alterations in lipid storage.

The function of actin is regulated by various posttranslational modifications. We have previously shown that in the kidneys of nonobese type 2 diabetes model Goto-Kakizaki rats, increased O-GlcNAcylation of β-actin protein is observed. It has also been reported that both O-GlcNAcylation and phosphorylation occur on Ser199of β-actin. However, their roles are not known. To elucidate their roles in diabetic nephropathy, we examined the rat kidney for changes in O-GlcNAcylation of Ser199(gS199)-actin and in the phosphorylation of Ser199(pS199)-actin. Both gS199- and pS199-actin molecules had an apparent molecular weight of 40 kDa and were localized as nonfilamentous actin in both the cytoplasm and nucleus. Compared with the normal kidney, the immunostaining intensity of gS199-actin increased in podocytes of the glomeruli and in proximal tubules of the diabetic kidney, whereas that of pS199-actin did not change in podocytes but decreased in proximal tubules. We confirmed that the same results could be observed in the glomeruli of the human diabetic kidney. In podocytes of glomeruli cultured in the presence of the O-GlcNAcase inhibitor Thiamet G, increased O-GlcNAcylation was accompanied by a concomitant decrease in the amount of filamentous actin and in morphological changes. Our present results demonstrate that dysregulation of O-GlcNAcylation and phosphorylation of Ser199occurred in diabetes, which may contribute partially to the causes of the morphological changes in the glomeruli and tubules. gS199- and pS199-actin will thus be useful for the pathological evaluation of diabetic nephropathy.

As a dynamic post-translational modification, O-linked β- N-acetylglucosamine ( O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-β-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.

O-linked β-d-N-acetyl glucosamine (O-GlcNAc) addition (O-GlcNAcylation), a post-translational modification of serine/threonine residues of proteins, is involved in diverse cellular metabolic and signaling pathways. Aberrant O-GlcNAcylation underlies the initiation and progression of multiple chronic diseases including diabetes, cancer, and neurodegenerative diseases. Numerous methods have been developed for the analysis of protein O-GlcNAcylation, but instead of discussing the classical biochemical techniques, this unit covers O-GlcNAc characterization by combining several enrichment methods and mass spectrometry detection techniques [including collision-induced dissociation (CID), higher energy collision dissociation (HCD), and electron transfer dissociation (ETD) mass spectrometry]. © 2017 by John Wiley & Sons, Inc. Keywords: BEMAD; CID; enrichment; ETD; HCD; mass spectrometry; O-GlcNAc; O-GlcNAcome; O-GlcNAcomics; site mapping

O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification on serine and threonine residues of many proteins, plays crucial regulatory roles in diverse biological events. As a nutrient sensor, O-GlcNAc modification (O-GlcNAcylation) on nuclear and cytoplasmic proteins underlies the pathology of diabetic complications including cardiomyopathy. However, mitochondrial O-GlcNAcylation, especially in response to chronic hyperglycemia in diabetes, has been poorly explored. We performed a comparative O-GlcNAc profiling of mitochondria from control and streptozotocin (STZ)-induced diabetic rat hearts by using an improved β-elimination/Michael addition with isotopic DTT reagents (BEMAD) followed by tandem mass spectrometric analysis. In total, 86 mitochondrial proteins, involved in diverse pathways, were O-GlcNAcylated. Among them, many proteins have site-specific alterations in O-GlcNAcylation in response to diabetes, which suggests that protein O-GlcNAcylation is a novel layer of regulation mediating adaptive changes in mitochondrial metabolism during the progression of diabetic cardiomyopathy.

At least 20 core ribosome proteins are modified by O-GlcNAc. O-GlcNAcase is localized to the nucleolus and O-GlcNAc transferase is excluded from the nucleolus. Both enzymes associate with active polysomes. Overexpression of OGT disrupts ribosomal subunit homeostasis. Data suggest that O-GlcNAc regulates translation and ribosome biogenesis., Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.

Contractile dysfunction and increased deposition of O-linked β-N-acetyl-d-glucosamine (O-GlcNAc) in cardiac proteins are a hallmark of the diabetic heart. However, whether and how this posttranslational alteration contributes to lower cardiac function remains unclear. Using a refined β-elimination/Michael addition with tandem mass tags (TMT)–labeling proteomic technique, we show that CpOGA, a bacterial analog of O-GlcNAcase (OGA) that cleaves O-GlcNAc in vivo, removes site-specific O-GlcNAcylation from myofilaments, restoring Ca2+ sensitivity in streptozotocin (STZ) diabetic cardiac muscles. We report that in control rat hearts, O-GlcNAc and O-GlcNAc transferase (OGT) are mainly localized at the Z-line, whereas OGA is at the A-band. Conversely, in diabetic hearts O-GlcNAc levels are increased and OGT and OGA delocalized. Consistent changes were found in human diabetic hearts. STZ diabetic hearts display increased physical interactions of OGA with α-actin, tropomyosin, and myosin light chain 1, along with reduced OGT and increased OGA activities. Our study is the first to reveal that specific removal of O-GlcNAcylation restores myofilament response to Ca2+ in diabetic hearts and that altered O-GlcNAcylation is due to the subcellular redistribution of OGT and OGA rather than to changes in their overall activities. Thus, preventing sarcomeric OGT and OGA displacement represents a new possible strategy for treating diabetic cardiomyopathy.

Glycans represent one of the four fundamental building blocks of life, and they are the most abundant biological molecules on our planet. Only in recent years, have we begun to appreciate how deeply glycan functions pervade all aspects of organismic biology, molecular biology and biochemistry. A recent National Academy Science report ( http://www.nap.edu/catalog.php?record_id=13446 ) has concluded that a better understanding of glycoscience is critical to advancing human health and for sustainability on this planet. The report further concludes that efforts to study this key area of biochemistry/biology have substantially lagged behind investments in other biological molecules, such as nucleic acids and proteins. The Journal of Biological Chemistry continues to be the leader in publication of glycoscience research. In this special issue of the Journal, five Mini-Reviews, authored by leaders in glycobiology, illustrate the remarkable diversity of the biological functions of glycans.

Recent evidence indicates that site-specific crosstalk between O-GlcNAcylation and phosphorylation and the O-GlcNAcylation of kinases play an important role in regulating cell signaling. However, relatively few kinases have been analyzed for O-GlcNAcylation. Here, we identify additional kinases that are substrates for O-GlcNAcylation using an in vitro OGT assay on a functional kinase array. Forty-two kinases were O-GlcNAcylated in vitro, representing 39% of the kinases on the array. In addition, we confirmed the in vivo O-GlcNAcylation of three identified kinases. Our results suggest that O-GlcNAcylation may directly regulate a substantial number of kinases and illustrates the increasingly complex relationship between O-GlcNAcylation and phosphorylation in cellular signaling.

O -linked N -acetylglucosamine ( O -GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O -GlcNAc transferase (OGT). O -GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O -GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O -GlcNAcylation in AD has been impeded by the difficulty in characterization of O -GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O -GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O -GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O -GlcNAc. Overall, 458 O -GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O -GlcNAcylation and phosphorylation. This study produced the most comprehensive O -GlcNAc proteome of mammalian brain tissue with both protein identification and O -GlcNAc site assignment. Interestingly, we observed O -β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O -GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1- O -Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3- N -acetylglucosaminyltransferases.

Hyaluronan, a high molecular mass polysaccharide on the vertebrate cell surface and extracellular matrix, is produced at the plasma membrane by hyaluronan synthases using UDP-GlcNAc and UDP-GlcUA as substrates. The availability of these UDP-sugar substrates can limit the synthesis rate of hyaluronan. In this study, we show that the cellular level of UDP-HexNAc also controls hyaluronan synthesis by modulating the expression of HAS2 (hyaluronan synthase 2). Increasing UDP-HexNAc in HaCaT keratinocytes by adding glucosamine down-regulated HAS2 gene expression, whereas a decrease in UDP-HexNAc, realized by mannose treatment or siRNA for GFAT1 (glutamine:fructose-6-phosphate amidotransferase 1), enhanced expression of the gene. Tracing the UDP-HexNAc-initiated signal to the HAS2 promoter revealed no change in the binding of STAT3, NF-κB, and cAMP response element-binding protein, shown previously to mediate growth factor and cytokine signals on HAS2 expression. Instead, altered binding of SP1 and YY1 to the promoter correlated with cellular UDP-HexNAc content and inhibition of HAS2 expression. siRNA silencing of YY1 and SP1 confirmed their inhibitory effects on HAS2 expression. Reduced and increased levels of O-GlcNAc-modified SP1 and YY1 proteins were associated with stimulation or inhibition of HAS2 expression, respectively. Our data are consistent with the hypothesis that, by regulating the level of protein O-GlcNAc modifications, cellular UDP-HexNAc content controls HAS2 transcription and decreases the effects on hyaluronan synthesis that would result from cellular fluctuations of this substrate.

The modification of Ser and Thr residues with O-linked b-N-acetyl glucosamine (O-GlcNAc) is a common and essential modification of nuclear and cytoplasmic proteins, and it is thought that O-GlcNAc performs a regulatory role in the cell. This unit concentrates on the techniques for the detection and analysis of proteins modified by O-GlcNAc as well as methods for the analysis of enzymes responsible for the addition and removal of this group.

Cells run on carbohydrates. Glycans, sequences of carbohydrates conjugated to proteins and lipids, are arguably the most abundant and structurally diverse class of molecules in nature. Recent advances in glycomics reveal the scope and scale of their functional roles and their impact on human disease.

The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assembles multiprotein complexes containing numerous transcription factors and cofactors, including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled-coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic-activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of the cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and the transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

The glycosylation state of envelope glycoproteins in Human and Simian Immunodeficiency Viruses (HIV/SIV) is critical to viral infectivity and tropism, viral protein processing, and in virus evasion of the immune system. Using a rapid fluorescent two-dimensional gel based method coupled with enzymatic pre-treatment of virus with PNGase F (Peptide: N-Glycosidase F) and fluorescent 2D gels or 2D gel Western blotting, we show significant differences in the glycosylation patterns of two SIV strains widely used in animal models of HIV disease and vaccine studies. We also demonstrate the modification of a host protein important in HIV biology (HLA-DR) by O-GlcNAc. Further, this experimental pipeline allows for the identification of the modified protein and the site of N-linked glycosylation by fluorescent two-dimensional gel electrophoresis coupled with mass spectrometry (MS) and the qualitative and semi-quantitative assessment of viral glycosylation. The method is fully compatible with downstream glycomics analysis. This approach will permit correlation of virus glycosylation status with pathological severity and may serve as a rapid screen of viruses from physiological samples for further study by more advanced MS methodology.

Author(s): Varki, Ajit P; Baum, Linda G; Bellis, Susan L; Cummings, Richard D; Esko, Jeffrey D; Hart, Gerald W; Linhardt, Robert J; Lowe, John B; McEver, Rodger P; Srivastava, Arun; Sarkar, Rita

Proteomic technologies, such as yeast two-hybrid, mass spectrometry (MS), protein/peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories.

Phosphorylation and O-GlcNAcylation of keratin 18 (K18) are highly dynamic and involve primarily independent K18 populations. We used in vitro phosphorylation and O-GlcNAcylation of wild-type, phospho-Ser52, glyco-Ser48, and Ser-to-Ala mutant 17mer peptides (K18 amino acids 40-56), which include the major K18 glycosylation (Ser48) and phosphorylation (Ser52) sites, to address whether each modification blocks the other. The glyco-K18 peptide blocks Ser52 phosphorylation by protein kinase C, an in vivo K18 kinase, while the phospho-K18 peptide blocks its O-GlcNAcylation. Our findings support the reciprocity of these two post-translational modifications. Therefore, regulation of protein Ser/Thr phosphorylation and glycosylation at proximal sites can be interdependent and provides a potential mechanism of counter regulation.

Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains of individuals with Alzheimer's disease (AD) and other tauopathies. Tau pathology is critical to pathogenesis and correlates to the severity of dementia. However, the mechanisms leading to abnormal hyperphosphorylation are unknown. Here, we demonstrate that human brain tau was modified by O-GlcNAcylation, a type of protein O-glycosylation by which the monosaccharide β- N -acetylglucosamine (GlcNAc) attaches to serine/threonine residues via an O-linked glycosidic bond. O-GlcNAcylation regulated phosphorylation of tau in a site-specific manner both in vitro and in vivo . At most of the phosphorylation sites, O-GlcNAcylation negatively regulated tau phosphorylation. In an animal model of starved mice, low glucose uptake/metabolism that mimicked those observed in AD brain produced a decrease in O-GlcNAcylation and consequent hyperphosphorylation of tau at the majority of the phosphorylation sites. The O-GlcNAcylation level in AD brain extracts was decreased as compared to that in controls. These results reveal a mechanism of regulation of tau phosphorylation and suggest that abnormal hyperphosphorylation of tau could result from decreased tau O-GlcNAcylation, which probably is induced by deficient brain glucose uptake/metabolism in AD and other tauopathies.

Nuclear and cytoplasmic protein glycosylation is a widespread and reversible posttranslational modification in eukaryotic cells. Intracellular glycosylation by the addition of N- acetylglucosamine (GlcNAc) to serine and threonine is catalyzed by the O-GlcNAc transferase (OGT). This “O-GlcNAcylation” of intracellular proteins can occur on phosphorylation sites, and has been implicated in controlling gene transcription, neurofilament assembly, and the emergence of diabetes and neurologic disease. To study OGT function in vivo , we have used gene-targeting approaches in male embryonic stem cells. We find that OGT mutagenesis requires a strategy that retains an intact OGT gene as accomplished by using Cre-loxP recombination, because a deletion in the OGT gene results in loss of embryonic stem cell viability. A single copy of the OGT gene is present in the male genome and resides on the X chromosome near the centromere in region D in the mouse spanning markers DxMit41 and DxMit95 , and in humans at Xq13, a region associated with neurologic disease. OGT RNA expression in mice is comparably high among most cell types, with lower levels in the pancreas. Segregation of OGT alleles in the mouse germ line with ZP3-Cre recombination in oocytes reveals that intact OGT alleles are required for completion of embryogenesis. These studies illustrate the necessity of conditional gene-targeting approaches in the mutagenesis and study of essential sex-linked genes, and indicate that OGT participation in intracellular glycosylation is essential for embryonic stem cell viability and for mouse ontogeny.

The intracellular transport of two closely related membrane glycoproteins was studied in the murine B cell lymphoma line, AKTB-1b. Using pulse-chase radiolabeling, the kinetics of appearance of the class I histocompatibility antigens, H-2Kk and H-2Dk, at the cell surface were compared and found to be remarkably different. Newly synthesized H-2Kk is transported rapidly such that all radiolabeled molecules reach the surface within 1 h. In contrast, the H-2Dk antigen is transported slowly with a half-time of 4-5 h. The rates of surface appearance for the two antigens closely resemble the rates at which their Asn-linked oligosaccharides mature from endoglucosaminidase H (endo H)-sensitive to endo H-resistant forms, a process that occurs in the Golgi apparatus. This suggests that the rate-limiting step in the transport of H-2Dk to the cell surface occurs before the formation of endo H-resistant oligosaccharides in the Golgi apparatus. Subcellular fractionation experiments confirmed this conclusion by identifying the endoplasmic reticulum (ER) as the site where the H-2Dk antigen accumulates. The retention of this glycoprotein in the ER does not appear to be due to a lack of solubility or an inability of the H-2Dk heavy chain to associate with beta 2-microglobulin. Our data is inconsistent with a passive membrane flow mechanism for the intracellular transport of membrane glycoproteins. Rather, it suggests that one or more receptors localized to the ER membrane may mediate the selective transport of membrane glycoproteins out of the ER to the Golgi apparatus. The fact that H-2Kk and H-2Dk are highly homologous (greater than or equal to 80%) indicates that this process can be strongly influenced by limited alterations in protein structure.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic post-translational modification more analogous to phosphorylation than to classical complex O-glycosylation. A large number of nuclear and cytosolic proteins are modified by O-GlcNAc. Proteins modified by O-GlcNAc include transcription factors, signaling components, and metabolic enzymes. While the modification has been known for almost 20 years, functions for the monosaccharide modification are just now emerging. In this review, we will focus on the cycling enzymes and emerging roles for this post-translational modification in regulating signal transduction and transcription. Finally, we will discuss future directions and the working model of O-GlcNAc serving as a nutrient sensor.

Background O-Linked β-N-acetylglucosamine (O-GlcNAc) is an enzyme-catalyzed posttranslational modification of serine or threonine side chains of nuclear and cytoplasmic proteins. O-GlcNAc is present in all metazoans and in viruses that infect eukaryotic cells. GlcNAcylation is dynamic and has a high cycling rate on many proteins in response to cellular metabolism and various environmental stimuli. The rapid cycling of O-GlcNAc modulates many biological processes, including transcriptional regulation, stress responses, cell cycle regulation, and protein synthesis and turnover. Rationale Despite the importance of O-GlcNAc, progress during the past two decades in this field has been slow. One of the major obstacles is the lack of simple and sensitive tools for efficient O-GlcNAc detection and localization. Recently developed O-GlcNAc derivatization and enrichment approaches, together with new techniques in mass spectrometric instrumentation and methods, have provided breakthroughs in O-GlcNAc site localization and site-specific quantitation. In this review, we will discuss how the current techniques are expanding our knowledge about O-GlcNAc proteomics/glycomics and functions.

We demonstrated that normal cardiac myofilaments contain 32 total O-GlcNAcylation sites on MHC, Actin, MLC 1, MLC2 and Tn I and that exposure of skinned muscles to GlcNAc induces myofilament Ca2+ desensitization. Actin O-GlcNAcylation is increased in myofilaments following GlcNAc exposure and in two in vivo Diabetes Mellitus (DM) models. Yet, the mechanisms of O-GlcNAc-induced myofilament Ca2+ desensitization remain unclear. We investigated the effect of O-GlcNAc removal by an engineered hexosaminidase (CPJ) on Ca2+ sensitivity of skinned muscles from hearts of control and DM type 1 rats. We found that 1 hour exposure to CPJ reversed myofilament Ca2+ desensitization in DM cardiac muscles (EC50 4.17±0.48 µM pre-CPJ vs 2.73±0.22 µM post-CPJ, n=5 vs n=4, p=0.029 ), but had no effect in control muscles (EC50 2.73±0.17 µM pre-CPJ vs 2.6±0.15 µM post-CPJ, n=6 vs n=5, p=n.s). CPJ activity against O-GlcNAc was verified by western blot on treated lysates. These results suggest that in diabetic muscle O-GlcNAcylation directly affects myofilament Ca2+ sensitivity. To address potential mechanism(s), we characterized O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) protein interactions in fresh whole heart homogenates from both groups by immunoprecipitation followed by Western blotting. Remarkably, we observed OGT and OGA interactions with members of thick (MyBP-C, MLC1, MLC2) and thin (Actin, TnI, Tm) myofilament proteins, which were differentially increased with actin, Tm and MLC1 in DM rat homogenates compared to control (n=4 vs n=4, p< 0.05). These results strongly suggest that in DM a specific increase in O-GlcNAcylation of cardiac myofilament proteins, and in OGT/OGA association with actin, tropomyosin and MLC1 leads to dysfunctional regulation of myocardial contractility.

O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) onto serine and threonine residues of proteins is an important post-translational modification (PTM), which is involved in many crucial biological processes including transcription, translation, proteasomal degradation, and signal transduction. Aberrant protein O-GlcNAcylation is directly linked to the pathological progression of chronic diseases including diabetes, cancer, and neurodegenerative disorders. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions. In this review, we mainly focus on technological developments regarding O-GlcNAc protein profiling. Specifically, on one hand, we show how these techniques are being used for the comprehensive characterization of certain targeted proteins in which biologists are most interested. On the other hand, we present several newly developed approaches for O-GlcNAcomic profiling as well as how they provide us with a systems perspective to crosstalk amongst different PTMs and complicated biological events. Promising technical trends are also highlighted to evoke more efforts by diverse laboratories, which would further expand our understanding of the physiological and pathological roles of protein O-GlcNAcylation in chronic diseases.

International audience; V(D)J recombination assembles immunoglobulin and T-cell receptor genes from the preexisting variable (V), diversity (D), and joining (J) gene segments by a cut and paste mechanism. While this receptor diversification strategy enables efficient immune responses against pathogens, it also poses a constant threat to the genome integrity. Tight regulation of this process at multiple levels is therefore critical to prevent genomic instability and neoplastic transformation. In this article, we discuss the temporal and spatial regulation of V(D)J recombination, in addition to the molecular mechanisms that regulate this process. Finally, we discuss the implication of V(D)J recombination in malignancies of the immune system. “RAGs are the end-point of the strategies developed through evolution to achieve the anticipatory “Promethean” recognition of all universal molecular forms”. – A.A. de Freitas, Tractus Immuno-Logicus.