Proteomic analysis and abrogated expression of O‐GlcNAcylated proteins associated with primary breast cancer

O‐GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O‐GlcNAc transferase (OGT) and O‐GlcNAcase, which catalyze the addition and removal of O‐GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor‐associated proteins are O‐GlcNAc modified, the total O‐GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O‐GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O‐GlcNAc immnoblotting and LC‐MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O‐GlcNAcylated or associated with O‐GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O‐GlcNAcylated or associated with O‐GlcNAcylation. Moreover, OGT knockdown showed that decreasing O‐GlcNAcylation was related to inhibition of the anchorage‐independent growth in vitro. These data indicate that aberrant protein O‐GlcNAcylation is associated with breast cancer. Abnormal modification of these O‐GlcNAc‐modified proteins might be one of the vital malignant characteristics of cancer.     

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)‐CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O‐GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O‐linked N‐acetylglucosamine (O‐GlcNAc). O‐GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O‐GlcNAcylation in metabolically active rat brain slices by O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidenamino) N‐phenylcarbamate (PUGNAc), an inhibitor of N‐acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non‐PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O‐GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O‐GlcNAcylation through intracerebroventricular injection of 6‐diazo‐5‐oxo‐l ‐norleucine (DON), the inhibitor of glutamine fructose‐6‐phosphate amidotransferase, suppressed PKA‐CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O‐GlcNAcylation is a novel mechanism that regulates PKA‐CREB signaling. Downregulation of O‐GlcNAcylation suppresses PKA‐CREB signaling and consequently causes learning and memory deficits in AD.     

Disruption of O‐GlcNAc homeostasis during mammalian oocyte meiotic maturation impacts fertilization

Meiotic maturation and fertilization are metabolically demanding processes, and thus the mammalian oocyte is highly susceptible to changes in nutrient availability. O‐GlcNAcylation—the addition of a single sugar residue (O‐linked β‐N‐acetylglucosamine) on proteins—is a posttranslational modification that acts as a cellular nutrient sensor and likely modulates the function of oocyte proteins. O‐GlcNAcylation is mediated by O‐GlcNAc transferase (OGT), which adds O‐GlcNAc onto proteins, and O‐GlcNAcase (OGA), which removes it. Here we investigated O‐GlcNAcylation dynamics in bovine and human oocytes during meiosis and determined the developmental sequelae of its perturbation. OGA, OGT, and multiple O‐GlcNAcylated proteins were expressed in bovine cumulus oocyte complexes (COCs), and they were localized throughout the gamete but were also enriched at specific subcellular sites. O‐GlcNAcylated proteins were concentrated at the nuclear envelope at prophase I, OGA at the cortex throughout meiosis, and OGT at the meiotic spindles. These expression patterns were evolutionarily conserved in human oocytes. To examine O‐GlcNAc function, we disrupted O‐GlcNAc cycling during meiotic maturation in bovine COCs using Thiamet‐G (TMG), a highly selective OGA inhibitor. Although TMG resulted in a dramatic increase in O‐GlcNAcylated substrates in both cumulus cells and the oocyte, there was no effect on cumulus expansion or meiotic progression. However, zygote development was significantly compromised following in vitro fertilization of COCs matured in TMG due to the effects on sperm penetration, sperm head decondensation, and pronuclear formation. Thus, proper O‐GlcNAc homeostasis during meiotic maturation is important for fertilization and pronuclear stage development.     

OGT Controls the Expression and the Glycosylation of E‐cadherin,and Affects Glycosphingolipid Structures in Human Colon Cell Lines

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongst the phenotypic changes exhibited by tumor cells, a wide range of glycosylation has been reported for colon cancer‐derived cell lines and CRC tissues. These aberrant modifications affect different aspects of glycosylation, including an increase in core fucosylation and GlcNAc branching on N‐glycans, alteration of O‐glycans, upregulated sialylation, and O‐GlcNAcylation. Although O‐GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O‐GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches on three human colon cell lines (HT29, HCT116 and CCD841CoN), it is herein reported that silencing O‐GlcNAc transferase (OGT, the sole enzyme driving O‐GlcNAcylation), only slightly affects overall N‐ and O‐glycosylation patterns. Interestingly, silencing of OGT in HT29 cells upregulates E‐cadherin (a major actor of epithelial‐to‐mesenchymal transition) and changes its glycosylation. On the other hand, OGT silencing perturbs biosynthesis of glycosphingolipids resulting in a decrease in gangliosides and an increase in globosides. Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O‐GlcNAcylation in colon cancer cells.     

PUGNAc induces protein ubiquitination in C2C12 myotube cells

O‐linked β‐N‐acetylglucosaminylation (O‐GlcNAcylation) regulates many cellular processes including the cell cycle, cell signaling, and protein trafficking. Dysregulation of O‐GlcNAcylation may be involved in the development of insulin resistance and type 2 diabetes. Therefore, it is necessary to identify cellular proteins that are induced by elevated O‐GlcNAcylation. Here, using adenosine 5′‐triphosphate affinity chromatography, we employed a proteomic approach in order to identify differentially expressed proteins in response to treatment with the O‐GlcNAcase inhibitor, O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), in mouse C2C12 myotube cells. Among 205 selected genes, we identified 68 nucleotide‐binding proteins, 14 proteins that have adenosinetriphosphatase activity, and 10 proteins with ligase activity. Upregulation of proteins, including ubiquitin‐activating enzyme E1, proteasome subunit 20S, cullin‐associated NEDD8‐dissociated protein 1, ezrin, and downregulation of the protein nucleoside diphosphate kinase B, were confirmed by western blot analysis. In particular, we found that the protein ubiquitination level in C2C12 cells was increased by PUGNAc treatment. This is the first report of quantitative proteomic profiles of myotube cells after treatment with PUGNAc, and our results demonstrate the potential to enhance understanding of the relationship between insulin resistance, O‐GlcNAc, and PUGNAc in the future. Copyright © 2015 John Wiley & Sons, Ltd.     

Oxidative stress induces transient O‐GlcNAc elevation and tau dephosphorylation in SH‐SY5Y cells

O‐linked β‐N‐acetlyglucosamine or O‐GlcNAc modification is a dynamic post‐translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O‐GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O‐GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH‐SY5Y we investigated the dynamic nature of O‐GlcNAc after treatment with 0.5 mM H₂O₂ for 30 min. to induce oxidative stress. We found that overall O‐GlcNAc quickly increased and reached peak level at around 2 hrs post‐stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O‐Glycosylation. In conclusion, our results show that temporary elevation in O‐GlcNAc modification after H₂O₂‐induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O‐GlcNAc and phosphorylation on tau proteins.     

Arabidopsis O‐GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering

Post‐translational modification of proteins by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is catalyzed by O‐GlcNAc transferases (OGTs). O‐GlcNAc modification of proteins regulates multiple important biological processes in metazoans. However, whether protein O‐GlcNAcylation is involved in epigenetic processes during plant development is largely unknown. Here, we show that loss of function of SECRET AGENT (SEC), an OGT in Arabidopsis, leads to an early flowering phenotype. This results from reduced histone H3 lysine 4 trimethylation (H3K4me3) of FLOWERING LOCUS C (FLC) locus, which encodes a key negative regulator of flowering. SEC activates ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1), a histone lysine methyltransferase (HKMT), through O‐GlcNAc modification to augment ATX1‐mediated H3K4me3 histone modification at FLC locus. SEC transfers an O‐GlcNAc group on Ser947 of ATX1, which resides in the SET domain, thereby activating ATX1. Taken together, these results uncover a novel post‐translational O‐GlcNAc modification‐mediated mechanism for regulation of HKMT activity and establish the function of O‐GlcNAc signaling in epigenetic processes in plants.     

Proteomics and PUGNAcity will overcome questioning of insulin resistance induction by nonselective inhibition of O‐GlcNAcase

PTMs are the ultimate elements that perfect the existence and the activity of proteins. Owing to PTM, not less than 500 millions biological activities arise from approximately 20 000 protein‐coding genes in human. Hundreds of PTM were characterized in living beings among which is a large variety of glycosylations. Many compounds have been developed to tentatively block each kind of glycosylation so as to study their biological functions but due to their complexity, many off‐target effects were reported. Insulin resistance exemplifies this problem. Several independent groups described that inhibiting the removal of O‐GlcNAc moieties using O‐(2‐acetamido‐2‐deoxy‐d‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), a nonselective inhibitor of the nuclear and cytoplasmic O‐GlcNAcase, induced insulin resistance both in vivo and ex vivo. The development of potent and highly selective O‐GlcNAcase inhibitors called into question that elevated O‐GlcNAcylation levels are responsible for insulin resistance; these compounds not recapitulating the insulin‐desensitizing effect of PUGNAc. To tackle this intriguing problem, a South Korean group recently combined ATP‐affinity chromatography and gel‐assisted digestion to identify proteins, differentially expressed upon treatment of 3T3‐L1 adipocytes with PUGNAc, involved in protein turnover and insulin signaling.     

Interactome analysis of herpes simplex virus 1 envelope glycoprotein H

Herpes simplex virus 1 (HSV‐1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV‐1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry‐based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive‐1 (GCN1), a trans‐acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV‐1‐infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV‐1‐infected cells and that the gH‐null mutation increases eIF2α in HSV‐1‐infected cells, whereas gH overexpression in the absence of other HSV‐1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV‐1‐infected cells and that the GCN1‐binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV‐1‐infected cells.     

Agents of change – concepts in Mycobacterium tuberculosis Ser/Thr/Tyr phosphosignalling

The flow of information from the outside to the inside of bacterial cells is largely directed by protein kinases. In addition to histidine/aspartate phosphorelays of two‐component response regulators, recent work in Mycobacterium tuberculosis (Mtb) reinforces the idea that phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) is central to bacterial physiology and pathogenesis, and that the corresponding phosphosystems are highly similar to those in eukaryotes. In this way, eukaryotes are a useful guide to understanding Ser/Thr/Tyr phosphorylation (O‐phosphorylation) in prokaryotes such as Mtb. However, as novel functions and components of bacterial O‐phosphorylation are identified, distinct differences between pro‐ and eukaryotic phosphosignalling systems become apparent. The emerging picture of O‐phosphorylation in Mtb is complicated, goes beyond the eukaryotic paradigms, and shows the limitations of viewing bacterial phosphosignalling within the confines of the ‘eukaryotic‐like’ model. Here, we summarize recent findings about Ser/Thr and the recently discovered Tyr phosphorylation pathways in Mtb, highlight the similarities and differences between eukaryotic and prokaryotic O‐phosphorylation, and pose additional questions about signalling components, pathway organization, and ultimately, the cellular roles of O‐phosphorylation in Mtb physiology and pathogenesis.     

ATM is involved in cell‐cycle control through the regulation of retinoblastoma protein phosphorylation

Ataxia telangiectasia mutated protein (ATM) is a member of the phosphatidylinositol‐3 kinase (PI3K) family, which has a role in the cellular response to DNA double‐strand breaks (DSBs). In the present study, we evaluated the role of ATM in cell‐cycle control in dopaminergic rat neuroblastoma B65 cells. For this purpose, ATM activity was either inhibited pharmacologically with the specific inhibitor KU‐55933, or the ATM gene was partially silenced by transfection with small interfering RNA (siRNA). Our data indicate that although ATM inhibition did not affect the cell cycle, both treatments specifically decreased the levels of cyclin A and retinoblastoma protein (pRb), phosphorylated at Ser780. Furthermore, ATM inhibition decreased the active form of p53, which is phosphorylated at Ser15, and also decreased Bax and p21 expression. Using H₂O₂ as a positive control of DSBs, caused a rapid pRb phosphorylation, this was prevented by KU‐55933 and siRNA treatment. Collectively, our data demonstrate how a new molecular network on ATM regulates the cell cycle through the control of pRb phosphorylation. These findings support a new target of ATM. J. Cell. Biochem. 110: 210–218, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of differentially expressed proteins by treatment with PUGNAc in 3T3‐L1 adipocytes through analysis of ATP‐binding proteome

O‐GlcNAc (2‐acetamino‐2‐deoxy‐β‐D‐glucopyranose), an important modification for cellular processes, is catalyzed by O‐GlcNAc transferase and O‐GlcNAcase. O‐(2‐acetamido‐2‐deoxy‐D‐glucopyranosylidene) amino‐N‐phenylcarbamate (PUGNAc) is a nonselective inhibitor of O‐GlcNAcase, which increases the level of protein O‐GlcNAcylation and is known to induce insulin‐resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNAc. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3T3‐L1 adipocytes and C2C12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP‐bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP‐binding protein families classified by PROSITE. The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3T3‐L1 adipocytes following treatment with PUGNAc. For label‐free quantitation, a gel‐assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data‐independent (671 proteins identified) and data‐dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide‐binding proteins and we focused on some nucleotide‐binding proteins, ubiquitin‐activation enzyme 1 (E1), Hsp70, vasolin‐containing protein (Vcp), and Hsp90, involved in ubiquitin‐proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNAc resulted in increased ubiquitination of proteins in a time‐dependent manner, and a decrease in both the amount of Akt and the level of phosphorylation of Akt, a key component in insulin signaling, through downregulation of Hsp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNAc. This result would provide insight into understanding functions of PUGNAc in 3T3‐L1 cells.     

Phosphorylation down-regulates the RNA binding function of the coat protein of potato virus A

Plant viruses encode movement proteins (MPs) to facilitate transport of their genomes from infected into neighboring healthy cells through plasmodesmata. Growing evidence suggests that specific phosphorylation events can regulate MP functions. The coat protein (CP) of potato virus A (PVA; genus Potyvirus) is a multifunctional protein involved both in virion assembly and virus movement. Labeling of PVA-infected tobacco leaves with [(33)P]orthophosphate demonstrated that PVA CP is phosphorylated in vivo. Competition assays established that PVA CP and the well characterized 30-kDa MP of tobacco mosaic virus (genus Tobamovirus) are phosphorylated in vitro by the same Ser/Thr kinase activity from tobacco leaves. This activity exhibits a strong preference for Mn(2+) over Mg(2+), can be inhibited by micromolar concentrations of Zn(2+) and Cd(2+), and is not Ca(2+)-dependent. Tryptic phosphopeptide mapping revealed that PVA CP was phosphorylated by this protein kinase activity on multiple sites. In contrast, PVA CP was not phosphorylated when packaged into virions, suggesting that the phosphorylation sites are located within the RNA binding domain and not exposed on the surface of the virion. Furthermore, two independent experimental approaches demonstrated that the RNA binding function of PVA CP is strongly inhibited by phosphorylation. From these findings, we suggest that protein phosphorylation represents a possible mechanism regulating formation and/or stability of viral ribonucleoproteins in planta.