O-GlcNAcylation-Inducing Treatments Inhibit Estrogen Receptor α Expression and Confer Resistance to 4-OH-Tamoxifen in Human Breast Cancer-Derived MCF-7 Cells.

PLoS OneInternational audienceO-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP3 production. We observed that these treatments stimulated PIP3 production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy

O-GlcNacylation Links TxNIP to Inflammasome Activation in Pancreatic β Cells

Thioredoxin interacting protein (TxNIP), which strongly responds to glucose, has emerged as a central mediator of glucotoxicity in pancreatic β cells. TxNIP is a scaffold protein interacting with target proteins to inhibit or stimulate their activity. Recent studies reported that high glucose stimulates the interaction of TxNIP with the inflammasome protein NLRP3 (NLR family, pyrin domain containing 3) to increase interleukin-1 β (IL1β) secretion by pancreatic β cells. To better understand the regulation of TxNIP by glucose in pancreatic β cells, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAcylation) in regulating TxNIP at the posttranslational level. O-GlcNAcylation of proteins is controlled by two enzymes: the O-GlcNAc transferase (OGT), which transfers a monosaccharide to serine/threonine residues on target proteins, and the O-GlcNAcase (OGA), which removes it. Our study shows that TxNIP is subjected to O-GlcNAcylation in response to high glucose concentrations in β cell lines. Modification of the O-GlcNAcylation pathway through manipulation of OGT or OGA expression or activity significantly modulates TxNIP O-GlcNAcylation in INS1 832/13 cells. Interestingly, expression and O-GlcNAcylation of TxNIP appeared to be increased in islets of diabetic rodents. At the mechanistic level, the induction of the O-GlcNAcylation pathway in human and rat islets promotes inflammasome activation as evidenced by enhanced cleaved IL1β. Overexpression of OGT in HEK293 or INS1 832/13 cells stimulates TxNIP and NLRP3 interaction, while reducing TxNIP O-GlcNAcylation through OGA overexpression destabilizes this interaction. Altogether, our study reveals that O-GlcNAcylation represents an important regulatory mechanism for TxNIP activity in β cells

O-GlcNAc glycosylation des protéines et régulation de la signalisation cellulaire : implication en physiopathologie

La O-GlcNAcylation des protéines cytosoliques ou nucléaires est une modification post-traductionnelle réversible, dynamique, analogue à la phosphorylation, qui contrôle les activités cellulaires en fonction de la disponibilité en glucose. Deux enzymes seulement, l’OGT (O-linked N-acetyl-glucosaminyltransferase) et l’OGA (O-linkedN-acetyl-β-D glucosaminidase), régulent l’addition ou le retrait du GlcNAc (N-Acétyl glucosamine) sur les sérines ou thréonines des protéines. La O-GlcNAcylation peut moduler l’état de phosphorylation, la localisation sub-cellulaire, la stabilité ou l’activité enzymatique des protéines. Des modifications de O-GlcNAc ont été décrites dans plusieurs pathologies humaines. Une diminution de O-GlcNAc dans le cerveau semble associée à la maladie d’Alzheimer, alors qu’une augmentation de O-GlcNAcylation des protéines a été décrite dans différents types de cancers. Par ailleurs, dans les situations d’hyperglycémie chronique, une augmentation de la O-GlcNAcylation de certains facteurs de transcription et des protéines de la signalisation de l’insuline pourrait participer au phénomène de glucotoxicité, en particulier en aggravant la résistance à l’insuline et les altérations β-pancréatiques. Alors que de nombreux biologistes ignorent encore jusqu’à l’existence de cette modification, les perturbations de O-GlcNAcylation observées dans des pathologies majeures soulignent l’urgence d’une meilleure compréhension de son rôle dans le fonctionnement de la cellule

Short O-GlcNAcase Is Targeted to the Mitochondria and Regulates Mitochondrial Reactive Oxygen Species Level

O-GlcNAcylation is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. Only two enzymes, OGT (O-GlcNAc transferase) and OGA (O-GlcNAcase), control the attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells and RAW 264.7 cells, as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a Bioluminescence Resonance Energy Transfer (BRET)-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we show that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA has no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis.</jats:p

Protein O-GlcNAcylation and the regulation of energy homeostasis: lessons from knock-out mouse models

International audienceAbstract O-GlcNAcylation corresponds to the addition of N-Acetylglucosamine (GlcNAc) on serine or threonine residues of cytosolic, nuclear and mitochondrial proteins. This reversible modification is catalysed by a unique couple of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). OGT uses UDP-GlcNAc produced in the hexosamine biosynthesis pathway, to modify proteins. UDP-GlcNAc is at the cross-roads of several cellular metabolisms, including glucose, amino acids and fatty acids. Therefore, OGT is considered as a metabolic sensor that post-translationally modifies proteins according to nutrient availability. O-GlcNAcylation can modulate protein–protein interactions and regulate protein enzymatic activities, stability or subcellular localization. In addition, it can compete with phosphorylation on the same serine or threonine residues, or regulate positively or negatively the phosphorylation of adjacent residues. As such, O-GlcNAcylation is a major actor in the regulation of cell signaling and has been implicated in numerous physiological and pathological processes. A large body of evidence have indicated that increased O-GlcNAcylation participates in the deleterious effects of glucose (glucotoxicity) in metabolic diseases. However, recent studies using mice models with OGT or OGA knock-out in different tissues have shown that O-GlcNAcylation protects against various cellular stresses, and indicate that both increase and decrease in O-GlcNAcylation have deleterious effects on the regulation of energy homeostasis

Short OGA is targeted to the mitochondria and regulates mitochondrial reactive oxygen species level

AbstractO-GlcNAcylation is a reversible post-translational modification involved the regulation of cytosolic, nuclear and mitochondrial proteins. Only two enzymes, OGT and OGA, control attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a BRET-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we showed that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA had no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis.</jats:p

Short O-GlcNAcase Is Targeted to the Mitochondria and Regulates Mitochondrial Reactive Oxygen Species Level

International audienceO-GlcNAcylation is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. Only two enzymes, OGT (O-GlcNAc transferase) and OGA (O-GlcNAcase), control the attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells and RAW 264.7 cells, as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a Bioluminescence Resonance Energy Transfer (BRET)-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we show that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA has no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis

Increased OGA Expression and Activity in Leukocytes from Patients with Diabetes: Correlation with Inflammation Markers

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