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

Study of the control of Type III secretion system expression, resulting from the inactivation of the gene yfgL coding an outer membrane lipoprotein, in Salmonella Enteritidis

Les salmonelles, responsables de fièvres typhoïdes et gastro-entérites, sont un problème majeur de santé publique. Les systèmes de sécrétion de type III (TTSS) sont des facteurs de virulence cruciaux de Salmonella. Nos travaux ont montré que délétion du cadre ouvert de lecture yfgL conduit à une faible transcription des gènes codant les 3 TTSS conduisant à une baisse importante de la virulence de Salmonella Enteritidis. Or, la délétion de ce gène, dont la protéine chez E.coli est en complexe avec les protéines YaeT, YfiO, NlpB et SmpA, conduit à une altération de la membrane externe. Chez S. Enteritidis, nous avons montré que le rôle du complexe « YaeT » dans l’assemblage des protéines de membrane externe est conservé. Or, seule l’inactivation d’yfiO, conduit une diminution de l’expression des TTSS. Nos travaux ont toutefois suggéré que le défaut d’assemblage des protéines de membrane externe n’est pas la cause de ce défaut d’expression des TTSS observés chez les mutants yfgL et yfiO.Salmonella, responsible for typhoid fever and gastroenteritis, is a worldwide health problem. Type three secretion system (TTSS) are crucial virulence factors in Salmonella. Our work showed that deletion of the open reading frame yfgL led to a transcriptional down-regulation of the genes encoding the proteins involved in the biosynthesis of the 3 TTSS in Salmonella Enteritidis. It was shown that inactivation of yfgL whose encoded protein is in complex with YaeT, YfiO, NlpB and SmpA in E. coli, causes an outer membrane alteration. In S. Enteritidis, we observed that the role of the “YaeT” complex in outer membrane protein assembly is conserved in S. Enteritidis. In addition, only yfiO inactivation resulted in a down-expression of the TTSS. However, we presented elements suggesting that the outer membrane protein targeting defect was not responsible for the TTSS down-expression observed in the yfgL and yfiO mutants

Étude du contrôle de l'expression des systèmes de sécrétion de type III, généré par l'inactivation du gène yfgL codant une lipoprotéine de la membrane externe, chez Salmonella Enteritidis

Les salmonelles, responsables de fièvres typhoïdes et gastro-entérites, sont un problème majeur de santé publique. Les systèmes de sécrétion de type III (TTSS) sont des facteurs de virulence cruciaux de Salmonella. Nos travaux ont montré que délétion du cadre ouvert de lecture yfgL conduit à une faible transcription des gènes codant les 3 TTSS conduisant à une baisse importante de la virulence de Salmonella Enteritidis. Or, la délétion de ce gène, dont la protéine chez E.coli est en complexe avec les protéines YaeT, YfiO, NlpB et SmpA, conduit à une altération de la membrane externe. Chez S. Enteritidis, nous avons montré que le rôle du complexe YaeT dans l assemblage des protéines de membrane externe est conservé. Or, seule l inactivation d yfiO, conduit une diminution de l expression des TTSS. Nos travaux ont toutefois suggéré que le défaut d assemblage des protéines de membrane externe n est pas la cause de ce défaut d expression des TTSS observés chez les mutants yfgL et yfiO.Salmonella, responsible for typhoid fever and gastroenteritis, is a worldwide health problem. Type three secretion system (TTSS) are crucial virulence factors in Salmonella. Our work showed that deletion of the open reading frame yfgL led to a transcriptional down-regulation of the genes encoding the proteins involved in the biosynthesis of the 3 TTSS in Salmonella Enteritidis. It was shown that inactivation of yfgL whose encoded protein is in complex with YaeT, YfiO, NlpB and SmpA in E. coli, causes an outer membrane alteration. In S. Enteritidis, we observed that the role of the YaeT complex in outer membrane protein assembly is conserved in S. Enteritidis. In addition, only yfiO inactivation resulted in a down-expression of the TTSS. However, we presented elements suggesting that the outer membrane protein targeting defect was not responsible for the TTSS down-expression observed in the yfgL and yfiO mutants.TOURS-Bibl.électronique (372610011) / SudocSudocFranceF

O-GlcNAcylation: A New Cancer Hallmark ?

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression

Transmission of Diverse Oral Bacteria to Murine Placenta: Evidence for the Oral Microbiome as a Potential Source of Intrauterine Infection▿ †

Microbial infection of the intrauterine environment is a major cause of preterm birth. The current paradigm indicates that intrauterine infections predominantly originate from the vaginal tract, with the organisms ascending into the sterile uterus. With the improvements in technology, an increasing number of bacterial species have been identified in intrauterine infections that do not belong to the vaginal microflora. We have demonstrated previously that intrauterine infections can originate from the oral cavity following hematogenous transmission. In this study, we begin to systemically examine what proportion of the oral microbiome can translocate to the placenta. Pooled saliva and pooled subgingival plaque samples were injected into pregnant mice through tail veins to mimic bacteremia, which occurs frequently during periodontal infections. The microbial species colonizing the murine placenta were detected using 16S rRNA gene-based PCR and clone analysis. A diverse group of bacterial species were identified, many of which have been associated with adverse pregnancy outcomes in humans although their sources of infection were not determined. Interestingly, the majority of these species were oral commensal organisms. This may be due to a dose effect but may also indicate a unique role of commensal species in intrauterine infection. In addition, a number of species were selectively “enriched” during the translocation, with a higher prevalence in the placenta than in the pooled saliva or subgingival plaque samples. These observations indicate that the placental translocation was species specific. This study provides the first insight into the diversity of oral bacteria associated with intrauterine infection

Regulatory O-GlcNAcylation sites on FoxO1 are yet to be identified

International audienceO-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified

Rôle de BamB et son interaction avec BamA dans la biogenèse de la membrane externe, l’expression des T3SS et la virulence chez <em>Salmonella</em>

National audienceLe repliement puis l’insertion des protéines à tonneaux β dans la membrane externe des bactéries est medié par le complexe Bam constitué par la protéine centrale BamA et par 4 lipoprotéines BamB-BamE. BamB interagit directement avec BamA et est impliquée dans plusieurs processus biologiques : l’assemblage des protéines de membrane externe (OMPs) et la perméabilité, le contrôle de l’expression de systèmes de sécrétion de type 3 (T3SS) et enfin la virulence chez Salmonella en modèles murin et aviaire. Pour déterminer s’il existe une relation entre ces différentes fonctions et si l’interaction entre BamB et BamA est requise pour chacune d’elle, des plasmides contenant des mutations ponctuelles dans bamB ont été introduits dans un mutant ΔbamB de Salmonella. Nous démontrons que l’association de résidus L173, L175 et R176 est essentielle à tous les rôles de BamB et à son interaction avec BamA. De plus, des résultats obtenus avec le variant BamB D229A qui est incapable d’immunoprécipiter BamA montrent que l’interaction entre BamA et BamB n’est pas requise pour l’assemblage des OMPs, l’expression des T3SS et la virulence. Enfin le variant BamB D227A restore complétement la virulence du mutant ΔbamB mais conserve les mêmes défauts de perméabilité membranaire que ce dernier indiquant que le défaut de virulence du mutant n’est pas relié à sa sensibilité accrue aux antimicrobiens. En conclusion, au moins certains rôles de BamB sont dissociables et l’association de BamB avec les complexe Bam n’est pas indispensable à son activité. Par ailleurs, de part leur essentialité, les résidus L173, L175 et R176 constituent un site privilégié pour le développement d’inhibiteurs de BamB comme alternative thérapeutique aux antibiotiques

Deciphering the roles of BamB and its interaction with BamA in outer membrane biogenesis, T3SS expression and virulence in Salmonella.

The folding and insertion of β-barrel proteins in the outer membrane of Gram-negative bacteria is mediated by the BAM complex, which is composed of the outer membrane protein BamA and four lipoproteins BamB to BamE. In Escherichia coli and/or Salmonella, the BamB lipoprotein is involved in (i) β-barrel protein assembly in the outer membrane, (ii) outer membrane permeability to antibiotics, (iii) the control of the expression of T3SS which are major virulence factors and (iv) the virulence of Salmonella. In E. coli, this protein has been shown to interact directly with BamA. In this study, we investigated the structure-function relationship of BamB in order to assess whether the roles of BamB in these phenotypes were inter-related and whether they require the interaction of BamB with BamA. For this purpose, recombinant plasmids harbouring point mutations in bamB were introduced in a ΔSalmonella bamB mutant. We demonstrated that the residues L173, L175 and R176 are crucial for all the roles of BamB and for the interaction of BamB with BamA. Moreover, the results obtained with a D229A BamB variant, which is unable to immunoprecipitate BamA, suggest that the interaction of BamB with BamA is not absolutely necessary for BamB function in outer-membrane protein assembly, T3SS expression and virulence. Finally, we showed that the virulence defect of the ΔbamB mutant is not related to its increased susceptibility to antimicrobials, as the D227A BamB variant fully restored the virulence of the mutant while having a similar antibiotic susceptibility to the ΔbamB strain. Overall, this study demonstrates that the different roles of BamB are not all inter-related and that L173, L175 and R176 amino-acids are privileged sites for the design of BamB inhibitors that could be used as alternative therapeutics to antibiotics, at least against Salmonella

O-GlcNAcylation of FoxO1 in pancreatic β cells promotes Akt inhibition through an IGFBP1-mediated autocrine mechanism

O-GlcNAcylation on serine/threonine is a post-translational modification that controls the activity of nucleocytoplasmic proteins according to glucose availability. We previously showed that O-GlcNAcylation of FoxO1 in liver cells increases its transcriptional activity. In the present study, we evaluated the potential involvement of FoxO1 O-GlcNAcylation in the context of pancreatic β-cell glucotoxicity. FoxO1 was O-GlcNAcylated in INS-1 832/13 β cells and isolated rat pancreatic islets. O-GlcNAcylation of FoxO1 resulted in a 2-fold increase in its transcriptional activity toward a FoxO1 reporter gene and a 3-fold increase in the expression of the insulin-like growth factor-binding protein 1 (Igfbp1) gene at the mRNA level, resulting in IGFBP1 protein oversecretion by the cells. Of note, increased IGFBP1 in the culture medium inhibited the activity of the insulin-like growth factor 1 receptor (IGF1R)/phosphatidyl inositol 3 kinase (PI3K)/Akt pathway. We reveal in this report a novel mechanism by which O-GlcNAcylation inhibits Akt activity through an autocrine mechanism. However, although inhibition of IGFBP1 expression using siRNA restored the PI3 kinase/Akt pathway, it did not rescue INS-1 832/13 cells from high-glucose- or O-glcNAcylation-induced cell death. In contrast, FoxO1 down-regulation by siRNA led to 30 to 60% protection of INS-1 832/13 cells from death mediated by glucotoxic conditions. Therefore, whereas FoxO1 O-GlcNAcylation inhibits Akt through an IGFBP1-mediated autocrine pathway, the deleterious effects of FoxO1 O-GlcNAcylation on cell survival appeared to be independent of this pathway.-Fardini, Y., Masson, E., Boudah, O., Ben Jouira, R., Cosson, C., Pierre-Eugene, C., Kuo, M.-S., Issad, T. O-GlcNAcylation of FoxO1 in pancreatic β cells promotes Akt inhibition through an IGFBP1-mediated autocrine mechanism