O-GlcNAc transferase associates with the MCM2-7 complex and its silencing destabilizes MCM-MCM interactions

International audienceO-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the Mini-Chromosome Maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2-7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2-7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilize MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2-7 complex and O-GlcNAcylation homeostasis might regulate MCM2-7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions

Cross-Dysregulation of O-GlcNAcylation and PI3K/AKT/mTOR Axis in Human Chronic Diseases

The hexosamine biosynthetic pathway (HBP) and the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway are considered as nutrient sensors that regulate several essential biological processes. The hexosamine biosynthetic pathway produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the substrate for O-GlcNAc transferase (OGT), the enzyme that O-GlcNAcylates proteins on serine (Ser) and threonine (Thr) residues. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) and phosphorylation are highly dynamic post-translational modifications occurring at the same or adjacent sites that regulate folding, stability, subcellular localization, partner interaction, or activity of target proteins. Here we review recent evidence of a cross-regulation of PI3K/AKT/mTOR signaling pathway and protein O-GlcNAcylation. Furthermore, we discuss their co-dysregulation in pathological conditions, e.g., cancer, type-2 diabetes (T2D), and cardiovascular, and neurodegenerative diseases

O-GlcNAcylation Is Involved in the Regulation of Stem Cell Markers Expression in Colon Cancer Cells

The dynamic O-linked-N-acetylglucosamine posttranslational modification of nucleocytoplasmic proteins has emerged as a key regulator of diverse cellular processes including several hallmarks of cancer. However, the role played by this modification in the establishment of CSC phenotype has been poorly studied so far and remains unclear. In this study we confirmed the previous reports showing that colon cancer cells exhibit higher O-GlcNAc basal levels than non-malignant cells, and investigated the role played by O-GlcNAcylation in the regulation of CSC phenotype. We found that the modification of O-GlcNAcylation levels by pharmacological inhibition of the O-GlcNAc-transferase enzyme that adds O-GlcNAc (OGT), but not of the enzyme that removes it (OGA), increased the expression of all stem cell markers tested in our colon malignant cell lines, and induced the appearance of a double positive (CD44+/CD133+) small stem cell-like subpopulation (which corresponded to 1–10%) that displayed very aggressive malignant phenotype such as increased clonogenicity and spheroid formation abilities in 3D culture. We reasoned that OGT inhibition would mimic in the tumor the presence of severe nutritional stress, and indeed, we demonstrated that nutritional stress reproduced in colon cancer cells the effects obtained with OGT inhibition. Thus, our data strongly suggests that stemness is regulated by HBP/O-GlcNAcylation nutrient sensing pathway, and that O-GlcNAc nutrient sensor represents an important survival mechanism in cancer cells under nutritional stressful conditions

Cyclin D1 Stability Is Partly Controlled by O-GlcNAcylation

Cyclin D1 is the regulatory partner of the cyclin-dependent kinases (CDKs) CDK4 or CDK6. Once associated and activated, the cyclin D1/CDK complexes drive the cell cycle entry and G1 phase progression in response to extracellular signals. To ensure their timely and accurate activation during cell cycle progression, cyclin D1 turnover is finely controlled by phosphorylation and ubiquitination. Here we show that the dynamic and reversible O-linked β-N-Acetyl-glucosaminylation (O-GlcNAcylation) regulates also cyclin D1 half-life. High O-GlcNAc levels increase the stability of cyclin D1, while reduction of O-GlcNAcylation strongly decreases it. Moreover, elevation of O-GlcNAc levels through O-GlcNAcase (OGA) inhibition significantly slows down the ubiquitination of cyclin D1. Finally, biochemical and cell imaging experiments in human cancer cells reveal that the O-GlcNAc transferase (OGT) binds to and glycosylates cyclin D1. We conclude that O-GlcNAcylation promotes the stability of cyclin D1 through modulating its ubiquitination

Proteomic analysis of nipple aspirate fluid to detect biologic markers of breast cancer.

The early detection of breast cancer is the best means to minimise disease-related mortality. Current screening techniques have limited sensitivity and specificity. Breast nipple aspirate fluid can be obtained noninvasively and contains proteins secreted from ductal and lobular epithelia. Nipple aspirate fluid proteins are breast specific and generally more concentrated than corresponding blood levels. Proteomic analysis of 1 microl of diluted nipple aspirate fluid over a 5-40 kDa range from 20 subjects with breast cancer and 13 with nondiseased breasts identified five differentially expressed proteins. The most sensitive and specific proteins were 6500 and 15 940 Da, found in 75-84% of samples from women with cancer but in only 0-9% of samples from normal women. These findings suggest that (1) differential expression of nipple aspirate fluid proteins exists between women with normal and diseased breasts, and (2) analysis of these proteins may predict the presence of breast cancer

Transcription profiles of non-immortalized breast cancer cell lines

BACKGROUND: Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. METHODS: Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs) were used in addition to commercially-available normal breast epithelial cells (HMECs), established breast cancer cell lines (T-est) and established normal breast cells (N-est). The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. RESULTS: According to Significance Analysis of Microarray (SAM) and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p < 0.01). Some of these genes have already been directly linked with breast cancer, metastasis and malignant progression, whilst others encode receptors linked to signal transduction pathways or are otherwise related to cell proliferation. Fifty genes showed at least a 2.5-fold difference between MSSMs and T-est cells according to AtlasImage, 2-fold according to SAM. Most of these classified as genes related to metabolism and cell communication. CONCLUSION: The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research

Apport de l'analyse protéomique à l'étude de la signalisation du fibroblast growth factor-2 dans les cellules de cancer du sein

Nous avons etudie la signalisation intracellulaire du fibroblast growth factor-2 dans les cellules de cancer du sein mcf-7 par analyse proteomique basee sur le couplage de l'electrophorese bidimensionnelle et de la spectrometrie de masse (maldi-tof et ms-ms). A l'aide d'inhibiteurs pharmacologiques et d'anticorps, nous avons montre que la stimulation des cellules mcf-7 par le fgf-2 declenche une augmentation de la tyrosine-phosphorylation du recepteur de haute affinite du fgf-2, du fgf receptor substrate-2 , de la kinase src, des p42/p44 mitogen-activated protein kinases (mapk), et d'une proteine de 30 kda. L'analyse en spectrometrie de masse maldi-tof nous a permis d'identifier cette proteine de 30 kda comme etant la cycline d2, element majeur de la regulation du cycle cellulaire. De plus, nos resultats suggerent que la phosphorylation de la cycline d2 est dependante de src mais pas des mapk. Nous avons ensuite etudie les modifications de synthese de proteines induites par le fgf-2 apres incorporation d'acides amines marques au 3 5s. Nous avons montre une augmentation de synthese de 4 proteines ( heat shock proteins hsp90 et hsp70, proliferating cell nuclear antigen (pcna) et transcriptionaly controlled tumor protein (tctp)) qui ont ete identifiees par spectrometrie de masse maldi-tof et ms-ms.De plus, ces 4 proteines sont constitutivement surexprimees dans les cellules mcf-7 transfectees par l'oncogene ras. L'inhibition de la proliferation induite par le fgf-2 par la geldanamycine souligne l'importance de l'hsp90 dans la croissance des cellules de cancer du sein et son emergence comme cible therapeutique potentielle. Enfin, l'analyse proteomique des cellules cancereuses mammaires nous a permis de mettre en evidence pour la premiere fois que la quantite de la proteine 14-3-3 sigma est diminuee dans les cellules cancereuses par rapport aux cellules normales. La 14-3-3 sigma ayant ete tres recemment impliquee dans le controle de la progression du cycle cellulaire, ces donnees suggerent que cette proteine pourrait constituer un nouveau marqueur de cancerisation des cellules du sein. L'ensemble de ces travaux contribue a une meilleure comprehension du mecanisme d'action intracellulaire du fgf-2 dans les cellules de cancer du sein et montre les potentialites de l'analyse proteomique pour l'etude de la signalisation et plus generalement pour la recherche biomedicale.LILLE1-BU (590092102) / SudocSudocFranceF

Etude de la dynamique de O-GlcNAcylation et identification de protéines différentiellement O-GlcNAcylées au cours de la transition G1/S du cycle cellulaire de cellules épithéliales humaines

La O-GlcNAcylation est une glycosylation dynamique et réversible sous le contrôle de la O-GlcNAc Transférase (OGT) qui transfère un résidu de GlcNAc sur les Ser/Thr de protéines intracellulaires, et de la O-GlcNAcase (OGA). Plusieurs travaux dont ceux de notre équipe ont montré l'importance de la dynamique de O-GlcNAcylation pour la progression normale du cycle cellulaire, et plus particulièrement de la mitose. L objectif de mes travaux de thèse était de comprendre comment la balance O-GlcNAc participe au contrôle des étapes précoces du cycle cellulaire. J ai d abord montré dans différentes lignées cellulaires que l entrée en phase S s accompagne d une baisse marquée du niveau de O-GlcNAc, corrélée à une augmentation de l expression et de l activité de l OGA endogène. Par protéomique, 58 protéines cytosoliques et nucléaires différentiellement O-GlcNAcylées à la transition G1/S ont ensuite été identifiées dans les cellules MCF7 synchronisées. Ces protéines interviennent dans des processus cellulaires essentiels à la phase G1 dont la régulation de la transcription, de la traduction et de la mise en conformation des protéines, et de la réplication de l ADN. Par immunoprécipitation, les variations O-GlcNAc dépendantes du cycle cellulaire ont été confirmées sur les protéines cytosoliques CK8, hnRNP K et Caprine 1, et sur les protéines nucléaires du complexe de pré-réplication, MCM-3, -4, -6, et -7. Ces travaux montrent donc que la transition G1/S est étroitement liée à la dynamique de O-GlcNAcylation et soulignent un rôle potentiel de cette glycosylation dans le contrôle de l initiation de la réplication de l ADN et par là même, dans le maintien de l'intégrité génomique.O-GlcNAcylation is a highly dynamic and reversible glycosylation which is governed by O-GlcNAc Transferase (OGT) that transfers the N-acetylglucosamine (GlcNAc) residue onto Ser/Thr of intracellular proteins, and O-GlcNAcase (OGA). Over the last decade, we and others have shown that dynamics of O-GlcNAcylation was important in regulating the cell cycle progression, and more particularly the mitosis events. The aim of my work was to explore how O-GlcNAc balance is implicated in the control of cellular proliferation by focusing on the early steps in the cell cycle. We highlighted in several cell lines that S-phase entry is associated with a marked decrease in the overall level of O-GlcNAcylated proteins, concordant with an increase in both the expression and activity of endogenous OGA. Then, using a proteomic approach we identified 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated between G0, G1 and S phases. These proteins are involved in key cellular functions that are essential for G1 and S progression, such as protein folding and translation, transcription or DNA replication. By immunoprecipitation, we further confirmed the cell cycle-dependent O-GlcNAc variations of CK8, hnRNP K, Caprin-1, and MCM -3, -4, -6, and -7 proteins which are part of the pre-replicative complex. To conclude, this study shows that there is a close link between the dynamics of O-GlcNAc and G1/S transition and provides a descriptive overview of differentially O-GlcNAcylated proteins at the G1/S transition, highlighting a potential role of O-GlcNAcylation in the initiation of DNA synthesis and therefore, in the maintenance of genome integrity.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Le Pollen de Pin d’Alep bio-Indicateur de la pollution atmosphérique

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