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1.
Front Endocrinol (Lausanne) ; 13: 949097, 2022.
Article in English | MEDLINE | ID: mdl-35992129

ABSTRACT

Pancreatic beta cell response to glucose is critical for the maintenance of normoglycemia. A strong transcriptional response was classically described in rodent models but, interestingly, not in human cells. In this study, we exposed human pancreatic beta cells to an increased concentration of glucose and analysed at a global level the mRNAs steady state levels and their translationalability. Polysome profiling analysis showed an early acute increase in protein synthesis and a specific translation regulation of more than 400 mRNAs, independently of their transcriptional regulation. We clustered the co-regulated mRNAs according to their behaviour in translation in response to glucose and discovered common structural and sequence mRNA features. Among them mTOR- and eIF2-sensitive elements have a predominant role to increase mostly the translation of mRNAs encoding for proteins of the translational machinery. Furthermore, we show that mTOR and eIF2α pathways are independently regulated in response to glucose, participating to a translational reshaping to adapt beta cell metabolism. The early acute increase in the translation machinery components prepare the beta cell for further protein demand due to glucose-mediated metabolism changes.


Subject(s)
Eukaryotic Initiation Factor-2 , Insulin-Secreting Cells , Blood Glucose/metabolism , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Glucose/metabolism , Glucose/pharmacology , Humans , Insulin-Secreting Cells/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
2.
FASEB J ; 34(1): 571-587, 2020 01.
Article in English | MEDLINE | ID: mdl-31914586

ABSTRACT

Beyond the study of its transcriptional target genes, the identification of the various interactors of a transcription factor (TF) is crucial to understand its diverse cellular roles. We focused on FOXL2, a winged-helix forkhead TF important for ovarian development and maintenance. FOXL2 has been implicated in diverse cellular processes, including apoptosis, the control of cell cycle or the regulation of steroid hormone synthesis. To reliably identify partners of endogenous FOXL2, we performed a proteome-wide analysis using co-immunoprecipitation in the murine granulosa cell-derived AT29c and the pituitary-derived alpha-T3 cell lines, using three antibodies targeting different parts of the protein. Following a stringent selection of mass spectrometry data on the basis of identification reliability and protein enrichment, we identified a core set of 255 partners common to both cell lines. Their analysis showed that we could co-precipitate several complexes involved in mRNA processing, chromatin remodeling and DNA replication and repair. We further validated (direct and/or indirect) interactions with selected partners, suggesting an unexpected role for FOXL2 in those processes. Overall, this comprehensive analysis of the endogenous FOXL2 interactome sheds light on its numerous and diverse interactors and unconventional cellular roles.


Subject(s)
Forkhead Box Protein L2/metabolism , Granulosa Cells/metabolism , Pituitary Gland/metabolism , Protein Interaction Maps , Proteome/metabolism , Animals , Cells, Cultured , Female , Granulosa Cells/cytology , Mice , Pituitary Gland/cytology , Proteome/analysis
3.
J Cell Sci ; 127(Pt 11): 2420-32, 2014 Jun 01.
Article in English | MEDLINE | ID: mdl-24652832

ABSTRACT

Epithelial junctions are fundamental determinants of tissue organization, subject to regulation by tyrosine phosphorylation. Homophilic binding of E-cadherin activates tyrosine kinases, such as Src, that control junctional integrity. Protein tyrosine phosphatases (PTPs) also contribute to cadherin-based adhesion and signaling, but little is known about their specific identity or functions at epithelial junctions. Here, we report that the receptor PTP RPTPα (human gene name PTPRA) is recruited to epithelial adherens junctions at the time of cell-cell contact, where it is in molecular proximity to E-cadherin. RPTPα is required for appropriate cadherin-dependent adhesion and for cyst architecture in three-dimensional culture. Loss of RPTPα impairs adherens junction integrity, as manifested by defective E-cadherin accumulation and peri-junctional F-actin density. These effects correlate with a role for RPTPα in cellular (c)-Src activation at sites of E-cadherin engagement. Mechanistically, RPTPα is required for appropriate tyrosine phosphorylation of cortactin, a major Src substrate and a cytoskeletal actin organizer. Expression of a phosphomimetic cortactin mutant in RPTPα-depleted cells partially rescues F-actin and E-cadherin accumulation at intercellular contacts. These findings indicate that RPTPα controls cadherin-mediated signaling by linking homophilic E-cadherin engagement to cortactin tyrosine phosphorylation through c-Src.


Subject(s)
Adherens Junctions/metabolism , Cadherins/metabolism , Epithelial Cells/physiology , Receptor-Like Protein Tyrosine Phosphatases, Class 4/metabolism , src-Family Kinases/metabolism , Actins/metabolism , Adherens Junctions/genetics , CSK Tyrosine-Protein Kinase , Caco-2 Cells , Cell Adhesion/genetics , Cortactin/genetics , Cortactin/metabolism , HEK293 Cells , Humans , Mutation/genetics , Organ Culture Techniques , Organogenesis/genetics , Phosphorylation/genetics , Protein Transport , RNA, Small Interfering/genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 4/genetics , Signal Transduction/genetics
4.
PLoS One ; 8(6): e65371, 2013.
Article in English | MEDLINE | ID: mdl-23785422

ABSTRACT

Alterations in function of the neurotrophin BDNF are associated with neurodegeneration, cognitive decline, and psychiatric disorders. BDNF promotes axonal outgrowth and branching, regulates dendritic tree morphology and is important for axonal regeneration after injury, responses that largely result from activation of its tyrosine kinase receptor TrkB. Although intracellular neurotrophin (NT) signaling presumably reflects the combined action of kinases and phosphatases, little is known about the contributions of the latter to TrkB regulation. The issue is complicated by the fact that phosphatases belong to multiple independently evolved families, which are rarely studied together. We undertook a loss-of-function RNA-interference-based screen of virtually all known (254) human phosphatases to understand their function in BDNF/TrkB-mediated neurite outgrowth in differentiated SH-SY5Y cells. This approach identified phosphatases from diverse families, which either positively or negatively modulate BDNF-TrkB-mediated neurite outgrowth, and most of which have little or no previously established function related to NT signaling. "Classical" protein tyrosine phosphatases (PTPs) accounted for 13% of the candidate regulatory phosphatases. The top classical PTP identified as a negative regulator of BDNF-TrkB-mediated neurite outgrowth was PTPN12 (also called PTP-PEST). Validation and follow-up studies showed that endogenous PTPN12 antagonizes tyrosine phosphorylation of TrkB itself, and the downstream activation of ERK1/2. We also found PTPN12 to negatively regulate phosphorylation of p130cas and FAK, proteins with previously described functions related to cell motility and growth cone behavior. Our data provide the first comprehensive survey of phosphatase function in NT signaling and neurite outgrowth. They reveal the complexity of phosphatase control, with several evolutionarily unrelated phosphatase families cooperating to affect this biological response, and hence the relevance of considering all phosphatase families when mining for potentially druggable targets.


Subject(s)
Neurites/physiology , Phosphoric Monoester Hydrolases/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 12/metabolism , Receptor, trkB/metabolism , Brain-Derived Neurotrophic Factor/pharmacology , Cell Differentiation/drug effects , Cell Line , Crk-Associated Substrate Protein/metabolism , Drug Evaluation, Preclinical/methods , Focal Adhesion Kinase 1/metabolism , Gene Knockdown Techniques , Humans , Neurites/drug effects , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Phenotype , Phosphoric Monoester Hydrolases/genetics , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 12/genetics , RNA Interference , Reproducibility of Results , Signal Transduction , Tretinoin/pharmacology
5.
Antivir Ther ; 12(2): 179-87, 2007.
Article in English | MEDLINE | ID: mdl-17503660

ABSTRACT

BACKGROUND: Zidovudine (3'-azido-3'-deoxythymidine, AZT), administered to pregnant women alone or in combination with other antiretroviral drugs, greatly reduces the mother-to-child transmission of HIV-1. The potential genotoxicity of these molecules is underestimated and wide-ranging evaluation of its biological and clinical consequences is required. METHODS: We investigated the nuclear organization of constitutive heterochromatin, a major domain participating in epigenetic regulation, in uninfected infants born to HIV-1-infected mothers treated with zidovudine and/or other nucleoside reverse transcriptase inhibitors (NRTIs) during pregnancy. We studied the organization of chromosome 1 heterochromatin (1q12) in peripheral leukocytes of 25 HIV-1-uninfected children (newborn to 9 years old): children born to HIV-1-infected mothers exposed to zidovudine and/or other NRTIs (n=15), children born to HIV-1-infected mothers not exposed to any NRTIs (n=6) and children born to HIV-1-uninfected mothers (n=4). RESULTS: Results differed significantly between NRTI-exposed and -unexposed children. By contrast, there was no difference between NRTI-unexposed children born to HIV-1-infected mothers and children born to HIV-uninfected mothers. The anomaly persisted in lymphocytes cultured for 48 h. There was no evidence of abnormal DNA methylation, a major feature of constitutive heterochromatin and associated with the loss of its structure. In a complementary sample of children, analysis of chromosome 11 and 16 heterochromatin suggests that the defect affects most of the other heterochromatic sites of the human genome. The heterochromatin defect persists long after the end of the exposure and appears in leukocytes of both myeloid and lymphoid lineages, suggesting that haematopoietic stem cells are affected.


Subject(s)
Anti-HIV Agents/adverse effects , HIV Infections/drug therapy , HIV-1 , Heterochromatin/drug effects , Leukocytes/drug effects , Prenatal Exposure Delayed Effects , Reverse Transcriptase Inhibitors/adverse effects , Zidovudine/adverse effects , Adolescent , Case-Control Studies , Cells, Cultured , Child , Child, Preschool , Chromatin Assembly and Disassembly/drug effects , Chromosomes, Human, Pair 1/drug effects , Chromosomes, Human, Pair 11/drug effects , Chromosomes, Human, Pair 16/drug effects , Cohort Studies , DNA Methylation/drug effects , Female , HIV Infections/virology , Humans , In Situ Hybridization, Fluorescence , Infant , Infant, Newborn , Pregnancy , Time Factors
6.
J Lipid Res ; 43(3): 453-62, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11893782

ABSTRACT

Hypercholesterolemia is characterized by elevated plasma levels of LDL in which the cholesteryl ester (CE)-rich LDL subclasses of light and intermediate density (LDL1+2 and LDL3, respectively) typically predominate. The molecular mechanisms implicated in oxidation of LDL particle subclasses in hypercholesterolemia are indeterminate. Lipid hydroperoxides (LOOH), primary oxidation products in LDL, are implicated in atherogenesis. LOOH formation was evaluated in light (LDL1+2), intermediate (LDL3), and dense (LDL4+5) LDL subclasses from hypercholesterolemic (HC) subjects (n = 7) during copper-mediated oxidative stress, and compared with that in corresponding subclasses from normolipidemic subjects (n = 7). HC LDL subclasses were distinguished by lower polyunsaturated phospholipid-alpha-tocopherol ratios (P < 0.02), lower contents of phosphatidyl choline (PC)16:0-18:0/18:2 and PC16:0-18:0/20:4+22:6 (P < 0.002), and higher surface phospholipid-free cholesterol ratios (P < 0.04). The LDL3, LDL4, and LDL5 subclasses in HC subjects displayed low-core polyunsaturated CE-alpha-tocopherol ratios (P < 0.05), despite similar PUFA CE content. These physicochemical differences did not modify the oxidative susceptibility of HC LDL but underlie the marked instability of cholesterol linoleate hydroperoxides in HC LDL1+2, LDL3, and LDL4 subclasses. Elevated concentrations of large, CE-rich, light, and intermediate LDL subclasses (LDL1+2, LDL3) in hypercholesterolemia may therefore act as an abundant proatherogenic source of highly unstable LOOH in the arterial wall.


Subject(s)
Hypercholesterolemia/blood , Lipoproteins, LDL/blood , Apolipoprotein B-100 , Apolipoproteins B/metabolism , Cholesterol Esters/blood , Cholesterol Esters/metabolism , Copper/metabolism , Esterification , Fatty Acids/blood , Fatty Acids, Unsaturated/blood , Fatty Acids, Unsaturated/metabolism , Female , Humans , Lipid Peroxides/blood , Lipid Peroxides/metabolism , Lipoproteins, LDL/chemistry , Lipoproteins, LDL/metabolism , Male , Oxidation-Reduction , Particle Size , Phosphatidylcholines/blood , Phosphatidylcholines/metabolism , Phospholipids/blood
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