Identification of Yeast and Human 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) Transporters

International audienc

Disruption of Nucleotide Homeostasis by the Antiproliferative Drug 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside Monophosphate (AICAR)

International audienceBackground: AICAR is a potent anti-proliferative compound, but the basis of its cytotoxicity is poorly understood. Results: AICAR affects NTP homeostasis in a carbon source-dependent way, in both yeast and human cells. Conclusion: AICAR balance with nucleotides triphosphate is critical for its in vivo effects. Significance: AICAR is significantly more cytotoxic on glucose and thus potentially targets cells prone to Warburg effect

Purification et etude comparee des facteurs de croissance EDFG I et FGF basique : mise en evidence et caracterisation partielle de leur recepteur commun

CNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

Etude du mecanisme d'action des facteurs de croissance "fibroplast growth factors" (FGFs)

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Rôle du capteur de stress ischémique IRE1a dans la croissance du glioblastome

Les glioblastomes (GBMs) sont les tumeurs cérébrales primaires les plus courantes chez l adulte avec un pronostic fatal dans les douze mois suivant le diagnostic. De nouvelles avancées dans la connaissance de la pathologie moléculaire des GBMs et de leurs régulateurs clés sont indispensables à l émergence de nouvelles pistes thérapeutiques. Inositol Requiring Enzyme 1 a (IRE1a) est une protéine résidente du réticulum endoplasmique (RE) agissant en tant que détecteur proximal de l Unfolded Protein Response (UPR) en conditions physiologiques ou pathologiques. IRE1a est une enzyme bivalente possédant une activité Ser/Thr kinase et endoribonucléasique (RNase). Récemment, des mutations ponctuelles dans le gène ERN1/IRE1a ont été détectées dans les cancers chez l homme, (en particulier dans les GBMs), et IRE1a a été proposé comme un régulateur majeur de la progression tumorale parmi les protéines kinases. Dans ce travail, nous avons montré que le blocage des deux activités Set/thr kinase et RNase dans les cellules U87-MG réprime fortement l angiogenèse tumorale, la perfusion des vaisseaux et l expression de facteurs pro-angiogéniques dans des modèles tumoraux de xénogreffes. Ce changement phénotypique est accompagné d une réponse dite d échappement des cellules tumorales. Celles-ci envahissent le tissu cérébral sain par migration de long des vaisseaux (mécanismes appelé co-option vasculaire). De plus, ce phénotype a été montré comme étant fonctionnellement associé au processus de transition mésenchymateuse. Par mutagenèse dirigée, nous avons montré qu IRE1a module les processus d angiogenèse et d invasion par ces deux domaines catalytiques : l activité Ser/Thr kinase d IRE1a est essentielle pour l angiogenèse alors que le domaine RNase d IRE1a contrôle le phénotype invasif et co-opté. IRE1a est ainsi identifié comme un régulateur clé de la croissance du glioblastome, agissant au carrefour de signalisations majeures dans le contrôle de l adaptation de la cellule tumorale à son micro-environnement.Glioblastomas (GBMs) are the most common primary brain tumors in humans and remain essentially incurable. New advances in the knowledge of GBM molecular pathology and their key regulators are crucial to identify new putative ways for GBM therapy. Inositol Requiring Enzyme 1 a (IRE1a) is a transmembrane Endoplasmic Reticulum (ER)-resident protein acting as proximal sensor of the Unfolded Protein Response (UPR) in both physiological and pathological situations. IRE1a is a bivalent enzyme, displaying Ser/Thr kinase and endoribonuclease (RNase) activities in its cytosolic side. Recently, single mutations in IRE1a gene were detected in human cancers, including GBM, and IRE1a was proposed as a major contributor to tumor progression among protein kinases. In this work, we have shown that blockade of both IRE1a Ser/Thr kinase and RNase activities in U87-MG cells highly repressed tumor angiogenesis, blood perfusion and the expression of pro-angiogenic factors in human xenograft tumor models. This phenotypic change is adversely associated to the so-called "evasive response" of tumors cells. The cells began to migrate along pre-existing brain capillaries and invade healthy tissue (a process named blood vessel co-option). Moreover, this phenotype was shown to be functionally linked to the mesenchymal differentiation process. By using site-directed mutagenesis, we demonstrated that IRE1a protein modulates both angiogenesis and invasive processes through its two catalytic domains: IRE1a Ser/Thr kinase domain was essential for IRE1-mediated angiogenesis, whereas IRE1's RNase domain drove the invasive, co-opted phenotype. IRE1a is therefore identified as a key regulator of glioma progression, acting at the crossroads of major signaling networks in the control of tumor cell adaptation to its microenvironment.BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

Blood vessel morphogenesis: Evidence for angiogenin receptors on bovine aortic smooth muscle cells

International audienc

Régulation de l activité biologique de la protéine IRE1 (rôle dans le développement des cancers)

Le Réticulum endoplasmique (RE) est le premier compartiment intracellulaire traversé par les protéines sécrétées. Au sein de cet organite, les protéines acquièrent une conformation native, et subissent de nombreuses modifications post-traductionnelles telles que la N-glycosylation ou la formation de ponts disulfures. Dans certaines conditions (stress réducteurs, hypoxie, privation en glucose ) des protéines anormalement conformées s accumulent au sein du RE ce qui conduit à l induction de l Unfolded Protein Response (UPR). Cette réponse va alors tout d abord induire l inhibition de la traduction, ce qui limite l entrée de nouvelles protéines dans le RE. En parallèle, un programme transcriptionnel spécifique conduit à l augmentation de l expression de protéines impliquées dans le repliement et la dégradation des protéines accumulées dans la lumière du RE. Cette réponse adaptative intégrée est contrôlée principalement par 3 protéines transmembranaires du RE : PERK (PKR-related ER kinase), ATF6 (Activating transcription factor) et enfin IRE1 (Inositol requiring kinase 1) sur laquelle porte notre étude. Au cours de ma thèse, j ai tout d abord participé à une étude démontrant que l activation des voies de signalisation dépendantes d IRE1 contribuait à la surexpression du VEGF-A in vitro et régulait l angiogenèse et la croissance tumorale in vivo dans un modèle de greffe orthotopique de cellules U87 dérivées de gliomes humains. Cette protéine pourrait donc constituer une cible thérapeutique potentielle. Ces résultats nous ont par conséquent amenés à identifier des modulateurs de l activité de la protéine IRE1. Pour cela nous avons développé un test in vitro permettant d évaluer l étape essentielle dans l activation de la protéine IRE1, sa dimérisation. Ce test nous a permis d identifier un peptide capable d interférer dans la formation des dimères de la protéine IRE1, mais aussi et de façon inattendue, d accroître son activité endoribonucléase in vitro et in vivo. Ainsi, nous proposons que ce peptide interfacial issu du domaine kinase de la protéine IRE1 pourrait promouvoir un changement conformationnel du domaine cytosolique de la protéine entière et par conséquent, potentialiserait de façon significative son activité endoribonucléasique. Ce modulateur identifié pourrait donc représenter un nouvel outil à potentiel thérapeutique utilisable par exemple dans des maladies conformationnelles.The endoplasmic Reticulum (ER) is the first intracellular compartment encountered by secretory proteins. In this organelle proteins acquire their correct conformation and undergo many post-translational modifications such as N-glycosylation or disulphide bond formation. Under specific environmental conditions (reductive stress, hypoxia, glucose deprivation ), protein folding is perturbed and uncorrectly folded proteins accumulate in the lumen of the ER. This leads to the activation of an adaptive response named the Unfolded Protein Response (UPR). The UPR consists in an attenuation of protein translation and an activation of a specific transcriptional program. This integrated adaptive response is mediated by 3 transmembrane ER resident proteins: PERK (PKR-related ER kinase), ATF6 (Activating transcription Factor) and IRE1 (Inositol requiring kinase 1) and we focused more particularly on IRE1. During my PhD thesis, I participated to a study that demonstrated the role of IRE1 signaling in the regulation of VEGF expression in vitro and tumor growth and angiogenesis in vivo. The latter was carried out using a ortotopic implantation model of human glioma-derived cells. As a consequence IRE1 could certainly constitute a potential therapeutic target. In an attempt to modulate IRE1 activity, we aimed at identifying artificial modulators of its activity. To this end, we designed an in vitro assay capable of monitoring the first essential step in IRE1 activation process, namely its dimerization. This assay allowed us to identify a peptide able to interfere with IRE1 dimer formation, but, unexpectedly, to also increase its RNAse activity in vitro and in vivo. We propose that this interfacial peptide, derived from IRE1 kinase domain could promote a conformational change in IRE1 cytosolic domain and consequently lead to an increase in its enzymatic activity. This modulator could represent a new tool with therapeutic potential that could then be used in protein misfolding diseases for instance.BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

Use of synchrotron-radiation-based FTIR imaging for characterizing changes in cell contents

FTIR imaging of individual cells is still limited by the low signal-to-noise ratio obtained from analysis of such weakly absorbing organic matter when using a Globar IR source. In this study, we used FTIR imaging with a synchrotron radiation source and a focal plane array detector to determine changes in the cellular contents of cryofixed cells after culture for 48 h on Si3N4 substrate. Several spectral differences were observed for cells deprived of glucose compared with control cells: a lower amide I-to-amide II ratio (P < 0.01); a different secondary structure profile of proteins (obtained from amide I spectral region curve fitting), with a significant increase in non-ordered structure components (P < 0.01); and a higher nu(C = C-H)/nu (as)(CH3) absorption ratio (P < 0.01), suggesting increased unsaturation of fatty acyl chains. Therefore, our study has shown that FTIR imaging with a synchrotron radiation source enables determination of several spectral changes of individual cells between two experimental conditions, which thus opens the way to cell biology studies with this vibrational spectroscopy technique

Blood and Synovial Distribution of Angiogenin in Rheumatic Diseases

International audienc

Hagedorn M: Inhibition of angiogenesis and the angiogenesis/invasion shift

Abstract Angiogenesis has become a major target in cancer therapy. However, current therapeutic strategies have their limitations and raise several problems. In most tumours, anti-angiogenesis treatment targeting VEGF (vascular endothelial growth factor) has only limited overall survival benefit compared with conventional chemotherapy alone, and reveals several specific forms of resistance to anti-VEGF treatment. There is growing evidence that anti-VEGF treatment may induce tumour cell invasion by selecting highly invasive tumour cells or hypoxia-resistant cells, or by up-regulating angiogenic alternative pathways such as FGFs (fibroblast growth factors) or genes triggering new invasive programmes. We have identified new genes upregulated during glioma growth on the chick CAM (chorioallantoic membrane). Our results indicate that antiangiogenesis treatment in the experimental glioma model drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. We have identified a molecular mechanism in tumour cells that allows the switch from an angiogenic to invasive programme. Furthermore, we are focusing our research on alternative inhibitors that act, in part, independently of VEGF. These are endogenous molecules that play a role in the control of tumour growth and may constitute a starting point for further development of novel therapeutic or diagnostic tools