Etude des conséquences physiologiques de l'utilisation de la thymidylate synthase ThyX.

The conversion of deoxyuridine 5'-monophosphate (dUMP) to deoxythymidine 5'-monophosphate (dTMP) by thymidylate synthases is one key step in the biosynthesis of pyrimidine deoxyribonucleotides. Two non homologous families of thymidylate synthases exist in the prokaryotic world (bacteria and archaea): ThyA and ThyX. The almost exclusive phylogenetic distribution of both enzyme families is complex and does not follow organismal phylogenetic relations. Proteins of both families do not share sequence or structural similarities, and the catalytic mechanisms and efficiencies are different. The work presented in this thesis demonstrates that ThyX activity is essential for the survival of the bacterium Rhodobacter capsulatus in absence of exogenous thymidine. Based upon the results of genetic complementation assays and a mathematical model of the folate metabolism, we showed that a very low dihydrofolate reductase activity is enough to allow survival of thyX containing organisms. Moreover, using a genetic approach and statistical analysis of more than 400 prokaryotic genomes, I was able to show that organisms using thymidylate synthase ThyX have slow DNA replication and growth rates, and mostly possess a small genome. Based upon these findings, I propose a model in which the weak activity of thymidylate synthases ThyX is limiting DNA replication and consequently genome expansion. Finally, I studied the pyrimidine metabolism in the human pathogenic bacterium Helicobacter pylori. In particular, I demonstrated that this bacterium is sensible to the anticancer drug ! 5-fluorouracil (5-FU) despite the absence of a uracil salvage pathway. I showed that the H. pylori orotate phosphoribosyltransferase is able to complement the uracil auxotrophy of an Escherichia coli strain, indicating that this enzyme could be responsible for the 5-FU sensitivity of H. pylori. The studies presented in this thesis allow a better understanding of the physiological consequences of the use of thymidylate synthase ThyX and provide an explanation for the apparent sporadic distribution of both thymidylate synthase families in the prokaryotic world.La conversion par les thymidylate synthases du désoxyuridine 5'-monophosphate (dUMP) en désoxythymidine 5'-monophosphate (dTMP) est une des étapes clés de la biosynthèse des désoxyribonucléotides pyrimidiques. Chez les procaryotes (bactéries et archées), il existe deux familles de thymidylate synthases non homologues : ThyA et ThyX. La distribution phylogénétique quasi-exclusive des deux familles est complexe et ne suit pas les relations phylogéniques entre les organismes. Les protéines de ces deux familles ne présentent aucune similarité de séquence, de structure et leurs mécanismes et efficacités catalytiques diffèrent. Les travaux présentés dans cette thèse ont permis de montrer que l'activité de ThyX est essentielle à la survie de la bactérie Rhodobacter capsulatus en absence de thymidine exogène. A partir des résultats de tests de complémentation génétique et de la modélisation mathématique du métabolisme des folates nous avons pu mettre en évidence que seulement une faible activité dihydrofolate réductase était nécessaire à la survie des organismes utilisant ThyX. D'autre part, j'ai pu montrer par des approches génétiques et par l'étude statistique de plus de 400 génomes procaryotes que les organismes utilisant ThyX ont une réplication et un taux de croissance lents, et possèdent majoritairement un petit génome. Je propose un modèle dans lequel la faible activité catalytique de ThyX limite la réplication de l'ADN e! t par conséquent l'expansion du génome. Enfin, j'ai étudié le métabolisme des pyrimidines chez la bactérie pathogène humaine, Helicobacter pylori. J'ai pu en particulier mettre en évidence qu'en absence de voie de récupération de l'uracile, cette bactérie est capable de métaboliser un analogue toxique le 5-fluorouracile (5FU), utilisé comme anticancéreux. J'ai par ailleurs montré que l'orotate phosphoribosyltransférase de H. pylori était capable de restaurer l'auxotrophie pour l'uracile d'une souche d'Escherichia coli indiquant que cette enzyme pourrait être responsable de la sensibilité de H. pylori au 5FU. Les travaux présentés dans cette thèse ont permis une meilleure compréhension des conséquences physiologiques de l'utilisation de la thymidylate synthase ThyX. Ils ont notamment permis d'expliquer la distribution apparemment sporadique des deux familles de thymidylate synthases dans le monde procaryote

Etude des conséquences physiologiques de l'utilisation de la thymidylate synthase ThyX

PALAISEAU-Polytechnique (914772301) / SudocSudocFranceF

Flavin-dependent thymidylate synthase X limits chromosomal DNA replication

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IGF-1 exacerbates the neurotoxicity of the mitochondrial inhibitor 3NP in rats

International audienceInsulin-like Growth Factor 1 (IGF-1) has broad-range neuroprotective effects and is a therapeutic candidate for Huntington’s disease (HD). IGF-1 protects striatal neurons from the toxicity of mutated huntingtin $in\ vitro$ and improves neuronal survival $in\ vivo$ in a phenotypic model of HD involving excitotoxic cell death. Because HD is a multifactorial disease, it is important to evaluate the neuroprotective role of IGF-1 in other pathological situations involved in HD progression. We have evaluated the neuroprotective effects of IGF-1 $in\ vivo$, using the 3-nitropropionic acid (3NP) rat model which replicates the mitochondrial dysfunction observed in HD. Continuous intracerebroventricular infusion of recombinant IGF-1 at a low dose (0.025 $\mu$g/h for 5 days) did not alleviate motor impairment and weight loss induced by 3NP treatment. In addition, histological evaluation and quantification of DNA fragmentation evidenced no improvement in neuronal survival. Of interest, we found that a higher concentration of IGF-1 (0.25 $\mu$g/h) resulted in an exacerbation of 3NP toxicity on striatal neurons. These results suggest that intracerebral delivery of IGF-1 may not provide a fully effective therapeutic strategy for HD or other disorders involving mitochondrial impairmen

Insulin growth factor-1 protects against excitotoxicity in the rat striatum

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Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria▿ †

Although flavin-dependent ThyX proteins show thymidylate synthase activity in vitro and functionally complement thyA defects in heterologous systems, direct proof of their cellular functions is missing. Using insertional mutagenesis of Rhodobacter capsulatus thyX, we constructed the first defined thyX inactivation mutant. Phenotypic analyses of the obtained mutant strain confirmed that R. capsulatus ThyX is required for de novo thymidylate synthesis. Full complementation of the R. capsulatus thyX::spec strain to thymidine prototrophy required not only the canonical thymidylate synthase ThyA but also the dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibited the growth of the different Rhodobacter strains used in this work. To rationalize these experimental results, we used a mathematical model of bacterial folate metabolism. This model suggests that a very low dihydrofolate reductase activity is enough to rescue significant thymidylate synthesis in the presence of ThyX proteins and is in agreement with the notion that intracellular accumulation of folates results in growth inhibition. In addition, our observations suggest that the presence of flavin-dependent thymidylate synthase X provides growth benefits under conditions in which the level of reduced folate derivatives is compromised

Reactive astrocytes mediate TSPO overexpression in response to sustained CNTF exposure in the rat striatum

International audienceThe 18 kDa translocator protein (TSPO) is a classical marker of neuroinflammation targeted for in vivo molecular imaging. Microglial cells were originally thought to be the only source of TSPO overexpression but astrocytes, neurons and endothelial cells can also up-regulate TSPO depending on the pathological context. This study aims to determine the cellular origin of TSPO overexpression in a simplified model of neuroinflammation and to identify the molecular pathways involved. This is essential to better interpret TSPO molecular imaging in preclinical and clinical settings. We used lentiviral vectors (LV) to overexpress the ciliary neurotrophic factor (CNTF) in the right striatum of 2-month-old Sprague Dawley rats. A LV encoding for β-Galactosidase (LV-LacZ) was used as control. One month later, TSPO expression was measured by single-photon emission computed tomography (SPECT) imaging using [ 125 I]CLINDE. The fluorescence-activated cell sorting to radioligand-treated tissue (FACS-RTT) method was used to quantify TSPO levels in acutely sorted astrocytes, microglia, neurons and endothelial cells. A second cohort was injected with LV-CNTF and a LV encoding suppressor of cytokine signaling 3 (SOCS3), to inhibit the JAK-STAT3 pathway specifically in astrocytes. GFAP and TSPO expressions were quantified by immunofluorescence. We measured a significant increase in TSPO signal in response to CNTF by SPECT imaging. Using FACS-RTT, we observed TSPO overexpression in reactive astrocytes (+ 153 ± 62%) but also in microglia (+ 2088 ± 500%) and neurons (+ 369 ± 117%), accompanied by an increase in TSPO binding sites per cell in those three cell populations. Endothelial cells did not contribute to TSPO signal increase. Importantly, LV-SOCS3 reduced CNTF-induced astrocyte reactivity and decreased global TSPO immunoreactivity (-71% ± 30%), suggesting that TSPO overexpression is primarily mediated by reactive astrocytes. Overall, this study reveals that CNTF induces TSPO in multiple cell types in the rat striatum, through the JAK2-STAT3 pathway in astrocytes, identifying this cell type as the primary mediator of CNTF effects neuroinflammatory processes. Our results highlight the difficulty to interpret TSPO imaging in term of cellular origin without addition cellular analysis by FACS-RTT or quantitative immunostainings. Consequently, TSPO should only be used as a global marker of neuroinflammation

In VivoIn\ Vivo Calpain/Caspase Cross-talk during 3-Nitropropionic Acid-induced Striatal Degeneration

International audienceThe role of caspases and calpains in neurodegeneration remains unclear. In this study, we focused on these proteases in a rat model of Huntington's disease using the mitochondrial toxin 3-nitropropionic acid (3NP). Results showed that 3NP-induced death of striatal neurons was preceded by cytochrome c redistribution, transient caspase-9 processing, and activation of calpain, whereas levels of the active/processed form of caspase-3 remained low and were even reduced as compared with control animals. We evidenced here that this decrease in active caspase-3 levels could be attributed to calpain activation. Several observations supported this conclusion. 1) Pharmacological blockade of calpain in 3NP-treated rats increased the levels of endogenous processed caspase-9 and caspase-3. 2) Cell-free extracts prepared from the striatum of 3NP-treated rats degraded $in\ vitro$ the p34 and p20 subunits of active recombinant caspase-9 and caspase-3, respectively. 3) This degradation of p34 and p20 could be mimicked by purified $\mu$-calpain and was prevented by calpain inhibitors. 4) $\mu$-Calpain produced a loss of the DEVDase (Asp-Glu-Val-Asp) activity of active caspase-3. 5) Western blot analysis and experiments with $^{35}$S-radiolabeled caspase-3 showed that $\mu$-calpain cleaved the p20 subunit of active caspase-3 near its catalytic site. 6) $\mu$-Calpain activity was selectively inhibited (IC$_{50}$ of 100 $\mu$M) by a 12 amino acid peptide corresponding to the C terminus of p20. Our results showed that calpain can down-regulate the caspase-9/caspase-3 cell death pathway during neurodegeneration due to chronic mitochondrial defects $in\ vivo$ and that this effect may involve, at least in part, direct cleavage of the caspase-3 p20 subunit