Utilisation du logiciel R pour l'identification de nouvelles cibles et régulateurs du protéasome

Utilisation du logiciel R pour l'identification de nouvelles cibles et régulateurs du protéasom

Etudes fonctionnelles et biophysiques de Hug1 ; une protéine intrinsèquement désordonnée impliquée dans le métabolisme des nucléotides

Face aux agressions constantes que subit l ADN, les cellules ont développé des mécanismes de protection, nommés checkpoints pour maintenir l intégrité de leur génome. Chez Saccharomyces cerevisiae, la kinase Rad53 joue un rôle central dans ces voies et son activation conduit à de nombreux effets cellulaires tels que le ralentissement du cycle cellulaire, le ralentissement de la réplication, l activation de la transcription de certains gènes, l activation de la réparation Lors d un crible transcriptomique, utilisant une souche exprimant une forme hyperactive de Rad53, nous avons identifié le gène HUG1 comme l un des gènes les plus transcrits suite à l activation de la voie RAD53. Cependant les fonctions de Hug1 demeurent énigmatiques.Pour mieux comprendre les fonctions de Hug1 dans la réponse aux dommages de l ADN, nous avons recherché ses partenaires physiques et avons identifié les protéines Rnr2 et Rnr4, les deux composants de la petite sous-unité de la Ribonucléotide Réductase (RNR). La RNR est un complexe enzymatique qui catalyse l étape limitante de synthèse des nucléotides. Nous avons alors cherché à caractériser cette interaction par diverses méthodes. Nous avons ainsi montré que Hug1 est une protéine intrinsèquement désordonnée capable d interagir physiquement avec la petite sous-unité de la RNR et qu au moins onze acides aminés de Hug1 sont impliqués dans son interaction avec la RNR. Lors de nos investigations, nous avons observé que le fait d étiqueter Rnr2 en position C-terminale sensibilisait les souches aux stress génotoxiques et que cette sensibilité était supprimée si on abrogeait la fonction de HUG1, faisant de Hug1 un nouvel inhibiteur de la RNR. Ainsi nous sommes parvenus à proposer un modèle de régulation de la RNR par Hug1.To maintain genome integrity, cells have developed protection mechanisms, called checkpoints, in response to DNA damage insults. In Saccharomyces cerevisiae, Rad53 protein kinase is one of the major actors in these mechanisms, and its activation triggers several cellular responses such as cell cycle delay, replication delay, transcription modifications, activation of DNA repair pathways Using an hyperactivative allele of RAD53, we identified HUG1, as one of the most induced gene in a transcriptomic analysis upon RAD53 pathway activation. However Hug1 s functions remains elusive.To better understand Hug1 s functions in DNA damage response, we searched for physical partners and identified Rnr2 and Rnr4 proteins, which are the two small subunits of Ribonucleotide Reductase (RNR). The RNR is an enzymatic complex that catalyses nucleotide reduction, a step limiting for dNTPs synthesis. We next experimentally tackled the Hug1-RNR interaction using various methods. We showed so that Hug1 is a small intrinsically disordered protein able to interact physically with the small RNR subunit and that at least eleven amino acids in Hug1 are involved in this interaction. During our investigations, we observed that C-terminal tagging of Rnr2 sensitizes strains to genotoxics stress and that this sensitivity was suppressed when HUG1 s function is abrogated. Hence, we showed that Hug1 is a negative RNR regulator and propose a model for Hug1 s function.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Identification et caractérisation de nouveaux facteurs d'assemblage du protéasome 26S chez la levure Saccharomyces cerevisiae

Chez S. cerevisiae, Rad53 occupe une place centrale au sein des checkpoints de l ADN qui coordonnent les réponses cellulaires aux dommages de l ADN et au blocage de la réplication. Afin d identifier de nouveaux activateurs ou substrats de Rad53, nous avons recherché, à l échelle du génome, les gènes qui suppriment la toxicité d un allèle dominant létal de RAD53 lorsqu ils sont inactivés. 110 gènes ont été isolés et classés en groupes fonctionnels. Un groupe composé de huit gènes dont l inactivation confère à la cellule une hyper-résistance à plusieurs stress génotoxiques a retenu notre attention. Trois de ces gènes codent des composants du protéasome 26S, l enzyme central du système de dégradation ubiquitine-dépendante des protéines. Le 26S est composé d une partie catalytique 20S associée au complexe régulateur 19S, lui même formé de 2 sous-complexes, la base et le couvercle. Avant le crible, un seul chaperon du protéasome était connu chez la levure, la protéine Ump1, impliquée dans la maturation du 20S. Par des analyses génétiques et biochimiques, nous avons caractérisé les cinq autres membres du groupe fonctionnel protéasome . Les gènes POC1-4 (Proteasome Chaperone) codent 4 protéines formant deux paires de chaperons du protéasome 20S (Poc1-Poc2 et Poc3-Poc4) agissant en amont de Ump1. HSM3 code la première protéine chaperon de la particule régulatrice du protéasome. Hsm3 assiste l assemblage de la base du 19S et régule l association du 19S en formation avec le protéasome 20S. Nous avons identifié les homologues mammifères de Poc1-4 (PAC1-4) et Hsm3 (S5b), révélant ainsi une remarquable conservation des facteurs d assemblage du protéasome au cours de l évolution.In S. cerevisiae, Rad53 plays a key role in DNA checkpoints which coordinate several processes of the DNA damage response. In order to identify new activators and substrates of Rad53, we carried out a genome-wide screen for genes whose deletion could suppress the toxicity of a dominant-lethal form of RAD53. 110 suppressor genes have been isolated and classified into functional groups. We further analyzed one of these groups, which is made of 8 genes that lead to hyperresistance to genotoxic stress when they are inactivated. Three genes encode components of the 26S proteasome, the central enzyme of ubiquitin-dependant proteolysis. Proteasome comprises the catalytic core particle (20S) and the regulatory particle (19S), itself composed of two subcomplexes, the base and the lid. At the time of our screen, only one proteasome chaperone was known in yeast, Ump1, which participates in 20S proteasome maturation. By combining genetical and biochemical analyses, we have assigned a molecular function to the five other members of the proteasome functional group. POC1-4 (Proteasome Chaperone) encode 4 proteins that form two pairs of chaperones of the 20S proteasome (Poc1-Poc2 and Poc3-Poc4) acting upstream of Ump1. HSM3 encodes the first chaperon protein of the regulatory particle. Hm3 associates with the base subcomplex of the 19S and is specifically required for its assembly. Hsm3 also modulates the association between the nascent 19S and the 20S proteasome. We have identified functional mammalian homologs of yeast Poc1-4 (PAC1-4) and Hsm3 (S5b) and provided evidence for a remarkable conservation of a chaperone-assisted proteasome assembly throughout evolution.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

A Novel Golgi Membrane Protein Is a Partner of the ARF Exchange Factors Gea1p and Gea2p

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking. The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors. However, these interactions remain largely unknown. Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs. Gmh1p is an evolutionarily conserved protein. We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p. This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins. A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain. We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER. Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function. These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins

The ArfGEF GBF-1 Is Required for ER Structure, Secretion and Endocytic Transport in C. elegans

Small GTPases of the Sar/Arf family are essential to generate transport containers that mediate communication between organelles of the secretory pathway. Guanine nucleotide exchange factor (GEFs) activate the small GTPases and help their anchorage in the membrane. Thus, GEFs in a way temporally and spatially control Sar1/Arf1 GTPase activation. We investigated the role of the ArfGEF GBF-1 in C. elegans oocytes and intestinal epithelial cells. GBF-1 localizes to the cis-Golgi and is part of the t-ER-Golgi elements. GBF-1 is required for secretion and Golgi integrity. In addition, gbf-1(RNAi) causes the ER reticular structure to become dispersed, without destroying ER exit sites (ERES) because the ERES protein SEC-16 was still localized in distinct punctae at t-ER-Golgi units. Moreover, GBF-1 plays a role in receptor-mediated endocytosis in oocytes, without affecting recycling pathways. We find that both the yolk receptor RME-2 and the recycling endosome-associated RAB-11 localize similarly in control and gbf-1(RNAi) oocytes. While RAB5-positive early endosomes appear to be less prominent and the RAB-5 levels are reduced by gbf-1(RNAi) in the intestine, RAB-7-positive late endosomes were more abundant and formed aggregates and tubular structures. Our data suggest a role for GBF-1 in ER structure and endosomal traffic

The ADP Ribosylation Factor-Nucleotide Exchange Factors Gea1p and Gea2p Have Overlapping, but Not Redundant Functions in Retrograde Transport from the Golgi to the Endoplasmic Reticulum

The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors. Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p. We identified GEA2 as a multicopy suppressor of a sec21-3 temperature-sensitive mutant. SEC21 encodes the γ-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat. GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable. Conditional mutants defective in both GEA1 and GEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions. The two genes do not serve completely overlapping functions because a Δgea1 Δarf1 mutant is not more sickly than a Δarf1 strain, whereas Δgea2 Δarf1 is inviable. Biochemical experiments revealed similar distributions and activities for the two proteins. Gea1p and Gea2p exist both in membrane-bound and in soluble forms. The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum. In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking