Orientador: Carlos Henrique Inacio Ramos / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T03:59:04Z (GMT). No. of bitstreams: 1 Peres_BarbaraRamalho_M.pdf: 4607988 bytes, checksum: e396f207db591110a7882ea1df2cfbd4 (MD5) Previous issue date: 2014 / Resumo: As proteínas Rvb1 e Rvb2 são DNA helicases dependentes de ATP pertencentes à família AAA+ ATPases (ATPases associadas com diversos processos celulares) que estão envolvidas na remodelação da cromatina, montagem da telomerase, mitose e biogênese de snoRNP ("small nucleolar ribonucleoprotein") e interagem com as co-chaperonas Tah1 (contém um domínio TPR associado com Hsp90) e Pih1 formando um complexo funcional, denominado R2TP. Este complexo já foi identificado em levedura e seres humanos, e desempenha um papel essencial na montagem de complexos multi-proteicos com DNA e RNA. Nosso grupo de pesquisa tem por objetivo identificar e caracterizar o complexo R2TP em Leishmania major pela importância deste parasita para medicina. Apresentamos o estudo das proteínas Rvb1 e Rvb2 de L. major. Os genes foram clonados e as proteínas recombinantes foram purificadas e caracterizadas quanto à conformação pelas técnicas de dicroísmo circular, gel filtração analítica e fluorescência intrínseca, mostrando que as proteínas foram purificadas enoveladas. Foram feitos ensaios funcionais de atividade ATPásica mostrando que estas possuem atividade ATPásica a qual é aumentada quando as proteínas foram incubadas com DNA. Espera-se que os resultados deste trabalho possam contribuir para o objetivo de compreensão do sistema R2TP e da regulação da homeostase do genoma de L. major. O conjunto destas pesquisas pode contribuir também para o desenvolvimento de possíveis estratégias para intervir no parasita causador de uma doença negligenciada / Abstract: Rvb1 and Rvb2 proteins are ATP-dependent DNA helicases belonging to the AAA + ATPases family (ATPases associated with diverse cellular processes) that are involved in chromatin remodeling, telomerase assembly, mitosis and snoRNP biogenesis ("small nucleolar ribonucleoprotein") and interact with co-chaperones Tah1 (wich contains a TPR domain associated with Hsp90) and Pih1 into a functional complex, named R2TP. The R2TP complex has been identified in yeast and humans, and has an essential role in the assembly of multi-protein complexes with DNA and RNA. Due to the medical importance of Leishmania major our research group has becoming interested in the identification and characterization of the R2TP complex of this parasite. We studied the Rvb1 and Rvb2 proteins of L. major. The genes were cloned, the recombinant proteins were purified and their conformation were characterized by circular dichroism, analytical gel filtration and intrinsic fluorescence techniques, indicating that the proteins were folded. Functional assays showed that Rvb1 and Rvb2 have ATPase activity and this activity increased when incubated with DNA. Hopefully, the results of this study may contribute to the goal of understanding the R2TP system and homeostasis of the L. major genome and together, may contribute to the development of possible strategies to intervene with a parasite that causes a neglected disease / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular

O complexo R2TP está presente em eucariotos, de leveduras a humanos, e está envolvido no correto dobramento de outras proteínas e montagem de complexos multiproteicos. R2TP é formado pelas proteínas Rvb1, Rvb2, Tah1 e Pih1/Nop17 em levedura, e direciona as chaperonas à proteínas alvo durante a montagem dos complexos. Os clusters Fe/S são sintetizados nas mitocôndrias e posteriormente transferidos para o citoplasma. Dre2 é uma proteína que contém cluster Fe/S, e está envolvida na transferência desses clusterspara outras proteínas citoplasmáticas. Nosso laboratório identificou a interação entre a subunidade Nop17 do complexo R2TP e Dre2 pelo método de duplo-híbrido, mas o papel desta interação ainda não foi elucidado. O objetivo deste trabalho foi o de estudar o papel funcional da interação entre Dre2 e Nop17 e identificar seus domínios de interação. Nossos resultados mostram que a porção N-terminal de Nop17 interage com a porção C-terminal de Dre2 e esta interação é necessária para a manutenção dos níveis de Dre2 na célula, indicando que o complexo R2TP atue na montagem do complexo CIA, de proteínas citosólicas Fe/S, do qual Dre2 faz parte. Dre2 também afeta a estabilidade de Nop17, sugerindo que Dre2 possa transferir um clusterFe/S para Nop17. Os dados mostrados aqui, portanto, indicam que a interação Nop17-Dre2 seja mutuamente importante para a estabilidade das duas proteínas / The R2TP protein complex is present in eukaryotes from yeast to humans, and is involved in the correct assembly of other protein or ribonucleoprotein complexes. R2TP is formed by proteins Rvb1, Rvb2, Tah1 and Pih1/Nop17 in yeast, and directs chaperones to target proteins during complexes assembly. Fe/S clusters are synthesized in mitochondria and later transferred to the cytoplasm. Dre2 is a Fe/S cluster protein, involved in transferring of Fe/S clusters to cytoplasmic proteins. Our laboratory has identified the interaction between the R2TP subunit Nop17 and Dre2 in the two-hybrid system. The aim of this work was to study the functional role of the interaction between Dre2 and Nop17, and to identify their domains of interaction. The results show that the N-terminal portion of Nop17 interacts with the C-terminal region of Dre2, and that this interaction is necessary for maintaining the levels of Dre2 in the cell, which suggests that the R2TP complex affects the cytosolic iron-sulfur protein assembly complex (CIA), of which Dre2 is a subunit. Dre2 also affects Nop17 stability, suggesting that Dre2 may transfer a Fe/S cluster to Nop17. The data here indicate that the interaction Nop17-Dre2 is mutually important for these proteins stabilities.

O complexo R2TP está presente em eucariotos, de leveduras a humanos, e está envolvido no correto dobramento de outras proteínas e montagem de complexos multiproteicos. R2TP é formado pelas proteínas Rvb1, Rvb2, Tah1 e Pih1/Nop17 em levedura, e direciona as chaperonas à proteínas alvo durante a montagem dos complexos. Os clusters Fe/S são sintetizados nas mitocôndrias e posteriormente transferidos para o citoplasma. Dre2 é uma proteína que contém cluster Fe/S, e está envolvida na transferência desses clusterspara outras proteínas citoplasmáticas. Nosso laboratório identificou a interação entre a subunidade Nop17 do complexo R2TP e Dre2 pelo método de duplo-híbrido, mas o papel desta interação ainda não foi elucidado. O objetivo deste trabalho foi o de estudar o papel funcional da interação entre Dre2 e Nop17 e identificar seus domínios de interação. Nossos resultados mostram que a porção N-terminal de Nop17 interage com a porção C-terminal de Dre2 e esta interação é necessária para a manutenção dos níveis de Dre2 na célula, indicando que o complexo R2TP atue na montagem do complexo CIA, de proteínas citosólicas Fe/S, do qual Dre2 faz parte. Dre2 também afeta a estabilidade de Nop17, sugerindo que Dre2 possa transferir um clusterFe/S para Nop17. Os dados mostrados aqui, portanto, indicam que a interação Nop17-Dre2 seja mutuamente importante para a estabilidade das duas proteínas / The R2TP protein complex is present in eukaryotes from yeast to humans, and is involved in the correct assembly of other protein or ribonucleoprotein complexes. R2TP is formed by proteins Rvb1, Rvb2, Tah1 and Pih1/Nop17 in yeast, and directs chaperones to target proteins during complexes assembly. Fe/S clusters are synthesized in mitochondria and later transferred to the cytoplasm. Dre2 is a Fe/S cluster protein, involved in transferring of Fe/S clusters to cytoplasmic proteins. Our laboratory has identified the interaction between the R2TP subunit Nop17 and Dre2 in the two-hybrid system. The aim of this work was to study the functional role of the interaction between Dre2 and Nop17, and to identify their domains of interaction. The results show that the N-terminal portion of Nop17 interacts with the C-terminal region of Dre2, and that this interaction is necessary for maintaining the levels of Dre2 in the cell, which suggests that the R2TP complex affects the cytosolic iron-sulfur protein assembly complex (CIA), of which Dre2 is a subunit. Dre2 also affects Nop17 stability, suggesting that Dre2 may transfer a Fe/S cluster to Nop17. The data here indicate that the interaction Nop17-Dre2 is mutually important for these proteins stabilities.

La protéine chaperon HSP90 a de nombreux substrats par les voies de signalisations. Son inhibition engendre un effet tumoral. L'identification du complexe R2TP,un nouveau cochaperon d'HSP90, a mis en évidence de nouveaux substrats. Le système HSP90/R2TP est impliqué dans l'assemblage de complexes macromoléculaires ( PIKKs, Télomérase RNP, ARN polyméras, snoRNP) jouant un rôle clé dans la prolifération cellulaire et la tumorigénèse. Le R2TP est composé de 4 protéines : RUVBL1, RUVBL2, RPAP3 et PIH1D1 dont certains sont surexprimés dans les cancers hépatocellulaires ou colorectaux. Afin d'étudier le rôle du R2TP dans l'homéostasie intestinale et la carcinogénèse, nous avons généré un modèle d'invalidation de RPAP3 chez la souris. L'invalidation de RPAP3 dans l'organisme entier est létale. Son invalidation à l'âge adulte uniquement dans l'intestin entraine une phénotype sévère en 8jours aboutissant à la mort des individus à 10jours. En parallèle, nous avons étudié le rôle potentiel de RPAP3 dans la tumorigénèse intestinale et colorectale avec deux modèles d'induction , génétique avec l'invalidation du gène APC et chimique avec le protocole AOM/DSS. / Many substrates of HSP90 are involved in signal transduction pathways and related to tumour progression. Inhibition of HSP90 has anti-tumoral effects. Identification of the R2TP, a new HSP90 co-chaperon, allowed the identification of a new set of HSP90 substrates. HSP90/R2TP is involved in the assembly of snoRNPs, telomerase RNP, the nuclear RNA polymerases and PIKKs, which play key functions in cellular proliferation and tumorigenesis. R2TP is formed of four proteins: RUVBL1, RUVBL2, PIH1D1 and RPAP3, some of which are overexpressed in hepatocellular and colorectal cancer. We thus hypothesize that the co-chaperone R2TP could be involved in colorectal carcinogenesis.To study the role of R2TP in intestinal homeostasis and carcinogenesis, we generated a conditional knock-out murine model for RPAP3. We showed that RPAP3 invalidation in whole organism or only in colon is lethal at embryonic stage. The invalidation of RPAP3 in adult intestine, using an inducible recombinase (RPAP3 fl; Villin>Cre-ERT2), leads to a drastic phenotype as soon as 8 days post-induction, resulting in death after 10 days. This phenotype is reminiscent of proliferative defects.In parallel, we address the possibility of a therapeutic window to target RPAP3 during intestinal tumorigenesis by using heterozygous animals (RPAP3 fl/+; Villin>Cre-ERT2 ) in which tumorignesis is induced (i) either by a chemical treatment : for this, we take advantage of the established AOM /DSS protocol, or, (ii) by a genetic one ( Apc LoxP/+).These ongoing experiments will address the role of R2TP in a tissue with a constant turnover and the relevance of R2TP in tumorigenesis.

Nop17p e Rsa1p são proteínas nucleolares em Saccharomyces cerevisiae, as quais foram identificadas pela sua associação a dois complexos celulares: os snoRNPs de box C/D, através de interação com as subunidades Nop58p e Snu13p, respectivamente, e o R2TP/Hsp90p. Nop17p parece ser responsável por direcionar a chaperona Hsp90p durante a montagem dos snoRNPs, e a associação de Rsa1p a estes complexos ainda não tem uma função estabelecida. Neste trabalho, nós mostramos que a ausência de ambas as proteínas afetam a estabilidade da proteína Nop58p dos snoRNPs e afetam a localização do snoRNA U3. Em relação à ordem de interação das proteínas do core de snoRNps de box C/D, Nop17p associa-se de maneira transiente a Nop1p/Snu13p, seguida da ligação de Nop58p ao complexo. Quanto à rede de interação do R2TP, obtivemos o mutante Nop17(N307S), que não mais interage com Tah1p. Este mutante interage com a subunidade Rvb1p do R2TP, mas não se associa com outras proteínas parceiras de Nop17p(WT). Apesar da importância da interação Nop17p-Tah1p, sua interrupção não afeta o crescimento celular, o que sugere a possibilidade de outro fator estar envolvido na associação entre Nop17p e Hsp90p. / Nop17p and Rsa1p are Saccharomyces cerevisiae nucleolar proteins, which were identified for its association with two cellular complexes: box C/D snoRNPs, through interaction with the core subunits Nop58p and Snu13p respectively, and the R2TP/Hsp90p. Nop17p seems to be responsible for directing Hsp90p to the assembly of snoRNPs. The Rsa1p association to these complexes still have no defined function. In this work, we showed that both proteins absence affect Nop58p stability and causes a mislocalization of the U3 snoRNA. Relativel to the order of assembly of the box C/D snoRNPs core proteins, Nop17p associates transiently with Nop1p/Snu13p, followed by the Nop58p joining to the complex. To study in more detail the protein interactions within the R2TP complex, we obtained the Nop17(N307S) mutant, which no longer interacts withTah1p, but still interacts withRvb1p, another R2TP subunit. Nop17(N307S) does not interact with other Nop17p(WT) partners. Despite the importance of the Nop17p-Tah1p association, the disruption of this interaction does not affect cell growth, suggesting the involvement of a second factor on the Nop17p and Hsp90p association.

Chez les eucaryotes, la biogenèse des ribosomes débute dans le nucléole par la maturation et la modification des ARN ribosomiques (ARNr), et fait intervenir des centaines de particules ribonucléoprotéiques (RNP) distinctes, comme les petites RNP nucléolaires (snoRNP) à boîtes C/D, qui portent une activité méthyl transférase ciblée sur la position 2’-OH des riboses. Leur biogenèse nécessite l’intervention transitoire de facteurs protéiques constituant une machinerie d’assemblage spécifique. Mon travail de thèse a visé à étudier le rôle fonctionnel de deux de ces facteurs chez la levure S. cerevisiae les protéines Hit1 et Bcd1. Hit1p avait été trouvée au laboratoire être impliquée dans la biogenèse des snoRNP à boîtes C/D, et il était connu que l’expression de Bcd1p est essentielle à la viabilité cellulaire et pour la stabilité des snoRNA à boîtes C/D. Lors de ce travail, nous avons retrouvé le domaine fonctionnel de Hit1p et identifié les acides aminés impliqués dans l’interaction avec Rsa1p, un autre facteur d’assemblage. Par une approche similaire, nous avons recherché les domaines nécessaires à la fonctionnalité de Bcd1p. Le mécanisme par lequel Bcd1p influence spécifiquement les taux de snoRNA à boîtes C/D reste inconnu, mais au cours de ce travail j’ai identifié un nouveau partenaire potentiel pour cette protéine - la chaperonne d’histone Rtt106p. La dernière partie de mon travail a visé à rechercher le lien fonctionnel entre Rtt106p et l’expression des snoRNA à boîtes C/D / In eukaryotes, ribosome biogenesis begins in the nucleolus, by maturation and modification of ribosomal RNAs (rRNA) and involves hundreds of distinct ribonucleoprotein particles, like box C/D small nucleolar RNPs (snoRNPs). Their assembly requires the transient intervention of protein factors constituting a specific assembly machinery. My PhD work aimed to investigate the functional role of two such factors, Bcd1p and Hit1p, in the yeast S. cerevisiae. Hit1p involvement in box C/D snoRNP biogenesis was revealed in our lab, and it was known that Bcd1p expression is essential to cell viability and box C/D snoRNA stability. During this work, we identified the functional domain of Hit1p, and the aminoacids involved in its interaction with Rsa1, another assembly factor. By a similar approach we identified the functional domains of Bcd1p. The mechanism by which Bcd1p specifically influences box C/D snoRNA levels is unknown. However, I identified a potentially new partner for this protein – the Rtt106p histone chaperone. The last part of my work aimed to search for a functional link between this histone chaperone and box C/D snoRNA expression

Nop17p e Rsa1p são proteínas nucleolares em Saccharomyces cerevisiae, as quais foram identificadas pela sua associação a dois complexos celulares: os snoRNPs de box C/D, através de interação com as subunidades Nop58p e Snu13p, respectivamente, e o R2TP/Hsp90p. Nop17p parece ser responsável por direcionar a chaperona Hsp90p durante a montagem dos snoRNPs, e a associação de Rsa1p a estes complexos ainda não tem uma função estabelecida. Neste trabalho, nós mostramos que a ausência de ambas as proteínas afetam a estabilidade da proteína Nop58p dos snoRNPs e afetam a localização do snoRNA U3. Em relação à ordem de interação das proteínas do core de snoRNps de box C/D, Nop17p associa-se de maneira transiente a Nop1p/Snu13p, seguida da ligação de Nop58p ao complexo. Quanto à rede de interação do R2TP, obtivemos o mutante Nop17(N307S), que não mais interage com Tah1p. Este mutante interage com a subunidade Rvb1p do R2TP, mas não se associa com outras proteínas parceiras de Nop17p(WT). Apesar da importância da interação Nop17p-Tah1p, sua interrupção não afeta o crescimento celular, o que sugere a possibilidade de outro fator estar envolvido na associação entre Nop17p e Hsp90p. / Nop17p and Rsa1p are Saccharomyces cerevisiae nucleolar proteins, which were identified for its association with two cellular complexes: box C/D snoRNPs, through interaction with the core subunits Nop58p and Snu13p respectively, and the R2TP/Hsp90p. Nop17p seems to be responsible for directing Hsp90p to the assembly of snoRNPs. The Rsa1p association to these complexes still have no defined function. In this work, we showed that both proteins absence affect Nop58p stability and causes a mislocalization of the U3 snoRNA. Relativel to the order of assembly of the box C/D snoRNPs core proteins, Nop17p associates transiently with Nop1p/Snu13p, followed by the Nop58p joining to the complex. To study in more detail the protein interactions within the R2TP complex, we obtained the Nop17(N307S) mutant, which no longer interacts withTah1p, but still interacts withRvb1p, another R2TP subunit. Nop17(N307S) does not interact with other Nop17p(WT) partners. Despite the importance of the Nop17p-Tah1p association, the disruption of this interaction does not affect cell growth, suggesting the involvement of a second factor on the Nop17p and Hsp90p association.

DNA double strand breaks are the most dangerous type of DNA damage. The MRN complex and 53BP1 have essential functions in the repair of DNA double strand breaks and are therefore important for maintaining genomic stability and preventing cancer. DNA double strand breaks are repaired by two main mechanisms - homologous recombination and non- homologous end joining. The MRN complex senses DNA double strand breaks and activates a cascade of posttranslational modifications that activates and recruits other effector proteins. In addition MRN mediated resection is important for removing adducts in non-homologous end joining and creating single stranded DNA required for homologous recombination. 53BP1 is recruited to DNA double strand breaks by site specific ubiquitinations and inhibits DNA resection, thereby promoting non-homologous end joining at the expense of homologous recombination. In this thesis we show that MRE11 binds to the R2TP chaperone complex through a CK2 mediated phosphorylation. Knockdown of R2TP or mutating the MRE11 binding site leads to decreased MRE11 levels and impaired DNA repair. Similar phenotype has been observed in cells from patients with ataxia-telangiectasia-like disorder (ATLD), containing MRE11 deletion mutation which is missing the R2TP complex binding site. Based on R2TP...

Les protéines de la famille L7Ae sont les constituants de nombreuses RNP essentielles. Chez les vertébrés, les particules snoRNP C/D et H/ACA sont impliquées dans la biogenèse des ribosomes, la UsnRNP U4 dans l'épissage des pré-ARNm, le complexe télomérase dans la réplication des télomères, et les mRNP SECIS dans la traduction des sélénoprotéines. Comme c'est le cas pour la majorité des RNP eucaryotes, leur assemblage, sous forme d'entités fonctionnelles, ne constitue pas un processus autonome et requiert l'intervention de facteurs spécialisés. En basant notre étude sur l'assemblage des snoRNP C/D, dans l'organisme modèle Saccharomyces cerevisiae, et en utilisant des approches de biologie moléculaire, de biochimie et de génétique, nous avons entrepris de caractériser ces événements. Nos travaux ont contribué à identifier un ensemble de protéines, agissant de façon coordonnée au sein d'une machinerie conservée entre la levure et l'homme. Cette dernière est composée de deux principales sous-unités : (i) Rsa1p/NUFIP, une protéine plate-forme, qui interagit avec certaines protéines de la famille L7Ae et facilite l'assemblage des RNP, (ii) le complexe R2TP (Rvb1p/TIP49, Rvb2p/TIP48, Pih1p/PIH1, Tah1p/SPAGH), qui pourrait opérer des remodelages conformationnels nécessaires à la formation des RNP matures. En plus de ces acteurs centraux, d'autres facteurs sont apparus intimement liés à ce mécanisme. La protéine Hit1p/TRIP3, interagit notamment avec Rsa1p/NUFIP et s'est avéré requise pour assurer sa stabilité chez la levure. La chaperonne HSP90, dont le rôle est prédominant chez l'homme, exerce son activité sur certains constituants des RNP. Enfin, la protéine Bcd1p/BCD1 pourrait être associée à cette machinerie dans le cadre spécifique de l'assemblage des snoRNP C/D / The L7Ae family proteins are essential components of many RNPs. In vertebrates, C/D and H/ACA snoRNPs are involved in ribosome biogenesis, the U4 snRNP in pre-mRNA splicing, the telomerase complex in telomeres replication, and mRNP SECIS in selenoproteins translation. Like most eukaryotic RNPs, assembly in functional entities is not an autonomous process and requires the intervention of specialized factors. Basing our study on the assembly of C/D snoRNP in the model organism Saccharomyces cerevisiae, and using approaches of molecular biology, biochemistry and genetics, we undertook to decipher these mechanisms. Our work has helped to identify a set of proteins, acting in a coordinated manner within a machinery conserved between yeast and human. This machinery consists of two major subunits: (i) Rsa1p/NUFIP, a platform protein that interacts with some proteins of the L7Ae family and facilitates the RNPs assembly, (ii) the R2TP complex (Rvb1p/TIP49, Rvb2p/TIP48, Pih1p/PIH1, Tah1p/SPAGH), which could induce conformational remodeling necessary for the formation of mature RNPs. In addition to these key players, other factors appeared closely linked to this mechanism. The Hit1p/TRIP3 protein interacts with Rsa1p/NUFIP and is required to ensure its stability in yeast. HSP90 chaperone, whose role is predominant in human, operates on some components of the RNPs. Finally, the Bcd1p/BCD1 protein is associated specifically with this machinery during C/D snoRNPs assembly