Plus de 200 facteurs pré-ribosomiques sont impliqués dans la maturation des ribosomes. La majorité de ces facteurs sont essentiels à la survie cellulaire, mais la fonction précise de la plupart d’entre eux demeure inconnue. Une des dernières étapes de maturation de la petite sous-unité du ribosome est le clivage du pré-ARNr 20S en ARNr 18S mature. Ce clivage est réalisé par l'endonucléase Nob1 et nécessite également la présence de la NTPase Fap7 ainsi que d’une pléthore d’autres facteurs pré-ribosomiques. La fonction de Fap7 est particulièrement intrigante, car l'homologue humain hCINAP possède une activité adénylate kinase, activité enzymatique qui n’est généralement pas liée à la biogenèse des particules ribonucléoprotéiques. En outre, la fonction de Fap7 est intimement liée à son interaction avec la protéine ribosomique Rps14. La partie C-terminale de Rps14 est essentielle pour le clivage au niveau du site D et est située à proximité de l’extrémité 3’ de l’ARNr 18S dans le ribosome mature. La suppression de cette protéine provoque le syndrome 5q qui est phénotypiquement proche de l’anémie de Diamond-Blackfan. Ces deux protéines interviennent également au niveau d’une voie de régulation de p53 qui est dérégulée dans de nombreux cancers. La combinaison d’études structurales par cristallographie aux rayons X, d’études enzymatiques sur des protéines recombinantes ainsi que des tests de maturation in vitro réalisés sur des pré-ribosomes purifiés, nous a permis de mieux appréhender la fonction de Fap7 au sein de la sous-unité pré-40S du ribosome. Nous avons également montré que l'interaction Fap7-Rps14 est impliquée dans un changement conformationnel majeur au cœur des pré-ribosomes et que cette réorganisation est nécessaire afin d'exposer le site D pour le clivage par l’endonucléase Nob1. / Over 200 pre-ribosomal factors involved in the maturation of ribosomes. Most of these factors are essential to cell survival, but the precise function of most of these factors remains elusive. One of the last steps of maturation of the small subunit of the ribosome is the cleavage of 20S pre-rRNA in 18S rRNA in the cytoplasm. This cleavage is carried out by the endonuclease Nob1 and also requires the presence of other factors such as the methyltransferase Dim1, and a plethora of NTPases including the Rio protein kinases, Prp43 and its cofactor Pfa1, the Ltv1 GTPase and the Fap7 NTPase. The function of Fap7 is especially intriguing since the human homologue bears Adenylate activity, an enzymatic activity not usually linked to ribonucleoprotein biogenesis. In addition, the function of Fap7 is intimately linked its interaction with the Rps14 ribosomal protein. The Rps14 C-terminal is essential of D site cleavage and is located in proximity to the 18S C-terminus in the mature ribosome. The deletion of this protein causes the 5q syndrome that is phenotypically close to Diamond Blackfan anemia. The link between the enzymatic activity of Fap7 and its role in ribosome biogenesis remains enigmatic. Using a combination of structural studies by X-ray crystallography, small angle X-ray scattering (SAXS) in solution, enzymatic studies on purified proteins, and in vitro D site cleavage reaction assays on purified pre-ribosomes, we were able to uncover the function of Fap7 within pre-40S ribosomes. We show that the Fap7/Rps14 interaction is involved in a major conformational change at the heart of the pre-ribosomes and that this structural rearrangement is necessary to expose the D-site for cleavage by the endonuclease Nob1.

Made available in DSpace on 2016-08-17T18:39:46Z (GMT). No. of bitstreams: 1 5245.pdf: 1568165 bytes, checksum: 9fc25ff9dc83339e50628c08854efd2c (MD5) Previous issue date: 2012-08-29 / Universidade Federal de Sao Carlos / Schistosimiasis mansoni is a neglected chronic parasitic disease that affects thousands of people worldwide, caused by the trematode Schistosoma mansoni. In the infected host the disease is characterized by the presence of granuloma, imunnopathological response of the cellular infiltration against egg antigens. Thus, the host-parasite relation favors hepatosplenomegaly, acite and hepatic fibrosis. Current chemotherapy is based on the use of Praziquantel (PZQ), used against all species of Schistosoma spp for over 30 years. The main issue is that the PZQ is practically inactive against immature schistosomula and favors the resistance growth of the existent lineages, which makes the study for new drugs and vaccines that can contribute to the control of this disease even more urgent. One of the paths on the search for new therapeutic targets is the study of essential enzymes to the S. mansoni. In particular, it is known that the enzymes Adenylate Kinase 1 and 2 (ADK), Uridine cytidine Kinase 1 and 2 (UCK), Hypoxanthine guanine phosphoribosyltransferase (HGPRT) e Purine nucleoside phosphorilase 1 (PNP) are found on the metabolic pathways of the parasite s nucleotide, participating in the metabolism of purines and pyrimidines. Our goals in this study were to assess the immunization with these enzymes, using the S. mansoni cercariae infected murine model, and subsequently analyze the acting in oviposition and growth of adult worms. Our results showed that the immunization in Balb/c mice with the UCK enzyme was capable of inducing a specific immune response, which favored a significant reduction of the parasitic load (adult worms). However, it was not possible to observe significant reduction in the number of eliminated eggs. Regarding the immunization with PNP and HGPRT enzymes, the mice had a reduction in the number of eggs per gram of feces. The data obtained are considered interesting and can be new targets for immunotherapy against schistosomiasis mansoni. Thereby, new assays must be made with different dosages of the enzymes for a better assessment on how these enzymes modulate the parasitic load through the eggs reduction, reduction in the adult worms retrieving, as well as the antibody levels during the murine infection by the S.mansoni. / A esquistossomose mansônica é uma doença parasitária, crônica e negligenciada que afeta milhares de pessoas ao redor do mundo, causada pelo trematódeo Schistosoma mansoni. No hospedeiro infectado a doença é caracterizada pela presença do granuloma, resultado imunopatológico do infiltrado celular contra antígenos dos ovos A quimioterapia atual é baseada no uso do Praziquantel (PZQ), usado contra todas as espécies de Schistosoma spp há mais de 30 anos. O principal problema é que o PZQ é praticamente inativo contra esquistossomulos imaturos e favorece o desenvolvimento de resistência das linhagens existentes. Um dos caminhos na busca por novos alvos terapêuticos é o estudo de enzimas que são essenciais para o S. mansoni. Em especial, sabe-se que as enzimas Adenilato Quinase 1 e 2 (ADK), Uridina Citidina Quinase 1 e 2 (UCK), Hipoxantina-guanina fosforibosiltransferase (HGPRT) e Purina Nucleosídeo Fosforilase 1 (PNP) são encontradas nas vias metabólicas de nucleotídeos do parasito, participando do metabolismo de purinas e pirimidinas. A estratégia de utilizar enzimas do parasito na esquistossomose mansônica murina foi de avaliar uma resposta induzida por estas enzimas quando aplicadas em camundongos BALB/c e desafiados com cercarias de S. mansoni. Desta forma, avaliamos a fase crônica de camundongos imunizados e infectados com S. mansoni, onde foram analisadas amostras parasitológicas, hematológicas, sorológicas e fluidos da cavidade peritoneal. Nosso objetivo neste estudo foi avaliar a imunização com essas enzimas, usando o modelo murino infectado com cercarias de S. mansoni e posteriormente avaliar a ação na oviposição e desenvolvimento de vermes adultos. Nossos resultados demonstraram que a imunização em camundongos Balb∕c com a enzima UCK foi capaz de induzir uma resposta imune específica, a qual favoreceu a diminuição significativa da carga parasitária (vermes adultos). No entanto, não foi possível observar redução significativa no número de ovos eliminados. Em relação à imunização com as enzimas PNP e HGPRT os camundongos que receberam as imunizações com PNP e HGPRT tiveram redução no número de ovos por grama de fezes. Os dados obtidos são considerados interessantes e podem ser considerados novos alvos para a imunoterapia contra a esquistosomose mansônica. Dessa forma, novos ensaios deverão ser realizados com diferentes doses das enzimas para melhor avaliar como essas enzimas modulam a carga parasitária através da redução de ovos, diminuição na recuperação de vermes adultos, assim como os níveis de anticorpos durante a infecção murina pelo S. mansoni.

No / Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-alpha4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.