Células eucarióticas contêm vários tipos de proteassomo que regulam o processo de degradação de proteína. Proteassomos são proteases multicatalíticas que são responsáveis pela maior parte de degradação não-lisossomal de proteínas em células eucarióticas. As três subunidades catalíticas do proteassomo são &beta;1, &beta;2 e &beta;5. Em condições de stress e resposta imune essas três subunidades são substituídas por &beta;1i, &beta;2i and &beta;5i, respectivamente, para formar o proteassomo imune. Estas três subunidades induzíveis, parecem alterar as especificidades de peptidase do proteassoma imune em células tratadas com IFN-<font face=\"symbol\">g. Nosso objetivo no presente trabalho foi caracterizar um modelo celular para a indução do proteassomo imune, e ainda investigar o repertório peptídeo intracelular produzido por esta forma particular do proteassoma, através da técnica de espectrometria de massas. Em resumo, os nossos dados mostraram um aumento de 3 vezes do peptídeo EL28 derivado da proteína RPT2 em células HeLa tratadas com o IFN-<font face=\"symbol\">g. O peptídeo EL28 pode ser de relevância clínica para o tratamento de distúrbios relacionados com a apresentação de antígenos, visto que ele parece ativar a atividade quimotripsina-like quando incubado com o extrato celular de células HeLa. / Eukaryotic cells contain several types of proteasome regulating the process of protein degradation. The proteasome are responsible for most non - lysosomal protein degradation in eukaryotic cells. The three catalytic subunits of the proteasome are &beta;1, &beta;2 and &beta;5. Under conditions of stress and immune response these three subunits are replaced by &beta;1i, &beta;2i and &beta;5i, respectively, to form the immune proteasome . These three inducible subunits, appear to alter the specificity of the immune proteasome peptidase in cells treated with IFN-<font face=\"symbol\">g. Our aim in this study was to characterize a cellular model for the induction of the immune proteasome, and even investigate the intracellular peptide repertoire produced by this particular form of the proteasome, through the technique of mass spectrometry. In summary, our data showed an increase of 3 times the peptide derived from RPT2 EL28 protein in HeLa cells treated with IFN-<font face=\"symbol\">g. The EL28 peptide may be of clinical relevance for the treatment of disorders related to antigen presentation, since it seems to activate the chymotrypsin-like activity when incubated with the cell extract of HeLa cells.

A atrofia está associada ao aumento da degradação protéica em doenças sistêmicas, sendo o sistema proteolítico ubiquitina proteassoma (SUP) uma das principais vias envolvidas. Contudo, pouco é conhecido sobre a contribuição do SUP à atrofia desencadeada pela insuficiência cardíaca (IC). Sabendo dos benefícios do treinamento físico aeróbico (TFA) e que os mecanismos moleculares envolvidos na atrofia na IC ainda não estão esclarecidos, nessa dissertação investigamos: 1) a contribuição do SUP para a atrofia associada à IC em 2 modelos experimentais: um modelo genético de camundongos com hiperatividade simpática (HS), e um modelo de infarto do miocárdio (IM) em ratos e 2) o efeito do TFA sobre a atrofia associada à IC e sobre o SUP. Na HS verificamos aumento da expressão das E3 ligases, da deubiquitinase USP28, das proteínas ubiquitinadas e da atividade do proteassoma no sítio quimiotripsina, sendo que o TFA reduziu a expressão dos componentes alterados. No IM, observamos disfunção cardíaca não associada à IC, porém, com aumento da expressão de Atrogin-1; enquanto o TFA não produziu efeitos significantes. Dessa forma, os dados sugerem a participação do SUP na atrofia desencadeada pela IC na HS e, que o TFA previne a atrofia por reduzir a expressão/atividade de alguns componentes do SUP; e, que no IM, o aumento da expressão de Atrogin-1 precedeu a perda de massa muscular / Skeletal muscle atrophy is associated with increased protein degradation in systemic diseases, which seems to be mainly related to ubiquitin-proteasome system (UPS). However, little is known about UPS contribution to the heart failure-induced muscle atrophy (HF-MA). Likewise, aerobic exercise training (AET) has been established as an adjuvant therapy for HF and molecular mechanisms underlying HF-MA has not been clarified yet. The objectives of the study were: 1) to verify UPS contribution for HF-MA in 2 experimental models: sympathetic hyperactivity-induced HF (&#945;2A/&#945;2CARKO) in mice, and myocardial infarction model (MI) in rats and 2) AET effects on HF-MA and UPS. In &#945;2A/&#945;2C ARKO mice, we observed activation of UPS characterized by increased mRNA levels of E3 ligases Atrogin-1 and E3-a, deubiquitinating enzyme USP28, increased levels of ubiquitinated proteins and chymotrypsin-like proteasome activity. AET prevented HF-MA in the &#945;2A/&#945;2C ARKO by reducing of UPS activity. In MI model, rats displayed cardiac dysfunction and exercise intolerance with no signs of atrophy. However, Atrogin-1 mRNA and protein levels were increased. Therefore, alterations in Atrogin-1expression might precede atrophy and HF in this model. In conclusion, our data provide evidence for skeletal muscle anti-atrophic effect upon AET in &#945;2A/&#945;2C ARKO that is related, at least in part, to a reduced UPS

Disfunções da glândula tireóide são, frequentemente, associadas a manifestações cardiovasculares e, em situações de hipertireoidismo, o coração hipertrofia. A hipertrofia cardíaca (HC) consiste em uma resposta adaptativa caracterizada pelo aumento de síntese de proteínas estruturais. O Sistema Ubiquitina Proteassoma (UPS) corresponde ao principal mecanismo de proteólise intracelular e crescentes evidências sugerem seu envolvimento no desenvolvimento da HC. O objetivo do presente estudo foi avaliar a modulação do UPS no tecido cardíaco de animais submetidos ao hipertireoidismo. Os resultados referentes ao aumento da atividade e expressão do proteassoma (PT) cardíaco apresenta-se mais contundente no grupo tratado por 7 dias, período em que a HC já encontra-se estável. Ao término de 14 e 21 dias, a modulação desse sistema tende à normalização. Os resultados obtidos atestam evidências da literatura que sugerem o aumento da atividade do PT cardíaco como resposta compensatória ao aumento de síntese proteica. / Thyroid gland disorders are often associated with cardiovascular events and hyperthyroidism state promotes cardiac hypertrophy (CH). CH consists in adaptive response characterized by increased synthesis of structural proteins. The Ubiquitin Proteasome System (UPS) is the major mechanism of intracellular proteolysis and increased evidences suggest its involvement in the development of CH. The aim of this study was to evaluate the modulation of UPS in cardiac tissue of animals subjected to hyperthyroidism. The results related to the increased proteasome (PT) activity and expression in the heart was more accentuated in the group treated for 7 days, when the CH process finds stable. At the end of 14 and 21 days of hyperthyroidism, the modulation of cardiac UPS achieves standard values. These results suggest an increased activity of cardiac PT as a compensatory response to protein synthesis induced by thyroid hormones.

No description available.

Das Ubiquitin-Proteasom-System (UPS) ist das wichtigste System für den Abbau von nicht mehr benötigten oder beschädigten Proteinen innerhalb der eukaryotischen Zelle und ist somit an der Aufrechterhaltung der zellulären Homöostase beteiligt. Ein Abfall der proteasomalen Aktivität führt zu intrazellulärem Stress. Die Zelle wirkt diesem Abfall entgegen, indem sie die proteasomalen Gene verstärkt exprimiert und dadurch die Neubildung von 26S Proteasomen bewirkt. Während in der Bäckerhefe Saccharomyces cerevisiae mit Rpn4 der Transkriptionsfaktor für die verstärkte Expression identifiziert wurde, war dieser in Säugetieren noch nicht bekannt. In der vorliegenden Arbeit konnte TCF11 (transcription factor 11) als der verantwortliche Transkriptionsfaktor identifiziert werden, der in der humanen Endothelzelllinie Ea.hy926 die Transkription der proteasomalen Gene nach Proteasominhibition induziert. Unter physiologischen Bedingungen ist TCF11 ein N-glykosyliertes ER-ständiges Membranprotein, welches durch die ER-assoziierte Protein Degradation, unter der Mitwirkung des E3-Enzyms HRD1 und der AAA-ATPase p97, schnell abgebaut wird. Nach der Proteasominhibition kommt es zur Akkumulation von oxidierten Proteinen, und TCF11 wird aktiviert und in den Zellkern transportiert. Im Zellkern bindet TCF11 an AREs (antioxidant response element) in den proteasomalen Promotoren und aktiviert dadurch die Transkription der proteasomalen Gene. Darüber hinaus reguliert TCF11 auch die Expression von zahlreichen Enzymen, die die Ubiquitinierung von Proteinen katalysieren. Dadurch wird die zelluläre Homöostase wiederhergestellt und TCF11 sehr wahrscheinlich durch die neu gebildeten Proteasomen abgebaut. Die Ergebnisse der vorliegenden Arbeit zeigen auf, dass die Integrität des UPS nach Proteasominhibition in der humanen Endolthelzelllinie Ea.hy926 über einen TCF11 abhängigen Rückkopplungsmechanismus aufrechterhalten wird. / The ubiquitin-proteasome-system (UPS) is the most important system for regulated protein degradation in eukaryotes. Therefore it is involved in the regulation of cellular homeostasis. Reduced proteasome activity results in proteotoxic stress. To counteract for reduced proteasome activity, eukaryotic cells enhance proteasome gene expression, which results in formation of new 26S proteasomes and recovery of physiological conditions. While in bakers yeast Saccharomyces cerevisiae the transcription factor Rpn4 is responsible for enhanced proteasome gene expression in response to proteasome inhibition, in mammals the responsible transcription factor was unknown. In this thesis, transcription factor TCF11 (transcription factor 11) was identified as a key regulator for 26S-proteasome formation in the human cell line Ea.hy926 to compensate for reduced proteolytic activity. Under non-inducing conditions N-glycosylated TCF11 resides in the endoplasmic reticulum (ER) membrane, where TCF11 is targeted to ER-associated protein degradation system requiring the E3-ubiquitin ligase HRD1 and the AAA-ATPase p97. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins, and promote the nuclear translocation of TCF11 from the ER, permitting activation of proteasome gene expression by binding of TCF11 to antioxidant response elements (ARE) in their promoter regions. Furthermore TCF11 controlls the expression of additional UPS-related genes. Thus the transcriptional feedback loop regulating human proteasome dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition was uncovered.

Le maintien de la masse musculaire est étroitement lié à la balance entre la synthèse et la dégradation des protéines. L'exercice physique est un puissant régulateur de la balance protéique et plus particulièrement l'exercice en résistance. S'intéresser à la balance protéique après un exercice s'inscrit dans une compréhension des mécanismes cellulaires et moléculaires conduisant aux phénomènes d'hypertrophie et/ou d'atrophie musculaire. Nos travaux mettent en évidence que l'hypertrophie obtenue dans le muscle FDP après 10 semaines d'un entraînement en résistance chez le rat, est en lien avec l'activation chronique de la voie IL-6/STAT3 après chaque exercice aigu, en partie au sein du pool de cellules satellites activées. En phase proliférative, les cellules dont la voie de signalisation STAT1/STAT3 est activée, répriment l'expression des facteurs myogéniques comme MyoD et retournent ainsi à l'état quiescent, concourant à augmenter le pool de réserve. Ces mécanismes participent à la synthèse protéique par l'apport de nouveau matériel génétique au sein des fibres musculaires conduisant à une augmentation de leur surface de section ainsi qu'à leur conversion phénotypique avec l'entraînement. L'exercice en résistance favorisant la protéolyse, nos travaux ont cherché à caractériser les systèmes protéolytiques (autophagique-lysosomal, ubiquitine-protéasome) impliqués dans la balance protéique post-exercice. Les marqueurs moléculaires étudiés (activités enzymatiques du protéasome et de la cathepsine L, expression protéique et génique de LC3B, des E3 ligases…) ne permettent pas d'expliquer clairement les +30% de protéolyse obtenus une heure après des contractions excentriques sur muscle EDL isolé de rat en condition à jeun. Des perspectives d'étude des systèmes des calpaines, des caspases et/ou des métalloprotéases matricielles sont alors à envisager. / The maintain of muscle mass is closely controlled by protein synthesis and degradation balance. Physical activity and mainly resistance exercise is a powerful stimulus to positive muscle protein balance. To understand how protein balance is regulated after exercise, cellular and molecular mechanisms leading to muscular hypertrophy and/or atrophy have to be elucidated. Our works point out that FDP muscular hypertrophy after 10 weeks of resistance training in rat is partly due to the chronically activation of IL-6/STAT3 signaling pathway, occurring in the activated satellite cell pool, after each single exercise bout. Once activated and engaged in the myogenic program, cells in which STAT1/STAT3 signaling pathway is activated, could downregulate MyoD and return to a quiescent state, leading to increase satellite cell reserve's pool. These events participate to enhance protein synthesis by the incorporation of new genetic material into muscle fiber leading to increase their cross sectional area and phenotypic shift after training. As resistance exercise increases proteolysis, our works attempt to characterize the proteolysis systems (lysosomal-autophagic, ubiquitin-proteasome) involved in protein balance after exercise. The molecular markers measured ( chymotrypsin-like and cathepsin L activities, protein and gene expressions of LC3B, E3 ligases…) could not explain the +30% of proteolysis obtained one hour after resistance eccentric contractions on EDL muscle of starved rats. Further studies based on calpains, caspases and metalloproteinase activities and/or expressions should bring us valuable information.

É proposto que vias de sinalização controlem a sobrevivência e adaptação do Plasmodium, nos diferentes hospedeiros. No presente trabalho buscamos por diferentes abordagens estudar a via de sinalização de melatonina em P. falciparum. Para isso, avaliamos os níveis de RNA mensageiro de genes do sistema-ubiquitina proteossomo (UPS) bem como o perfil de ubiquitinação resultante do tratamento de parasitas com melatonina. Mostramos que a proteína quinase 7 de P. falciparum (PfPK7) atua na modulação dos genes do UPS em resposta a melatonina. Avaliamos também se o parasita é responsivo ao ácido indol-3-acético (AIA). Sabendo-se da importância de receptores de membrana na regulação de diversas funções celulares incluindo a percepção do meio externo, buscamos caracterizar um receptor serpentino putativo identificado previamente pelo grupo. Pudemos concluir que a via de sinalização por melatonina em P. falciparum envolve a participação da PfPK7, uma vez que em parasitas nocautes para pfpk7 são irresponsivos à melatonina quando comparados ao parental. / It is proposed that signaling pathways can control the parasite survival and adaptation into the hosts. In the present work we inquire about to study the melatonin signaling pathway trhough different metodologies. For this purpose we have analized post-translational modification of melatonin signaling, through ubiquitin-proteasome system (UPS) mRNA levels as well as the profile of ubiquitination resulted of melatonin treatment when compared with control. Moreover, we have found here that the P. falciparum protein kinase 7 (PfPK7) plays a major role in ubiquitin-proteasome system mRNA modulation in response to melatonin since parasites knockout to pfpk7 gene do not upregulate the UPS genes in response to melatonin. As for melatonin we have evaluated if P. falciparum parasites were responsive to indoleacetic acid. Last but not least, we made an effort to characterize a putative serpentine receptor previously identified by our group. We conclude that melatonin signaling pathway involves PK7 participation since pfpk- parasites are irresponsives to melatonin.

No description available.

Le VIH-1 a développé plusieurs mécanismes menant à la dégradation de son récepteur cellulaire, la molécule CD4, dans le but d’augmenter la relâche de particules virales infectieuses et d’éviter que la cellule soit surinfectée. L’un de ces mécanismes est la dégradation, induite par la protéine virale Vpu, du CD4 nouvellement synthétisé au niveau du réticulum endoplasmique (RE). Vpu doit lier CD4 et recruter l’ubiquitine ligase cellulaire SCFβ-TrCP, via sa liaison à β-TrCP, afin de dégrader CD4. Puisque CD4 doit être retenu au RE pour permettre à Vpu d’induire sa dégradation via le système ubiquitine-protéasome, il a été suggéré que ce processus implique un mécanisme semblable à une voie cellulaire de dégradation des protéines mal-repliées appelée ERAD (« endoplasmic reticulum-associated degradation »). La dégradation par ERAD implique généralement la dislocation des protéines du RE vers le cytoplasme afin de permettre leur poly-ubiquitination et leur dégradation par le protéasome. Nous avons démontré que Vpu induit la poly-ubiquitination de CD4 dans des cellules humaines. Nos résultats suggèrent aussi que CD4 doit subir une dislocation afin d’être dégradé par le protéasome en présence de Vpu. De plus, un mutant transdominant négatif de l’ATPase p97, qui est impliquée dans la dislocation des substrats ERAD, inhibe complètement la dégradation de CD4 par Vpu. Enfin, nos résultats ont montré que l’ubiquitination sur des résidus accepteurs de l’ubiquitine (lysines) de la queue cytoplasmique de CD4 n’était pas essentielle, mais que la mutation des lysines ralentit le processus de dégradation de CD4. Ce résultat suggère que l’ubiquitination de la queue cytosolique de CD4 pourrait représenter un événement important dans le processus de dégradation induit par Vpu. L’attachement de l’ubiquitine a généralement lieu sur les lysines de la protéine ciblée. Toutefois, l’ubiquitination sur des résidus non-lysine (sérine, thréonine et cystéine) a aussi été démontrée. Nous avons démontré que la mutation de tous les sites potentiels d’ubiquitination cytoplasmiques de CD4 (K, C, S et T) inhibe la dégradation par Vpu. De plus, la présence de cystéines dans la queue cytoplasmique apparaît suffisante pour rendre CD4 sensible à Vpu en absence de lysine, sérine et thréonine. Afin d’expliquer ces résultats, nous proposons un modèle dans lequel l’ubiquitination de la queue cytosolique de CD4 serait nécessaire à sa dégradation et où les sites d’ubiquitination de CD4 seraient sélectionnés de façon non spécifique par l’ubiquitine ligase recrutée par Vpu. Enfin, nous avons observé que la co-expression d’une protéine Vpu incapable de recruter β-TrCP (Vpu S52,56/D) semble stabiliser le CD4 qui est retenu au RE. De plus, d’autres mutants de Vpu qui semblent capables de recruter β-TrCP et CD4 sont toutefois incapables d’induire sa dégradation. Ces résultats suggèrent que l’association de Vpu à CD4 et β-TrCP est essentielle mais pas suffisante pour induire la dégradation de CD4. Par conséquent, ces résultats soulèvent la possibilité que Vpu puisse recruter d’autres facteurs cellulaires pour induire la dégradation de CD4. Les résultats présentés ont permis de mieux définir le mécanisme de dégradation de CD4 par Vpu dans des cellules humaines. De plus, ces résultats nous ont permis d’élaborer un modèle dans lequel l’ubiquitine ligase cellulaire SCFβ-TrCP démontre de la flexibilité dans le choix des résidus à ubiquitiner afin d’induire la dégradation de CD4. Enfin, ces études jettent un oeil nouveau sur le rôle de Vpu dans ce processus puisque nos résultats suggèrent que Vpu doive recruter d’autres partenaires cellulaires, mis à part β-TrCP, pour induire la dégradation de CD4. / HIV-1 has developed many mechanisms leading to the down-regulation of its cellular receptor, the CD4 molecule, in order to increase the release of infectious viral particles and to inhibit superinfection of the target cell. One of these mechanisms is the HIV-1 Vpu-mediated degradation of newly synthesized CD4 at the level of endoplasmic reticulum (ER). Vpu must interact with CD4 and recruit the cellular ubiquitin ligase SCFβ-TrCP, via its binding to β-TrCP, in order to induce CD4 degradation. Because CD4 has to be retained in the ER to allow Vpu to induce its degradation via the ubiquitin-proteasome system, it has been suggested that this process involves a mechanism reminiscent of a cellular degradation pathway involved in the proteolysis of unfolded proteins called ERAD (endoplasmic reticulum-associated degradation). The ERAD degradation usually involves the dislocation of proteins from the ER to the cytoplasm in order to induce their poly-ubiquitination and subsequent degradation by the proteasome. We demonstrated that Vpu induces the poly-ubiquitination of CD4 in human cells. Our results also suggest that CD4 has to be dislocated in order to be degraded by the proteasome in presence of Vpu. Furthermore, the expression of a transdominant negative mutant of the ATPase p97, that is involved in the dislocation of ERAD substrates, inhibits completely the Vpu-mediated CD4 degradation process. Finally, our results demonstrated that the ubiquitination of putative ubiquitin acceptor residues (lysines) in the cytosolic tail of CD4 is not essential but the mutation of these lysines slowed down the process of CD4 degradation induced by Vpu. This results suggests that ubiquitination of CD4 cytosolic tail could represent an important step during Vpu-mediated CD4 degradation. Ubiquitin is usually attached on lysine residues in the targetted protein. However, the ubiquitination on non-lysine residues (S, T and C) has also been demonstrated. We demonstrated that the mutation of all cytosolic potential ubiquitination sites (K, C, S and T) of CD4 abolishes Vpu-mediated degradation. In addition, the presence of cysteines in the cytosolic tail of CD4 appeared sufficient to render CD4 sensitive to Vpu in absence of lysine, serine or threonine. In order to explain these results, we propose a model in which CD4 cytosolic tail ubiquitination is necessary for its degradation and where ubiquitination sites are selected non specifically by the ubiquitin ligase recruited by Vpu. Finally, we observed that co-expression of a phosphorylation mutant of Vpu unable to interact with β-TrCP (Vpu S52,56/D) appears to stabilize ER-retained CD4 molecules. In addition, other Vpu mutants seem able to recruit β-TrCP and CD4 without inducing CD4 degradation. These results suggest that Vpu association with CD4 and β-TrCP is essential but not sufficient for CD4 degradation. Consequently, these results raised the possibility that other cellular factors could be recruited by Vpu in order to induce CD4 degradation. The results presented here allowed us to better define the mechanism underlying Vpu-mediated CD4 degradation. In addition, these results allowed us to elaborate a model in which the ubiquitin ligase SCFβ-TrCP show some flexibility in the choice of ubiquitination sites in order to induce CD4 degradation. Finally, theses studies shed a new light on the role of Vpu in the CD4 degradation process because our results suggest that Vpu could recruit, in addition of β-TrCP, other cellular partners in order to induce CD4 degradation.

Disfunções da glândula tireóide são, frequentemente, associadas a manifestações cardiovasculares e, em situações de hipertireoidismo, o coração hipertrofia. A hipertrofia cardíaca (HC) consiste em uma resposta adaptativa caracterizada pelo aumento de síntese de proteínas estruturais. O Sistema Ubiquitina Proteassoma (UPS) corresponde ao principal mecanismo de proteólise intracelular e crescentes evidências sugerem seu envolvimento no desenvolvimento da HC. O objetivo do presente estudo foi avaliar a modulação do UPS no tecido cardíaco de animais submetidos ao hipertireoidismo. Os resultados referentes ao aumento da atividade e expressão do proteassoma (PT) cardíaco apresenta-se mais contundente no grupo tratado por 7 dias, período em que a HC já encontra-se estável. Ao término de 14 e 21 dias, a modulação desse sistema tende à normalização. Os resultados obtidos atestam evidências da literatura que sugerem o aumento da atividade do PT cardíaco como resposta compensatória ao aumento de síntese proteica. / Thyroid gland disorders are often associated with cardiovascular events and hyperthyroidism state promotes cardiac hypertrophy (CH). CH consists in adaptive response characterized by increased synthesis of structural proteins. The Ubiquitin Proteasome System (UPS) is the major mechanism of intracellular proteolysis and increased evidences suggest its involvement in the development of CH. The aim of this study was to evaluate the modulation of UPS in cardiac tissue of animals subjected to hyperthyroidism. The results related to the increased proteasome (PT) activity and expression in the heart was more accentuated in the group treated for 7 days, when the CH process finds stable. At the end of 14 and 21 days of hyperthyroidism, the modulation of cardiac UPS achieves standard values. These results suggest an increased activity of cardiac PT as a compensatory response to protein synthesis induced by thyroid hormones.