Yes / Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects. However, the DUBs that play a role in this process have not been identified. In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5. We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance.

Deubiquitinating enzymes (DUBs) constitute a diverse protein family. The specificities and functions of the majority of DUBs are unknown, although their impact on many biological and pathological processes is widely appreciated. This dissertation entails a detailed characterization of otubain 1 (OTUB1), an ovarian tumor domain-containing DUB. The presented work describes OTUB1’s specificity, localization, protein interactions, importance in infection with Yersinia, and proposes a novel model of regulation of its enzymatic activity. I first discuss the structural and biochemical properties of OTUB1, demonstrating its selectivity towards ubiquitin and NEDD8. Moreover, I show that OTUB1 cleaves lys48- but not lys63-linked polyubiquitin, emphasizing its role in ubiquitin-mediated proteasomal degradation. Mass spectrometric identification of interaction partners and localization studies suggest possible involvement of OTUB1 in RNA processing and cell morphology. Furthermore, I demonstrate that invasion of the host cells by the enterobacteria Yersinia can be altered by changing OTUB1 expression. This effect is dependent on the catalytic activity of OTUB1 and its ability to stabilize RhoA-GTP prior to infection. YpkA and OTUB1 modulate RhoA-GTP stability in opposing ways, leading to cytoskeletal rearrangements that may be involved in bacterial invasion. Moreover, OTUB1 is post-translationally modified by phosphorylation that modulates its ability to stabilize RhoA-GTP and counteracts its effect on bacterial invasion. These findings provide a novel entry point for the manipulation of the host—pathogen interactions. Lastly, a kinase screen revealed that FER, an oncogenic kinase with a role in cell morphology, phosphorylates OTUB1, as demonstrated by overexpression, siRNA and in vitro studies. The phosphorylated site was mapped to tyr26 and the activity-based labeling revealed that this modification interferes with the deubiquitinating activity of OTUB1. In summary, the results presented in this thesis confirm that OTUB1 exerts properties of a “classical DUB” and uncover some of its physiological functions.

O proteassoma 26S é uma estrutura em forma de barril com um núcleo catalítico 20S que é flanqueado por tampas 19S em ambos os lados. Nosso grupo tem demonstrado que o proteassoma 26S é crítico para o desenvolvimento e sobrevivência do Schistosoma mansoni, sendo que mais de 95% dos casais de vermes tratados com o inibidor de proteassoma MG132 apresentaram alteração na postura dos ovos e viabilidade. O tampão 19S é o complexo regulador e funciona no desdobramento e na desubiquitinação das proteínas, antes de sua entrada no complexo 20S, usando enzimas desubiquitinadoras (DUBS). Recentemente, foi demonstrado que a inibição das DUBS, UCHL5 e USP14, que estão reversivelmente ligadas à partícula 19S do complexo 26S, chamada b-AP15, resulta em autofagia, seguida de morte celular, pois leva uma variedade de células de mamíferos ao estresse oxidativo. O objetivo geral deste trabalho foi avaliar o efeito da droga sobre o desenvolvimento reprodutivo de S. mansoni e como possível alvo terapêutico, tendo como objetivos específicos determinar os seus efeitos: na inibição da oviposição in vitro de casais de parasitas Schistosoma mansoni através da contagem dos ovos e expressão do gene p14; avaliar se os órgãos reprodutores dos parasitas sofreram alguma modificação estrutural; se a substância utilizada leva a autofagia; se o tratamento dos casais de parasita com a droga leva a apoptose, ocasionada pela ativação de caspase-3. Este estudo é o primeiro a documentar o papel da droga b-AP15 como um agente esquistossomicida, pois desencadeia alterações ultraestruturais em casais de vermes de S. mansoni. Os seguintes métodos foram utilizados para analisar as alterações: Microscopia Eletrônica de Transmissão (MET), Varredura (MEV) e Confocal; ensaio quantitativo colorimétrico baseado no brometo de 3- (4,5-dimetiltiazol-2-il) -2,5-difenil tetrazólio (MTT), atividade do proteassoma através do substrato Suc-Leu-Le-Al-Tyr-AMC específico para atividade de quimotripsina-like; western blotting; Reação de Polimerase em Cadeia em tempo real e TUNEL. Foram testadas várias concentrações do fármaco (0,2; 0,4; 0,8; 1,6; 3,2 até 50 µM), sendo que a partir de 1,6 µM de b-AP15 ocorreu a inibição da produção de ovos dos casais de parasitas tratados in vitro por 24 h, não havendo alteração da viabilidade, mas mostrou alterações a partir da dosagem de 3,2 µM. A partir de 1,6 µM ocorreram alterações celulares e tegumentares e vermes adultos tratados com 50 µM estavam mortos. O Western blotting mostrou acúmulo de proteínas poliubiquitinadas de alto peso molecular na presença de 1,6µM, havendo mudanças na atividade quimotripsina-like do proteassoma 20S. Os resultados mostraram que o b-AP15 altera a oviposição, a viabilidade e leva à morte de casais de parasitas, reforçando a hipótese de que o sistema ubiquitina/ proteassoma e que as enzimas deubiquitinadoras ligadas a partícula 19S são essenciais para a biologia de S. mansoni. / The 26S proteasome is a barrel structure with a catalytic core 20S that is flanked by 19S caps on both sides. Our group has been showed that 26S proteasomes are critical for Schistosoma mansoni development and survival, being that more than 95% of worms pairs treated with the proteasome inhibitor MG132 showed alteration on egg laying and viability. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14, which are reversibly bound to the 19S particle of the 26S complex, called b-AP15 , results in cell death because it leads to autophagy, followed by cell death in a variety of mammalian cells. The general objective of this work was to evaluate the effect of the drug on the reproductive development of S. mansoni and as a possible therapeutic target. The specific objectives to determine its effects: in inhibiting the in vitro oviposition of couples of Schistosoma mansoni parasites by counting eggs and p14 gene expression; evaluate whether the reproductive organs of the parasites have undergone some structural modification; if the substance used leads to autophagy; if the treatment of the parasite couples with the drug leads to apoptosis, caused by the activation of caspase-3. This study is the first to document the role of the b-AP15 drug as a schistosomicidal agent, as it triggers ultrastructural changes in couples of S. mansoni worms. The following methods were used to analyze the changes: Transmission Electron Microscopy (MET), Scanning (SEM) and Confocal; Colorimetric assay based on 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), proteasome activity through Suc-Leu-Le-Al-Tyr-AMC substrate specific for chymotrypsin-like activity; western blotting; Polymerase chain reaction in real time and TUNEL. Various concentrations of the drug (0.2, 0.4, 0.8, 1.6, 3.2 to 50 ?M) were tested. 1.6 ?M b-AP15 occurred the inhibition of egg production of couples of parasites treated in vitro for 24 h, with no change in viability, but showed changes from the dosage of 3.2 ?M. Cell and tegumentary changes occurred in 1.6 ?M and adult worms treated with 50 ?M were dead. Western blotting showed accumulation of high molecular weight polyubiquitinated proteins in the presence of 1.6?M, with changes in the chymotrypsin-like activity of the 20S proteasome. The results showed that b-AP15 alters the oviposition, viability and leads to the death of couples of parasites, reinforcing the hypothesis that the ubiquitin / proteasome system and the deubiquitinating enzymes bound to the 19S particle are essential for the biology of S. mansoni.

O proteassoma 26S é uma estrutura em forma de barril com um núcleo catalítico 20S que é flanqueado por tampas 19S em ambos os lados. Nosso grupo tem demonstrado que o proteassoma 26S é crítico para o desenvolvimento e sobrevivência do Schistosoma mansoni, sendo que mais de 95% dos casais de vermes tratados com o inibidor de proteassoma MG132 apresentaram alteração na postura dos ovos e viabilidade. O tampão 19S é o complexo regulador e funciona no desdobramento e na desubiquitinação das proteínas, antes de sua entrada no complexo 20S, usando enzimas desubiquitinadoras (DUBS). Recentemente, foi demonstrado que a inibição das DUBS, UCHL5 e USP14, que estão reversivelmente ligadas à partícula 19S do complexo 26S, chamada b-AP15, resulta em autofagia, seguida de morte celular, pois leva uma variedade de células de mamíferos ao estresse oxidativo. O objetivo geral deste trabalho foi avaliar o efeito da droga sobre o desenvolvimento reprodutivo de S. mansoni e como possível alvo terapêutico, tendo como objetivos específicos determinar os seus efeitos: na inibição da oviposição in vitro de casais de parasitas Schistosoma mansoni através da contagem dos ovos e expressão do gene p14; avaliar se os órgãos reprodutores dos parasitas sofreram alguma modificação estrutural; se a substância utilizada leva a autofagia; se o tratamento dos casais de parasita com a droga leva a apoptose, ocasionada pela ativação de caspase-3. Este estudo é o primeiro a documentar o papel da droga b-AP15 como um agente esquistossomicida, pois desencadeia alterações ultraestruturais em casais de vermes de S. mansoni. Os seguintes métodos foram utilizados para analisar as alterações: Microscopia Eletrônica de Transmissão (MET), Varredura (MEV) e Confocal; ensaio quantitativo colorimétrico baseado no brometo de 3- (4,5-dimetiltiazol-2-il) -2,5-difenil tetrazólio (MTT), atividade do proteassoma através do substrato Suc-Leu-Le-Al-Tyr-AMC específico para atividade de quimotripsina-like; western blotting; Reação de Polimerase em Cadeia em tempo real e TUNEL. Foram testadas várias concentrações do fármaco (0,2; 0,4; 0,8; 1,6; 3,2 até 50 µM), sendo que a partir de 1,6 µM de b-AP15 ocorreu a inibição da produção de ovos dos casais de parasitas tratados in vitro por 24 h, não havendo alteração da viabilidade, mas mostrou alterações a partir da dosagem de 3,2 µM. A partir de 1,6 µM ocorreram alterações celulares e tegumentares e vermes adultos tratados com 50 µM estavam mortos. O Western blotting mostrou acúmulo de proteínas poliubiquitinadas de alto peso molecular na presença de 1,6µM, havendo mudanças na atividade quimotripsina-like do proteassoma 20S. Os resultados mostraram que o b-AP15 altera a oviposição, a viabilidade e leva à morte de casais de parasitas, reforçando a hipótese de que o sistema ubiquitina/ proteassoma e que as enzimas deubiquitinadoras ligadas a partícula 19S são essenciais para a biologia de S. mansoni. / The 26S proteasome is a barrel structure with a catalytic core 20S that is flanked by 19S caps on both sides. Our group has been showed that 26S proteasomes are critical for Schistosoma mansoni development and survival, being that more than 95% of worms pairs treated with the proteasome inhibitor MG132 showed alteration on egg laying and viability. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14, which are reversibly bound to the 19S particle of the 26S complex, called b-AP15 , results in cell death because it leads to autophagy, followed by cell death in a variety of mammalian cells. The general objective of this work was to evaluate the effect of the drug on the reproductive development of S. mansoni and as a possible therapeutic target. The specific objectives to determine its effects: in inhibiting the in vitro oviposition of couples of Schistosoma mansoni parasites by counting eggs and p14 gene expression; evaluate whether the reproductive organs of the parasites have undergone some structural modification; if the substance used leads to autophagy; if the treatment of the parasite couples with the drug leads to apoptosis, caused by the activation of caspase-3. This study is the first to document the role of the b-AP15 drug as a schistosomicidal agent, as it triggers ultrastructural changes in couples of S. mansoni worms. The following methods were used to analyze the changes: Transmission Electron Microscopy (MET), Scanning (SEM) and Confocal; Colorimetric assay based on 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), proteasome activity through Suc-Leu-Le-Al-Tyr-AMC substrate specific for chymotrypsin-like activity; western blotting; Polymerase chain reaction in real time and TUNEL. Various concentrations of the drug (0.2, 0.4, 0.8, 1.6, 3.2 to 50 ?M) were tested. 1.6 ?M b-AP15 occurred the inhibition of egg production of couples of parasites treated in vitro for 24 h, with no change in viability, but showed changes from the dosage of 3.2 ?M. Cell and tegumentary changes occurred in 1.6 ?M and adult worms treated with 50 ?M were dead. Western blotting showed accumulation of high molecular weight polyubiquitinated proteins in the presence of 1.6?M, with changes in the chymotrypsin-like activity of the 20S proteasome. The results showed that b-AP15 alters the oviposition, viability and leads to the death of couples of parasites, reinforcing the hypothesis that the ubiquitin / proteasome system and the deubiquitinating enzymes bound to the 19S particle are essential for the biology of S. mansoni.

No description available.

Ubiquitin (Ub) wird meist in Kettenform an Lysine von anderen Proteinen gekoppelt und bestimmt somit, ob das Substrat beispielsweise vom Proteasom abgebaut oder an der Reparatur des Erbguts beteiligt wird. Die Regulierung dieses Signals ist entscheidend für die zelluläre Homöostase, Fehler führen zu Krankheiten wie Parkinson und Krebs. Die wohl wichtigsten Regulatoren sind deubiquitinierende Enzyme (DUBs). Ein tiefes Verständnis über deren enzymatischen Mechanismus ist daher unerlässlich für die Entwicklung von therapeutischen Ansätzen. Biochemische Methoden ermöglichten bisher grundlegende Erkenntnisse darüber, wie DUBs Ubiquitinketten prozessieren. Allerdings werden dabei viele überlappende Reaktionen analysiert, wodurch viele relevante Details schwer oder gar nicht erkennbar sind. Diese Arbeit bietet durch die Etablierung einer Einzelmolekül-Mikroskopie-Methode eine neue Perspektive auf die Interaktion zwischen DUBs und Ubiquitinketten. Dafür wurden Messkammern mit Ubiquitinketten bestückt. Verschiedene DUBs und die Ubiquitinketten wurden mit Fluorophoren markiert und mit Hilfe von interner Totalreflexions-Fluoreszenz-Mikroskopie analysiert. Am Beispiel des DUBs USP5 konnten dadurch erstmals die Bindung sowie das darauffolgende Schneiden einer Ubiquitinkette direkt und in Echtzeit beobachtet werden. Anschließende Auswertungen gewährten neue Einblicke in die Unterschiede zwischen produktiven und unproduktiven Bindungen und auch auf das heterogene Bindungsverhalten von USP5 vor dem Schneiden der Kette. Mit Anpassungen am Versuchsaufbau konnten am Beispiel von USP2 und drei verschiedenen Ubiquitinketten diverse kinetische Parameter bestimmt werden. Diese Ergebnisse unterstützen ein bestehendes Modell, nach dem USP2 nach der Bindung eine Konformationsänderung durchlaufen muss, um Ubiquitinketten zu schneiden. Zusätzlich weisen die Messungen darauf hin, dass diese Konformationsänderung der entscheidende Faktor für die Substratspezifität von USP2 ist. / Ubiquitin (Ub) is a post-translational modification attached to substrate proteins, often in the form of Ub chains. Depending on the length and linkage type of those chains the substrates can, for example, be targeted for proteasomal degradation or initiate DNA repair mechanisms. Regulation of this signal is crucial for cellular homeostasis and errors cause diseases like Parkinson´s or cancer. The main regulators are deubiquitinating enzymes (DUBs) which cleave Ub chains. Deeply understanding their enzymatic mechanism is necessary for the development of medical intervention strategies. Biochemical methods have enabled a fundamental understanding about Ub cleavage. However, this results from population measurements, where individual mechanistic details are hard or impossible to be determined. By establishing a single-molecule microscopy method, the work in hand offers a new perspective on the enzymatic reactions between DUBs and Ub chains. Here, self-made imaging chambers were decorated with Ub chains. These Ub chains and a selection of DUBs were labelled with fluorescent dyes and analysed by total internal reflection fluorescence microscopy in real-time. Using USP5, this set-up enabled a DUB-Ub chain interaction with subsequent cleavage reaction to be directly observed for the first time. In-depth analyses revealed several insights into the enzymatic mechanism, including a clear difference between productive and unproductive binding events, as well as two populations of USP5 with different binding times before Ub chain cleavage. By adapting the experimental set-up, several kinetic parameters were determined on the example of USP2 and three differently linked Ub chains. These measurements provided evidence for a published model proposing that USP2 must undergo a conformational change after Ub chain binding to enable processing. The analyses further indicated this conformational change to be the crucial factor for USP2 substrate specificity.