The Hap2/3/4/5 complex is a heme-activated, CCAATT binding, global transcriptional activator of genes involved in respiration and mitochondrial biogenesis in the yeast species Saccharomyces cerevisiae. Hap4 is the regulatory subunit of the complex and its levelsdetermine the activity of the complex. Hap4 is known to play a signaling role in response toenvironmental conditions; however, little is known about the regulation of Hap4 levels or how it responses to a cell’s functional state. The activity of the Hap2-5 complex is known to be reduced in respiratory-deficient cells. In Liu Lab, it has previously been found that a link between Hap4 stability, mediated through 26S proteasome-dependent degradation, and dependence on mitochondrial functional state plays a regulatory role on downstream targets of the Hap complex. However, the mechanism behind this regulation is still largely unknown. In normally functioning yeast cells, Hap4 is a highly unstable protein with a half-life of ~10 min. We have observed that loss of mitochondrial DNA in respiratory deficient rho 0 cells has a role in the further destabilization of Hap4 to a half-life of ~4 min through the ubiquitin-proteasome pathway. Through the screening of a collection of mutants defective in E2 ubiquitin-conjugating enzymes, we show that Hap4 is greatly stabilized in ubc1Δubc4Δ double mutant cells. We also show that Hap4 stabilization in the ubc1Δubc4Δ mutant leads to increased activity of the Hap2-5 complex, indicating that mitochondrial biogenesis in yeast is regulated by the functional state of mitochondria through ubiquitin/proteasome-dependent degradation of Hap4. Furthermore, studies on Hap4 mutants involving two highly conserved cysteine residues led to a proposed mechanism behind the regulation of Ubc4 activity towards Hap4 in response to changes in the cellular redox state.

Câncer se destaca no contexto de patologias por ser uma das doenças que mais acometem mortes por ano, sendo caracterizada como um conjunto de doenças multifatoriais que tem em comum o crescimento desordenado de células que invadem tecidos e órgãos, podendo espalhar-se para outras regiões do corpo, dando origem às metástases. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem carácter invasivo e migratório, além de se tornarem mais resistentes às drogas. Durante este processo, ocorrem inúmeras alterações celulares que modificam a estabilidade proteica e/ou promovem sua translocação subcelular, o transporte de proteínas para a membrana, alterações no citoesqueleto e incluindo o envio de proteínas para degradação pelo proteassoma. A desregulação de fatores de transcrição e modificação pós traducional de proteínas são fatores que podem levar à EMT. Após a eficiente indução da EMT in vitro utilizando o inibidor de histonas deacetilase (SAHA) em células de adenocarcinoma de mama MCF-7, foram realizadas análises proteômicas envolvimento os inibidores relacionados ao sistema ubiquitina proteassoma, MG132 e P5091. A modulação por inibição de USP7 resultou em variação da expressão de diversas proteínas biomarcadoras da EMT (SNAIL, ?-Catenina, CDK1) e proteínas envolvidas no ciclo celular (P53 e CDK1). O estudo proteômico permitiu a correlação do processo da EMT por SAHA com as vias de modificações pós traducionais relacionadas ao sistema ubiquitina proteassoma, e ainda propõe USP7 como alvo de estudos detalhados para EMT com potencial proposta terapêutica / Cancer stands out in the context of pathologies because it is one of the diseases that most affect deaths per year, being characterized as a set of multifactorial diseases that has in common the disordered growth of cells that invade tissues and organs, being able to spread to other regions of the body, giving rise to metastases. An important step in the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of the epithelial phenotype and acquisition of the mesenchymal phenotype by the tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. During this process, numerous cellular alterations occur that modify the protein stability and/or promote its subcellular translocation, the transport of proteins to the membrane, changes in the cytoskeleton and including the sending of proteins for degradation by the proteasome. Deregulation of transcription factors and posttranslational modification of proteins are factors that can lead to EMT. After an efficient induction of EMT using the histone deacetylase inhibitor (SAHA) in MCF-7 breast adenocarcinoma cells, proteomic analyzes were performed involving inhibitors related to the ubiquitin proteasome system, MG132 and P5091. Modulation by inhibition of USP7 resulted in varying expression of various EMT biomarker proteins (SNAIL, ?-Catenina, CDK1) and cell cycle (P53 e CDK1). The proteomic study allowed the correlation of the SAHA EMT process with the posttranslational modifications pathways related to the ubiquitin proteasome system and also proposes USP7 as the target of detailed studies for EMT with potential therapeutic proposal

A esquistossomose mansônica é a segunda maior endemia parasitária do mundo em termos de extensão das áreas endêmicas e do número de pessoas infectadas com 200 milhões de pessoas acometidas. Esta doença é causada pelo parasito trematódeo Schistosoma mansoni, o qual apresenta adequados mecanismos de resposta ao estresse envolvendo a regulação da expressão gênica e proteica, reparo ou substituição de moléculas danificadas, recuperação do balanço redox, controle do ciclo celular e apoptose. O sistema ubiquitina- proteassoma é importante para manter a homeostase proteica durante o estresse celular. Inibidores do proteassoma podem interferir em processos como crescimento, progressão do ciclo celular e replicação, e os seus efeitos vem sendo caracterizados em muitos parasitos. Nosso laboratório demonstrou que MG132 reduz o número de esquistossômulos, a carga parasitária e a ovoposição em camundongos infectados com S. mansoni. Neste trabalho, são descritos os efeitos in vitro do estresse oxidativo, choque térmico e estresse químico em vermes adultos de S. mansoni. Observou-se alteração no perfil de expressão proteica durante estresse oxidativo e térmico, sendo identificadas dezoito proteínas upreguladas nestas condições. Estas proteínas estão envolvidas em muitas vias intracelulares como dobramento de proteínas, proteólise, ligação a íons cálcio, regulação de proteínas e resposta a estresse. Além disso, o estresse oxidativo gerou mudanças em vermes adultos de S. mansoni em processos como produção de ovos, motilidade, morfologia do tegumento, viabilidade e pareamento dos vermes. O estresse químico induzido com Curcumina, IBMX e MG132 aumentou a produção de ROS intracelular e alterou o perfil de expressão de enzimas antioxidantes em S. mansoni. As enzimas SmGPx1 e SmPGx2 tiveram a expressão aumentada no estresse com Curcumina e IBMX, enquanto que SmSOD e SmTGR foram induzidas no estresse com Curcumina. As enzimas do proteassoma SmHul5 e SmUbp6 tiveram a expressão modulada durante o estresse oxidativo, choque térmico e estresse químico. Em adição, a análise de expressão no ciclo de vida de S. mansoni revelou que estes genes apresentam um nível alto de expressão em esporocistos, esquistossômulos e miracídios. Estes resultados sugerem que estas proteínas acessórias do proteassoma participam da resposta ao estresse e desenvolvimento do parasito. O nível de expressão de SmHul5 e SmUbp6 foi cerca 9 e 16 vezes menor em relação ao controle no estresse químico induzido com IBMX, respectivamente, sugerindo a desmontagem do proteassoma. Por outro lado, Curcumina, MG132, estresse oxidativo e choque térmico aumentaram o nível de expressão de SmHul5 e SmUbp6. Além disso, o nível de expressão da proteína de maturação do proteassoma (SmPOMP) aumentou no estresse com Curcumina, MG132 e estresse oxidativo, sugerindo a síntese de novas populações de proteassoma. Em relação ao estresse oxidativo, nós demonstramos o aumento no nível proteico de proteassoma 20S e da subunidade alfa-3 do proteassoma sugerindo que em S. mansoni as proteínas oxidadas são degradadas pelo proteassoma 20S. Além do mais, nós observamos que vermes adultos de S. mansoni parecem utilizar mecanismos de resposta similares para diferentes estresses. Nossos resultados demonstraram que o estresse oxidativo, choque térmico e estresse químico modificam o perfil de expressão de genes relacionados ao sistema ubiquitina-proteassoma e sugerem que o proteassoma é importante para as respostas celulares ao estresse neste parasito. / Schistosomiasis is a neglected tropical disease caused by blood flukes (genus Schistosoma) and affecting 200 million people worldwide. This disease continues to rank, following malaria, at the second position of the world\'s parasitic diseases in terms of the extent of endemic areas and the number of infected people. There are different types of stress and the organisms have many mechanisms to respond to these stressor agents. The responses involve the regulation of gene and protein expression and consist in events such as repair or substitution of damaged molecules, recovery of redox balance, cell cycle control and apoptosis. The proteasomal system is important to support the protein homeostasis during the cellular stress. Effect of proteasome inhibitors has been described in many protozoans, either inhibiting growth or cell cycle progression, or blocking replication. Our laboratory\'s results have shown that MG132 reduces the number of lung stage schistosomula, the worm burden and consequently decreases oviposition in S. mansoni-infected mice. Here, we describe the in vitro effects of oxidative stress, heat shock and chemical stress in S. mansoni adult worms. We report that the oxidative stress and heat shock cause drastic changes in the protein profile of S. mansoni adult worms, and we identified a total of eighteen upregulated proteins in these conditions. These proteins are involved with many intracellular pathways as protein folding, proteolysis, calcium ion binding, regulator proteins and stress response. In addition, oxidative stress induced with H2O2 generated significative changes in the adult worms concerning process such as egg production, motor activity, tegument morphology, viability and pairing of worms. Chemical stress induced with Curcumin, IBMX and MG132 increases ROS production and changes the gene expression profile of antioxidant enzymes of S. mansoni adult worms. The enzymes SmGPx1 and SmGPx2 were upregulated in Curcumin and IBMXinduced chemical stress, and both SmSOD and SmTGR were upregulated- Curcumin. The proteasomal enzymes SmHul5 and SmUbp6 had their gene expression modified during oxidative stress, heat shock and chemical stress. Besides of, expression analyses in the S. mansoni life cycle indicate that genes are different express in sporocyst, schistosomula and miracidia. These results suggest these accessory proteins proteasome participates of stress response and parasite development. The expression level of SmHul5 and SmUbp6 were 16 and 9 times less than the control in chemical stress induced by IBMX, and we suggest that these results are due to the proteasome disassembling. On the other hand, Curcumin, MG132, oxidative stress e heat shock increases the expression of SmHul5 and SmUbp6. Furthermore, the expression level of maturation proteasome protein (SmPOMP) increases in stress induced by Curcumin, MG132 and oxidative stress suggesting new proteasome synthesis. In addition, we demonstrate increase the both 20S level and alpha-3 subunit proteasome in the oxidative stress, suggesting that in S. mansoni oxidized protein formed due to oxidative damage are degrade by proteasome 20S. We observed that S. mansoni adult worms utilize similar mechanisms to respond different stresses. Ours results demonstrate that oxidative stress, heat shock and chemical stress modified the expression profile of genes related with the ubiquitinproteasome system and suggest that the proteasome is important to responses the cellular stresses in the parasite.

The work in this thesis is divided into two parts; the first is the synthesis of a ‘switch-on’ fluorophore to measure cell viability, and the second is the development of a fluorescent detection method for protein−peptide affinity assays applied in the identification of protein-protein inhibitors. Tetrazolium salts are often used in cytotoxicity assays as indicators of cell viability as they are reduced to deeply coloured formazans exclusively in healthy cells. However, measuring the absorbance of the formazan is prone to bias from other coloured species in the cell media, requires solubilisation and can be difficult to quantify. A preferable method of detection is direct fluorescence as it is easily quantified, more sensitive and would ideally remove the need to solubilise the insoluble dye. The aim of this project was to synthesise a tetrazolium salt that could be reduced to a soluble fluorescent formazan in healthy cells as an indicator of cell viability. A number of fluorescent formazans were synthesised by incorporation of a fluorophore. The corresponding tetrazolium salts were non-fluorescent and could be reduced to the formazan in vitro. Several formazans were synthesised to attempt to increase the emission wavelength and intensity to overcome cellular autofluorescence. Protein-protein interactions have been implicated in the pathogenesis of many human diseases but until recently were considered undruggable. However, peptides have emerged as ideal compounds for targeting the large and relatively featureless protein interfaces. Work focussed on the discovery of peptide inhibitors for the E3 ubiquitin ligase stationary-phase kinase associated protein (Skp2). Potential peptide inhibitors were identified using CelluSpot synthesis and array technology to screen peptide libraries. Qualitative analysis of the protein affinity assay results by enhanced chemiluminescent detection was found to be misleading, and so a quantifiable and more sensitive fluorescent detection method was developed.

L’apoptose ou mort cellulaire programmée joue un rôle prépondérant dans l’homéostasie cellulaire. Ce processus est très finement régulé par les protéines de la famille BCL-2 qui contrôlent la perméabilité de membrane mitochondriale externe et la libération du cytochrome c, deux événements majeurs précédant la mort cellulaire. Les protéines anti-apoptotiques de la famille BCL-2 contribuent à la tumorigenèse et sont impliquées dans la résistance des cancers aux molécules chimiothérapeutiques ; à ce titre, elles représentent des cibles importantes pour le développement de nouvelles thérapies. BCL2A1 est un membre anti-apoptotique de la famille BCL-2 impliqué dans la chimiorésistance de nombreuses tumeurs. La protéine BCL2A1 a pour caractéristique d’avoir une demi-vie courte due à sa dégradation constitutive par le système ubiquitine-protéasome. Ceci régule la stabilité et la fonction anti-apoptotique de BCL2A1 et représente un mécanisme suppresseur de tumeur majeur. Cependant, les enzymes qui contrôlent les modifications post-traductionnelles impliquées dans l’ubiquitination et la dégradation de BCL2A1 demeurent, à ce jour, inconnues. Dans la présente thèse, nous donnons un aperçu des acteurs et des mécanismes impliqués dans la régulation de l’ubiquitination de BCL2A1. Nous présentons des preuves que TRIM28 est une E3 ubiquitine-ligase pour BCL2A1. En effet, les protéines TRIM28 et BCL2A1 endogènes interagissent ensemble au niveau des mitochondries et la déplétion de TRIM28 diminue l’ubiquitination de BCL2A1. Nous montrons aussi que TRIM17 stabilise BCL2A1 en empêchant son interaction avec TRIM28 et son ubiquitination médiée par TRIM28, et que l’activité de GSK3 est impliquée dans l’inhibition de la dégradation de BCL2A1. Ainsi, BCL2A1 et son proche homologue MCL-1 sont régulés par des facteurs communs mais de façon opposé. Finalement, la surexpression de TRIM28 ou l’inactivation de TRIM17 diminue le niveau protéique de BCL2A1 et restaure la sensibilité des cellules de mélanomes aux thérapies utilisant des inhibiteurs de la kinase BRAF. Globalement, nos résultats décrivent un rhéostat moléculaire au sein duquel deux protéines de la famille TRIM régulent de façon antagoniste la stabilité de BCL2A1 et modulent ainsi la mort cellulaire. / Apoptosis or programmed cell death plays a crucial role in tissue homeostasis and is regulated by the Bcl-2 proteins, which control mitochondria membrane permeability and cytochrome c release, two events that precede cell demise. Anti-apoptotic Bcl-2 family members can contribute to tumorigenesis and cause resistance to anti-cancer regimens, therefore representing important targets for novel therapeutics. BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.Sommaire

Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR

The ubiquitin proteasome system (UPS) depends on three enzymes called E1, E2, and E3 to ubiquitinate proteins and several isopeptidases to de-ubiquitinate them. Ubiquitination serves as a post-translational modification that either tags proteins for degradation by the proteasome or serves to modulate their function. This dynamic system plays a role in synaptic plasticity and dysfunction of the UPS is associated a variety of neurodegenerative diseases. In this study, three inhibitors the UPS, ziram, clasto-lactacystin β-lactone (lactacystin) and G5 were employed to illuminate involvement of the UPS in long-term and short term plasticity in area CA1 of rat hippocampal slices. Ziram, lactacystin and G5 inhibits the E1 ubiquitin-activating enzyme, the proteasome and isopeptidases, respectively. It was found that UPS inhibition enhanced long-term plasticity, by specifically increasing the magnitude of long-term depression (LTD) and altered short term plasticity, measured with paired pulse facilitation (PPF), to varying degrees. These findings establish that the UPS may play a regulatory role in LTD and PPF, and the changes in PPF further indicate that the UPS may be acting presynaptically. Overall, the results suggest ubiquitination and proteasome-mediated proteolysis are important in both long-term and short-term plasticity.

Brain stem cardiovascular regulatory dysfunction during brain stem death is underpinned by an upregulation of nitric oxide synthase II (NOS II) in rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from blood pressure of comatose patients that disappears before brain stem death ensues. At the same time, the ubiquitin-proteasome system (UPS) is involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain stem death by engaging in both synthesis and degradation of NOS II in RVLM. In a clinically relevant experimental model of brain stem death using Sprague-Dawley rats, pretreatment by microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) antagonized the hypotension and reduction in the life-and-death signal elicited by intravenous administration of Escherichia coli lipopolysaccharide (LPS). On the other hand, pretreatment with an inhibitor of ubiquitin-recycling or UCH-L1 potentiated the elicited hypotension and blunted the prevalence of the life-and-death signal. Real-time polymerase chain reaction, Western blot, electrophoresis mobility shift assay, chromatin immunoprecipitation and co-immunoprecipitation experiments further showed that the proteasome inhibitors antagonized the augmented nuclear presence of NF-£eB or binding between NF-£eB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated I£eB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-£eB in RVLM. In animals pretreated with UCH-L1 inhibitor and died before significant increase in nos II mRNA occurred, NOS II protein expression in RVLM was considerably elevated. We conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain stem death by engaging in both synthesis and degradation of NOS II at RVLM. Our results provide information on new therapeutic initiatives against this fatal eventuality.

The ubiquitin proteasome system (UPS) depends on three enzymes called E1, E2, and E3 to ubiquitinate proteins and several isopeptidases to de-ubiquitinate them. Ubiquitination serves as a post-translational modification that either tags proteins for degradation by the proteasome or serves to modulate their function. This dynamic system plays a role in synaptic plasticity and dysfunction of the UPS is associated a variety of neurodegenerative diseases. In this study, three inhibitors the UPS, ziram, clasto-lactacystin β-lactone (lactacystin) and G5 were employed to illuminate involvement of the UPS in long-term and short term plasticity in area CA1 of rat hippocampal slices. Ziram, lactacystin and G5 inhibits the E1 ubiquitin-activating enzyme, the proteasome and isopeptidases, respectively. It was found that UPS inhibition enhanced long-term plasticity, by specifically increasing the magnitude of long-term depression (LTD) and altered short term plasticity, measured with paired pulse facilitation (PPF), to varying degrees. These findings establish that the UPS may play a regulatory role in LTD and PPF, and the changes in PPF further indicate that the UPS may be acting presynaptically. Overall, the results suggest ubiquitination and proteasome-mediated proteolysis are important in both long-term and short-term plasticity.

No description available.