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‣ Proteasome-dependent regulation of signal transduction in retinal pigment epithelial cells

Fernandes, Alexandre F.; Guo, Weimin; Zhang, Xinyu; Gallagher, Matthew; Ivan, Mircea; Taylor, Allen; Pereira, Paulo; Shang, Fu
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Artigo de Revista Científica Formato: aplication/PDF
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As in many other types of cells, retinal pigment epithelial (RPE) cells have an active ubiquitin-proteasome pathway (UPP). However, the function of the UPP in RPE remains to be elucidated. The objective of this study is to determine the role of the UPP in controlling the levels and activities of transcription factors hypoxia-inducible factor (HIF) and NF-[kappa]B. We inhibited the UPP with proteasome-specific inhibitors and determined the activation of HIF and NF-[kappa]B as well as the expression and secretion of pro-angiogenic factors. HIF-1[alpha] was not detectable in ARPE-19 cells under normal culture conditions. However, when proteasome activity was inhibited, HIF-1[alpha] accumulated in RPE in a time-dependent manner. Consistent with accumulation of HIF-1[alpha] in the cells, levels of mRNA for vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) in RPE were up to 7-fold higher upon inhibition of the proteasome. Proteasome inhibition was also associated with a 2-fold increase in levels of mRNA for angiopoietin-1 (Ang-1). ARPE-19 cells secrete significant levels of VEGF under normal culture conditions. Inhibition of proteasome activity increased the secretion of VEGF by 2-fold. In contrast to the increase in HIF activity...

‣ Interplay between the Ubiquitin-Proteasome System and mechanisms of neurodegeneration in the context of Machado-Joseph disease (Tese corrigida)

Baptista, Márcio
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Tese de Doutorado
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O Sistema Ubiquitina-Proteosoma (SUP) é um dos maiores sistemas de degradação regulada de proteínas. O sistema é essencial para a manutenção da homeostase e sinalização celulares. O tipo de ligação entre ubiquitinas e o seu comprimento vão definir o destino dos substratos ubiquitinados. A ubiquitinação de substratos pode ser revertida pela acção das enzimas desubiquitinadoras (DUBs). As DUBs podem actuar através da edição ou desmontagem das cadeias de poli-ubiquitina, são responsáveis pela clivagem dos precursores de ubiquitina, por evitar a degradação de substratos-alvo, pela edição das cadeias de ubiquitina à entrada do proteossoma e também pela manutenção das reservas celulares de ubiquitina livre. O papel do SUP nas doenças do sistema nervoso tem sido caracterizado ao longo dos últimos anos, e em particular nas doença de expansão de poliglutaminas, tendo começado por evidências de que componentes do SUP faziam parte dos agregados de proteínas desnaturadas. A ataxina 3 (ATXN3) é uma DUB presente em todos ou quase todos os tecidos do corpo humano e do murganho. As funções da ATXN3 vão desde a edição das proteínas poli-ubiquitinadas, ao sequestro de proteínas agregadas em agregossomas, à regulação da degradação proteica e resposta ao stress...

‣ Aging and calorie restriction modulate yeast redox state, oxidized protein removal, and the ubiquitin-proteasome system

CUNHA, Fernanda Marques da; DEMASI, Marilene; KOWALTOWSKI, Alicia J.
Fonte: ELSEVIER SCIENCE INC Publicador: ELSEVIER SCIENCE INC
Tipo: Artigo de Revista Científica
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The ubiquitin-proteasome system governs the half-life of most cellular proteins. Calorie restriction (CR) extends the maximum life span of a variety of species and prevents oxidized protein accumulation. We studied the effects of CR on the ubiquitin-proteasome system and protein turnover in aging Saccharomyces cerevisiae. CR increased chronological life span as well as proteasome activity compared to control cells. The levels of protein carbonyls, a marker of protein oxidation, and those of polyubiquitinated proteins were modulated by CR. Controls, but not CR cells, exhibited a significant increase in oxidized proteins. In keeping with decreased proteasome activity, polyubiquitinated proteins were increased in young control cells compared to time-matched CR cells, but were profoundly decreased in aged control cells despite decreased proteasomal activity. This finding is related to a decreased polyubiquitination ability due to the impairment of the ubiquitin-activating enzyme in aged control cells, probably related to a more oxidative microenvironment. CR preserves the ubiquitin-proteasome system activity. Overall, we found that aging and CR modulate many aspects of protein modification and turnover. (C) 2011 Elsevier Inc. All rights reserved.; FAPESP Fundacao de Amparo a Pesquisa do Estado De Sao Paulo; CNPq Conselho Nacional de Desenvolvimento Cientifico e Tecnologico; Instituto Nacional de Ciencia e Tecnologia de Processos Redox em Biomedicina (INCT)

‣ The ubiquitin-proteasome system in Strongyloididae. Biochemical evidence for developmentally regulated proteolysis in Strongyloides venezuelensis

PAULA, Fabiana M. de; CASTRO-BORGES, William; PEREIRA JUNIOR, Olavo S.; GOMES, Matheus de Souza; UETA, Marlene T.; RODRIGUES, Vanderlei
Fonte: SPRINGER Publicador: SPRINGER
Tipo: Artigo de Revista Científica
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Nematode parasites from the genus Strongyloides spp. are important pathogens of the intestinal mucosa of animals and humans. Their complex life cycles involve alternating developmental adaptations between larvae stages and the adult parthenogenetic female. Here, we report, primarily through homology-based searching, the existence of the major components of the ubiquitin-proteasome system in this genus, using the available EST data from S. ratti, S. stercoralis, and Parastrongyloides trichosuri. In this study, S. venezuelensis was used as our model organism for detection of proteasome activity and ubiquitinated substrates in cytosolic preparations from the L3 larvae and the adult female. Marked differences in proteasome capabilities were found when these two stages were compared. A preference for degradation of chymotryptic synthetic peptides was found in both stages with the adult exhibiting a higher rate of hydrolysis compared to the larvae. Due to the high evolutionary conservation of proteasome alpha subunits, an anti-human proteasome antibody was able to recognize proteasome subunits in these preparations by Western blotting, supporting the proposal that the activity of the ubiqutin-proteasome system is developmentally regulated in this nematode.; Conselho Nacional de Pesquisa e Desenvolvimento (CNPq)

‣ Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome

SILVA, Gustavo M.; NETTO, Luis E. S.; DISCOLA, Karen F.; PIASSA-FILHO, Gilberto M.; PIMENTA, Daniel C.; BARCENA, Jose A.; DEMASI, Marilene
Fonte: WILEY-BLACKWELL Publicador: WILEY-BLACKWELL
Tipo: Artigo de Revista Científica
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The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys-SH) into cysteine sulfenic acid (Cys-SOH), followed by S-glutathionylation (Cys-S-SG). Proteasome S-glutathionylation promotes partial loss of chymotrypsin-like activity and post-acidic cleavage without alteration of the trypsin-like proteasomal activity. Here we show that the 20S proteasome purified from stationary-phase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-glutathionylated 20S proteasome, allowing the recovery of chymotrypsin-like activity and post-acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S-glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non-stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS-PAGE and mass spectrometry, and S-glutathionylation was evaluated by either western blot analyses with anti-glutathione IgG or by spectrophotometry with the thiol reactant 7-chloro-4-nitrobenzo-2-oxa-1...

‣ Redox regulation of the proteasome via S-glutathionylation

Demasi, Marilene; Netto, Luis Eduardo Soares; Silva, Gustavo M.; Hand, Adrian; Oliveira, Cristiano Luis Pinto de; Bicev, Renata Naporano; Gozzo, Fabio; Barros, Mário Henrique de; Leme, Janaina de Moraes Maciel; Ohara, Erina
Fonte: Elsevier; Amsterdam Publicador: Elsevier; Amsterdam
Tipo: Artigo de Revista Científica
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The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, various signaling processes, antigen presentation, and control of protein synthesis. The central catalytic complex of the proteasome is called the 20S core particle. The majority of these are flanked on one or both sides by regulatory units. Most common among these units is the 19S regulatory unit. When coupled to the 19S unit, the complex is termed the asymmetric or symmetric 26S proteasome depending on whether one or both sides are coupled to the 19S unit, respectively. The 26S proteasome recognizes poly-ubiquitinylated substrates targeted for proteolysis. Targeted proteins interact with the 19S unit where they are deubiquitinylated, unfolded, and translocated to the 20S catalytic chamber for degradation. The 26S proteasome is responsible for the degradation of major proteins involved in the regulation of the cellular cycle, antigen presentation and control of protein synthesis. Alternatively, the proteasome is also active when dissociated from regulatory units. This free pool of 20S proteasome is described in yeast to mammalian cells. The free 20S proteasome degrades proteins by a process independent of poly-ubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20S proteasome comprises two central heptamers (β-rings) where the catalytic sites are located and two external heptamers (α-rings) that are responsible for proteasomal gating. Because the 20S proteasome lacks regulatory units...

‣ Estudo e caracterização do processo de glutatiolação e desglutatiolação da unidade 20S do proteassomo da levedura Saccharomyces cerevisiae: Implicações na regulação do metabolismo redox intracelular e na geração de peptídeos; Study and characterization of the S-glutathiolation and deglutathiolation of the 20S proteasome core from the yeast Saccharomyces cerevisiae: Implications on the intracellular redox metabolism and peptide generation.

Silva, Gustavo Monteiro
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 15/10/2010 Português
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O proteassomo é o componente do sistema Ubiquitina-Proteassomo (UPS), responsável pela degradação de proteínas intracelulares marcadas com cauda de ubiquitina. No entanto, a unidade catalítica do proteassomo (20SPT), destituída de unidades regulatórias, é capaz de degradar proteínas de maneira ubiquitina-independente. Diversas modificações pós-traducionais já foram descritas para o 20SPT, incluindo a S-glutatiolação. De acordo com Demasi e col., (2003) o 20SPT da levedura Saccharomyces cerevisiae possui a atividade tipo-quimiotripsina modulada por glutationa e o mecanismo de glutatiolação implica na formação do intermediário ácido sulfênico. No presente trabalho, identificamos por espectrometria de massas (MS/MS) um total de sete resíduos diferentes de cisteína glutatiolados no 20SPT, sendo seis in vitro por incubação com GSH e três in vivo, extraído de células crescidas até atingir fase estacionária tardia em meio rico. Analisando a estrutura 3D do 20SPT, observou-se que os resíduos de cisteína glutatiolados não estão localizados na entrada da câmara catalítica nem próximos aos sítios-ativos, indicando um mecanismo alostérico da modulação da atividade proteassomal. O proteassomo glutatiolado extraído de leveduras é capaz de degradar proteínas oxidadas de maneira mais eficiente que o proteassomo reduzido por DTT...

‣ Síntese de derivados da L-cistina e L-cisteína para aplicação em estudos de inibição do proteassomo 20S; Synthesis of L-cystine and L-cysteine derivatives for use in studies of 20S proteasome inhibition

Paula, Priscila Milani de
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 21/10/2011 Português
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Neste trabalho foi realizada a síntese de amidas, bem como de ácidos e ésteres borônicos, derivados dos aminoácidos L-cistina e L-cisteína, através de rota sintética simples, curta e de baixo custo, com o intuito de busca e a identificação de novo(s) inibidor(es) do proteassomo 20S. Esta classe de compostos possui estrutura que permite a inserção de diversos grupos funcionais, o que confere versatilidade e a construção de biblioteca de compostos que contém partes hidrofílicas e hidrofóbicas importantes para posterior avaliação inibitória. Para tanto, empregou-se rota sintética química convencional e rota biocatalisada para a formação da ligação amida. Os compostos derivados de L-cisteína foram obtidos via síntese clássica de peptídeos a qual forneceu os compostos desejados em rendimentos de até 85%. Por outro lado, tentativas de obtenção das amidas via biocatálise não se mostraram efetivas. Já amidas derivadas de L-cistina foram obtidas em rendimentos de até 79%, via síntese tradicional e até 100% de conversão através de rota biocatalítica. A inserção do átomo de boro nas estruturas se deu utilizando-se metodologias sintéticas já bem estabelecidas na literatura. Os ésteres borônicos derivados de L-cisteína foram obtidos em bons rendimentos (até 78%)...

‣ Caracterização do repertório peptídico intracelular de células expressando o proteassomo imune.; Characterization of intracellular peptide repertoire of cells expressing the immune proteasome.

Silva, Elisabete Rodrigues do Monte
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 18/03/2014 Português
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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 β1, β2 e β5. Em condições de stress e resposta imune essas três subunidades são substituídas por β1i, β2i and β5i, respectivamente, para formar o proteassomo imune. Estas três subunidades induzíveis, parecem alterar as especificidades de peptidase do proteassoma imune em células tratadas com IFN-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-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 β1...

‣ Studies on terrein as a new class of proteasome inhibitors

Demasi,M.; Felicio,A. L.; Pacheco,A. O.; Leite,H. G.; Lima,C.; Andrade,L. H.
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2010 Português
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The proteasome is an intracellular multicatalytic protease involved in the cell cycle regulation, signaling response, antigen presentation and apoptosis. Since proteasome inhibitors promote cell death by apoptosis, they have been proposed as new anti-tumoral drugs. Terrein, a secondary metabolite secreted by the fungus Aspergillus terreus, was firstly described in 1935. In the present work we report that terrein isolated through the screening for inhibitors of the 20S proteasome showed inhibitory effect upon both chymotrypsin- and trypsin-like activities of the multicatalytic core particle, the 20S proteasome. Despite of the high inhibitory concentration determined in vitro, that verified by incubating cells (fibroblasts and a pulmonary tumor cell line) in the presence of terrein was 4-fold lower indicating the proteasome as a selective intracellular target. Moreover, terrein promoted apoptotic cell death on both fibroblasts and pulmonary tumor cell line tested. Although terrein concentrations (mM range) necessary to elicit apoptosis in the cellular models herein tried were high when compared to those (μM and nM range) of other inhibitors recently described, its chemical structure is not correlated to any other inhibitor reported thus far. Therefore...

‣ Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

Sandu, Cristinel; Chandramouli, Nagaranjan; Glickman, Joseph Fraser; Molina, Henrik; Kuo, Chueh-Ling; Kukushkin, Nikolay; Goldberg, Alfred L; Steller, Hermann
Fonte: John Wiley & Sons, Ltd Publicador: John Wiley & Sons, Ltd
Tipo: Artigo de Revista Científica
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Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together...

‣ Proteasome proteolytic profile is linked to Bcr-Abl expression

Crawford, L.; Windrum, P.; Magill, L.; Vaz de Melo, J.; McCallum, L.; McMullin, M.; Ovaa, H.; Walker, B.; Irvine, A.
Fonte: Elsevier Science Inc Publicador: Elsevier Science Inc
Tipo: Artigo de Revista Científica
Publicado em //2009 Português
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OBJECTIVE: We have previously demonstrated that proteasome activity is higher in bone marrow from patients with chronic myeloid leukemia (CML) than normal controls. This study investigates whether there is any relationship between Bcr-Abl expression and proteasome activity. MATERIALS AND METHODS: Fluorogenic substrate assays and an activity-based probe were used to profile proteasome activity in CML cell-line models and the effect of the proteasome inhibitor BzLLLCOCHO on these cell-line models and primary CML cells was investigated. RESULTS: We have demonstrated that oncogenic transformation by BCR-ABL is associated with an increase in proteasome proteolytic activity. Furthermore, small interfering RNA targeted against BCR-ABL reduces proteasome activity. In addition, we have found that Bcr-Abl-positive cells are more sensitive than Bcr-Abl-negative cells to induction of apoptosis by the proteasome inhibitor BzLLLCOCHO, and that sequential addition of imatinib followed by BzLLLCOCHO has an additive effect on the induction of apoptosis in Bcr-Abl-positive cells. Finally, we demonstrate that cell lines that become resistant to imatinib remain sensitive to proteasome inhibition. CONCLUSION: This is the first time that a direct relationship has been demonstrated between BCR-ABL transformation and the enzymatic activity of the proteasome. Our results suggest that the proteasome might provide a useful therapeutic target in CML...

‣ Impaired methylation as a novel mechanism for proteasome suppression in liver cells

Osna, N.; White, R.; Donohue, T.; Beard, M.; Tuma, D.; Kharbanda, K.
Fonte: Academic Press Inc Publicador: Academic Press Inc
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
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The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional...

‣ New 26S proteasome inhibitors with high selectivity for chymotrypsin-like activity and p53-dependent cytotoxicity

Neilsen, P.; Pehere, A.; Pishas, K.; Callen, D.; Abell, A.
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
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The 26S proteasome has emerged over the past decade as an attractive therapeutic target in the treatment of cancers. Here, we report new tripeptide aldehydes that are highly specific for the chymotrypsin-like catalytic activity of the proteasome. These new specific proteasome inhibitors demonstrated high potency and specificity for sarcoma cells, with therapeutic windows superior to those observed for benchmark proteasome inhibitors, MG132 and Bortezomib. Constraining the peptide backbone into the β-strand geometry, known to favor binding to a protease, resulted in decreased activity in vitro and reduced anticancer activity. Using these new proteasome inhibitors, we show that the presence of an intact p53 pathway significantly enhances cytotoxic activity, thus suggesting that this tumor suppressor is a critical downstream mediator of cell death following proteasomal inhibition.; Paul M. Neilsen, Ashok D. Pehere, Kathleen I. Pishas, David F. Callen, and Andrew D. Abell

‣ Identifikation des HIV-Proteaseinhibitors Ritonavir als Medikament mit potentieller Wirksamkeit in der Therapie der Akuten Myeloischen Leukämie in Kombination mit Proteasominhibitoren; Identification of the HIV protease inhibitor ritonavir as a drug with potential effect in the therapy of acute myeloid leukemia in combination with proteasome inhibitors

Müller-Ide, Hendrik
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Gegenstand dieser experimentellen Dissertation ist es, synergistische zytotoxische Effekte von Protease- und Proteasominhibitoren mit anderen Substanzgruppen und insbesondere mit bereits als Medikament verfügbaren Substanzen am Modell einer AML-Zelllinie sowie bei primären AML-Blasten zu identifizieren und zu charakterisieren. Mit Hilfe der von einem kovalent bindenden Proteasominhibitor abgeleiteten Affinitätssonde DansylAhx3L3VS (DALVS) werden in dieser Arbeit erstmals selektiv die aktiven Proteasomuntereinheiten in vitalen malignen humanen hämatopoetischen Zellen dargestellt. Die selektive Wirkung von Bortezomib auf die beta1/beta5 katalytischen Untereinheiten des Proteasoms konnten bestätigt werden. Da postuliert wurde, dass auch der HIV-Proteaseinhibitor Ritonavir eine Proteasom-inhibierende Aktivität hat (und dieses Medikament damit potentiell sehr interessant für die Therapie hämatologischer Neoplasien sein könnte), wurde der Effekt von Ritonavir auf AML-Zelllinien sowie primäre AML-Blasten mittels Affinitätsmarkierung untersucht. Dabei konnte gezeigt werden, dass Ritonavir in den klinisch relevanten Konzentrationen die Proliferation von AML-Zelllinien und primären humanen Blasten zwar hemmt, jedoch nicht über eine Proteasominhibition sondern vielmehr (ähnlich wie Bortezomib) zu einer der Proteasominhibition nachgeschalteten Aktivierung der Unfolded Protein Response (UPR) und ER-Stress führt...

‣ The role of the proteasome in cellular protein degradation

Driscoll, J.
Fonte: Murcia : F. Hernández Publicador: Murcia : F. Hernández
Tipo: Artigo de Revista Científica Formato: application/pdf
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Eukaryotic cells contain a major intracellular proteolytic activity known as the proteasome. The proteasome is a strongly conserved cylindrical structure of high molecular weight (650 kDa, -20 S) and desmonstrates multiple endopeptidase activities. The general structural, biochemical and genetic features of the proteasonie are conserved from archaebacteria through yeast to humans. This structure fulfills an essential role by functioning as the proteolytic core of a 26 S multienzyme complex responsible for the energydependent degradation of ubiquitinated proteins. The bulk of intracellular proteolysis appears to be through the ubiquitin-dependent pathway. Incorporation of the proteasome into the 26 S niultienzyme complex appears to confer both a specificity for ubiquitinated proteins as well as a nieans to tightly regulate proteolytic activity. Thus, one function of the proteasome is required for the degradation of either abnormal or certain regulatory proteins by the ubiquitin pathway. Proteasoine subunits appear to be encoded by a related gene family as defined by extensive sequence similarities. The gene products are confined to either of two general classes: a-type which appear to be structural and 8-type which may be catalytic. Genes encoding at least two proteasome subunits map to the Ma-jor Histocompatibility Complex. Accurnulating evidence points to the proteasome (or a specialized form) participatirig in the cytosolic degradation of these viral proteins upon cellular infection. Through a previously unforseen mechanisin. it appears that the products from the digestion of the viral proteins may be rescued from further digestion to amino acids and shuttled from the cytoplasm through the endoplasniic reticulum to the cell surface where they serve as antigenic peptides for recognition by the immune system. The proteasome may have been recruited by the immune system to serve as the cytosolic activity responsible for generating these antigenic peptides. The proteasome may function in the ubiquitindependent degradation of not only certain self-proteins but may fulfill a second essential role in the degradation of proteins originating from viral infection

‣ DOES PROTEASOME INHIBITION PRODUCE REM SLEEP BEHAVIOUR DISORDER LEADING TO PARKINSON’S DISEASE? EXAMINING A PROGRESSIVE MODEL OF PARKINSON’S DISEASE

McGilvray, Mark
Fonte: Quens University Publicador: Quens University
Tipo: Tese de Doutorado
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A recent model of Parkinson’s disease (PD) suggests that the neuropathological, behavioural and cognitive symptoms progress in stages. There is substantial evidence for a prodromal stage of PD, during which time pre-motor symptoms develop. Rapid eye movement (REM) sleep behaviour disorder (RBD) is a risk factor for developing PD and may be part of the pre-motor stage. In both disorders, neuropathological α-synuclein aggregates are thought to be a direct cause of the resulting symptoms. One model has shown that in rats, proteasome inhibition produced by systemic exposure to environmental toxins results in α-synuclein pathology and motor behaviour dysfunction that mimics the progression of PD in humans. The present study examined the hypothesis that the systemic proteasome inhibition model would produce pre-Parkinsonian RBD-like pathology in rats. It was expected that sleep disturbances would be seen prior to behavioural disturbances in rats treated systemically with PSI (a proteasome inhibitor). Following baseline sleep recording and training on the inclined beam-traverse task, rats were injected with PSI (a proteasome inhibitor) or ethanol (control), 6 times over 2 wk. Sleep recording over 8 wk and behavioural testing over 16 wk provided no evidence of sleep disturbances or motor dysfunction. Post-mortem immunohistochemical analyses of brain tissue provided no evidence of PSI-associated α-synuclein aggregates in the locus coeruleus...

‣ Ubiquitin Recognition by the Proteasome

Shi, Yuan
Fonte: Harvard University Publicador: Harvard University
Tipo: Thesis or Dissertation
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Ubiquitin proteasome pathway is an important cellular pathway that affects the fate of almost all intracellular proteins. Misregulation of this pathway has been found to be associated with a broad range of human diseases, such as cancer, neurodegenerative diseases, as well as viral infections. Ubiquitin recognition by the proteasome is of central importance to this pathway. So far, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors. An alternative pathway is mediated by shuttling factors. In yeast, three shuttling factors, known as UBL-UBA proteins, have been found. A UBL receptor activity of the proteasome has been attributed to Rpn1. However, yeast cell mutated all five proteasomal ubiquitin receptors is still viable. To identify the additional proteasomal ubiquitin receptor in cells, I first obtained and characterized a new Rpn13 mutant allele. This Rpn13 mutant completely abolished its ubiquitin binding activity, and functionally resembles a null allele. Rpn13 substrate pool has also been sought in this mutant cells. In the second part of this dissertation, I reported a novel ubiquitin binding site on proteasomal subunit Rpn1. With the help of NMR analysis, Rpn1's ubiquitin and UBL binding surfaces were resolved at high resolution and found to substantially overlap. A specific Rpn1 mutation that disrupts both ubiquitin and UBL binding while not compromising the folding of Rpn1 was obtained. This mutant allele shows a pleiotropic proteasomal defect in vivo. Moreover...

‣ Induction of Tumor Cell Apoptosis by a Proteasome Deubiquitinase Inhibitor Is Associated with Oxidative Stress

Brnjic, Slavica; Mazurkiewicz, Magdalena; Fryknäs, Mårten; Sun, Chao; Zhang, Xiaonan; Larsson, Rolf; D'Arcy, Pádraig; Linder, Stig
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica
Publicado em 10/12/2014 Português
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Aims: b-AP15 is a recently described inhibitor of the USP14/UCHL5 deubiquitinases (DUBs) of the 19S proteasome. Exposure to b-AP15 results in blocking of proteasome function and accumulation of polyubiquitinated protein substrates in cells. This novel mechanism of proteasome inhibition may potentially be exploited for cancer therapy, in particular for treatment of malignancies resistant to currently used proteasome inhibitors. The aim of the present study was to characterize the cellular response to b-AP15-mediated proteasome DUB inhibition. Results: We report that b-AP15 elicits a similar, but yet distinct, cellular response as the clinically used proteasome inhibitor bortezomib. b-AP15 induces a rapid apoptotic response, associated with enhanced induction of oxidative stress and rapid activation of Jun-N-terminal kinase 1/2 (JNK)/activating protein-1 signaling. Scavenging of reactive oxygen species and pharmacological inhibition of JNK reduced b-AP15-induced apoptosis. We further report that endoplasmic reticulum (ER) stress is induced by b-AP15 and is involved in apoptosis induction. In contrast to bortezomib, ER stress is associated with induction of α-subunit of eukaryotic initiation factor 2 phosphorylation. Innovation: The findings establish that different modes of proteasome inhibition result in distinct cellular responses...

‣ The proteasome distinguishes between heterotypic and homotypic lysine-11 linked polyubiquitin chains

Grice, Guinevere L.; Lobb, Ian T.; Weekes, Michael P.; Gygi, Steven P.; Antrobus, Robin; Nathan, James A.
Fonte: Elsevier Publicador: Elsevier
Tipo: Article; published version
Português
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This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.celrep.2015.06.061; [Graphical abstract - see article] Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome can also bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here, we identify that pure homotypic lysine-11 linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell cycle regulator, cyclin B1. Thus, while heterotypic lysine-11 linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrates the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains.; This work is supported by a Wellcome Trust Senior Clinical Research Fellowship to JAN (102770/Z/13/Z)...