A Leishmaniose é uma doença infecciosa causada por protozoários flagelados do gênero Leishmania. Os parasitas como a Leishmania braziliensis, sofrem várias mudanças morfológicas durante seu ciclo de vida, incluindo a troca de organismo hospedeiro. Durante essas mudanças, proteínas de choque térmico ou chaperones moleculares, como, por exemplo, a Hsp70, são expressas em grande quantidade. A função da Hsp70 é auxiliar no processo de enovelamento protéico, no transporte de proteínas entre as membranas e em muitas outras importantes funções celulares. A Hsp70 é auxiliada por várias proteínas denominadas como co-chaperone e a Hep1 (do inglês Hsp70-escort protein 1) é uma delas. Essa co-chaperone tem seu papel descrito principalmente em mitocôndrias como estabilizadoras da Hsp70 capazes de prevenir a sua agregação. O objetivo deste trabalho foi clonar, expressar, purificar e caracterizar as proteínas Hsp70 e Hep1 de L. braziliensis (LbHsp70 e LbHep1). Os ensaios preliminares mostraram que a LbHsp70 foi expressa de forma insolúvel, sendo necessário expressar a proteína em corpos de inclusão para tentativas de reenovelamento, afim de obter a mesma na fração solúvel. Apesar da LbHsp70 se apresentar na fração solúvel após o reenovelamento, a mesma foi purificada como agregado. Ainda na tentativa de obter a LbHsp70 na forma solúvel, a mesma foi co-expressa com a LbHep1 (expressa na forma solúvel), porém a LbHsp70 continuou na fração insolúvel do lisado bacteriano. Como a LbHep1 não apresentou a atividade esperada quando co-expressa com a LbHsp70 citoplasmática, foram feitos ensaios de co-expressão da LbHep1 com a Hsp70 mitocondrial humana, que é heterologamente expressa na forma de agregados, com o intuito de confirmar a atividade estabilizadora das Hep1 sobre as Hsp70 mitocondriais. Este experimento possibilitou a obtenção de ambas proteínas na fração solúvel, de acordo com dados apresentados na literatura para este sistema em outros organismos. Uma vez mostrada à funcionalidade da LbHep1, foi feita a caracterização desta proteína por métodos biofísicos como dicroísmo circular, espectrometria de fluorescência, cromatografia de exclusão molecular analítica e ultracentrifugação analítica. Os experimentos mostraram que a LbHep1 apresenta estrutura secundária composta principalmente de folhas-β pregueadas e que o único triptofano está parcialmente exposto ao solvente. As análises hidrodinâmicas mostraram que a LbHep1 é assimétrica e em equilíbrio entre monômeros e dímeros. Por fim, dados de ultracentrifugação analítica indicam que a LbHep1 está em equilíbrio monômero-dímero. / Leishmaniasis is an infectious disease caused by flagellate protozoa of the genus Leishmania. The parasites such as Leishmania braziliensis undergo various morphological changes during its life cycle, including the exchange of the host organism. During these changes, heat shock proteins or molecular chaperones like Hsp70, for example, are expressed in large amounts. The function of Hsp70 is to assist in the process of protein folding, protein transport between the membranes and many other important cellular functions. The Hsp70 is assisted by several proteins called co-chaperones and the Hsp70-escort protein (Hep1) is one of them. This co-chaperone has been described based on its role as a stabilizer of mitochondrial Hsp70 preventing their aggregation. The objective of this study was to clone, express, purify and characterize the Hsp70 and Hep1 ortologues of Leishmania braziliensis (LbHsp70 and LbHep1). The preliminary tests showed that LbHsp70 was expressed in the insoluble form, being necessary to express the protein in inclusion bodies to attempt its refolding in order to get it in the soluble fraction. Despite LbHsp70 was obtained in the soluble fraction after refolding, it was purified as aggregates. Still trying to get the LbHsp70 in the soluble form, it was co-expressed with LbHep1 (always expressed in the soluble form), but LbHsp70 remained in the insoluble fraction of the bacterial lysate. As LbHep1 showed no expected activity when co-expressed with LbHsp70, which is citoplasmatic, we tested if LbHep1 was able to act on human mitochondrial Hsp70 which is expressed as aggregates in bacterial heterologous systems. Then, we co-expressed LbHep1 with human mitochondrial Hsp70 which allowed obtaining both proteins in the soluble fraction, in according to data presented in the literature. Once the functionality of LbHep1 was showed, we characterize this protein by biophysical methods such as circular dichroism, fluorescence spectrometry, molecular exclusion chromatography and analytical ultracentrifugation analysis. The experiments showed that the secondary structure features LbHep1 composed mainly of β-sheets and that the only tryptophan is partially exposed to solvent. Hydrodynamic analysis showed that the protein is asymmetric and in equilibrium between monomers and dimers. Finally, analytical ultracentrifugation data indicate that LbHep1 is a system in equilibrium monomer-dimer.

A Leishmaniose é uma doença infecciosa causada por protozoários flagelados do gênero Leishmania. Os parasitas como a Leishmania braziliensis, sofrem várias mudanças morfológicas durante seu ciclo de vida, incluindo a troca de organismo hospedeiro. Durante essas mudanças, proteínas de choque térmico ou chaperones moleculares, como, por exemplo, a Hsp70, são expressas em grande quantidade. A função da Hsp70 é auxiliar no processo de enovelamento protéico, no transporte de proteínas entre as membranas e em muitas outras importantes funções celulares. A Hsp70 é auxiliada por várias proteínas denominadas como co-chaperone e a Hep1 (do inglês Hsp70-escort protein 1) é uma delas. Essa co-chaperone tem seu papel descrito principalmente em mitocôndrias como estabilizadoras da Hsp70 capazes de prevenir a sua agregação. O objetivo deste trabalho foi clonar, expressar, purificar e caracterizar as proteínas Hsp70 e Hep1 de L. braziliensis (LbHsp70 e LbHep1). Os ensaios preliminares mostraram que a LbHsp70 foi expressa de forma insolúvel, sendo necessário expressar a proteína em corpos de inclusão para tentativas de reenovelamento, afim de obter a mesma na fração solúvel. Apesar da LbHsp70 se apresentar na fração solúvel após o reenovelamento, a mesma foi purificada como agregado. Ainda na tentativa de obter a LbHsp70 na forma solúvel, a mesma foi co-expressa com a LbHep1 (expressa na forma solúvel), porém a LbHsp70 continuou na fração insolúvel do lisado bacteriano. Como a LbHep1 não apresentou a atividade esperada quando co-expressa com a LbHsp70 citoplasmática, foram feitos ensaios de co-expressão da LbHep1 com a Hsp70 mitocondrial humana, que é heterologamente expressa na forma de agregados, com o intuito de confirmar a atividade estabilizadora das Hep1 sobre as Hsp70 mitocondriais. Este experimento possibilitou a obtenção de ambas proteínas na fração solúvel, de acordo com dados apresentados na literatura para este sistema em outros organismos. Uma vez mostrada à funcionalidade da LbHep1, foi feita a caracterização desta proteína por métodos biofísicos como dicroísmo circular, espectrometria de fluorescência, cromatografia de exclusão molecular analítica e ultracentrifugação analítica. Os experimentos mostraram que a LbHep1 apresenta estrutura secundária composta principalmente de folhas-β pregueadas e que o único triptofano está parcialmente exposto ao solvente. As análises hidrodinâmicas mostraram que a LbHep1 é assimétrica e em equilíbrio entre monômeros e dímeros. Por fim, dados de ultracentrifugação analítica indicam que a LbHep1 está em equilíbrio monômero-dímero. / Leishmaniasis is an infectious disease caused by flagellate protozoa of the genus Leishmania. The parasites such as Leishmania braziliensis undergo various morphological changes during its life cycle, including the exchange of the host organism. During these changes, heat shock proteins or molecular chaperones like Hsp70, for example, are expressed in large amounts. The function of Hsp70 is to assist in the process of protein folding, protein transport between the membranes and many other important cellular functions. The Hsp70 is assisted by several proteins called co-chaperones and the Hsp70-escort protein (Hep1) is one of them. This co-chaperone has been described based on its role as a stabilizer of mitochondrial Hsp70 preventing their aggregation. The objective of this study was to clone, express, purify and characterize the Hsp70 and Hep1 ortologues of Leishmania braziliensis (LbHsp70 and LbHep1). The preliminary tests showed that LbHsp70 was expressed in the insoluble form, being necessary to express the protein in inclusion bodies to attempt its refolding in order to get it in the soluble fraction. Despite LbHsp70 was obtained in the soluble fraction after refolding, it was purified as aggregates. Still trying to get the LbHsp70 in the soluble form, it was co-expressed with LbHep1 (always expressed in the soluble form), but LbHsp70 remained in the insoluble fraction of the bacterial lysate. As LbHep1 showed no expected activity when co-expressed with LbHsp70, which is citoplasmatic, we tested if LbHep1 was able to act on human mitochondrial Hsp70 which is expressed as aggregates in bacterial heterologous systems. Then, we co-expressed LbHep1 with human mitochondrial Hsp70 which allowed obtaining both proteins in the soluble fraction, in according to data presented in the literature. Once the functionality of LbHep1 was showed, we characterize this protein by biophysical methods such as circular dichroism, fluorescence spectrometry, molecular exclusion chromatography and analytical ultracentrifugation analysis. The experiments showed that the secondary structure features LbHep1 composed mainly of β-sheets and that the only tryptophan is partially exposed to solvent. Hydrodynamic analysis showed that the protein is asymmetric and in equilibrium between monomers and dimers. Finally, analytical ultracentrifugation data indicate that LbHep1 is a system in equilibrium monomer-dimer.

No description available.

Recent studies have focused their attention on the structure and function of histone chaperones involved in different aspects of somatic chromatin assembly and disassembly. However, one of the most dramatic chromatin remodeling processes takes place immediately after fertilization and is mediated by proteins in metazoan eggs that function as histone chaperones and histone storage proteins. These include members of the nucleoplasmin (NPM) family and the nuclear autoantigenic sperm protein (NASP) families. While it had been know for some time that these proteins function as “histone sinks”, new studies are shedding light on their role as histone chaperones. NASP was first identified in Xenopus laevis eggs where it accumulated in the nucleoplasm and was found to bind histones H3 and H4 at which time it believed to act simply as a histone storage protein. Interestingly, in addition to binding and providing storage to H3-H4 in the egg and in somatic cells, our studies have shown NASP to be the first characterized chaperone for histone H1. The members of the histone H1 family (linker histones) are essential to maintaining the structure of chromatin with respect to the folding of the chromatin fiber, nucleosome spacing, chromatin remodeling, gene transcription and progression through the cell cycle. Until recently there has been no histone H1 chaperone characterized and no evidence of a storage protein to which linker histones are bound, when not associated with DNA of NCPs. By using recombinant NASP, to incorporate linker histones onto the nucleosome arrays in a chromatin fiber, we studied the dynamics and conformation of chromatin in a more biologically relevant and precise method than presented in any previous chromatin research. Like NASP, nucleoplasmin was identified as a factor in X. laevis eggs that binds histones and loads them onto DNA. Subsequently, the nucleoplasmin protein family has been to be universally represented throughout metazoans where it plays a similar role in chromatin metabolism. Members of this family include nucleophosmin (NPM1), nucleoplasmin (NPM2, NPM or Np), the newly characterized NPM3 and nucleoplasmin-like proteins (NPM-like or NLP). We have been able to demonstrate that NPM2 that is highly phosphorylated in X. laevis eggs can unfold sperm and somatic chromatin by facilitating the removal of linker histones and other chromosomal proteins from linker DNA regions between nucleosomes in the absence of any stable interaction with the nucleosome core particle (NCP) itself. In addition, our studies reveal that NPM2 is a pentameric chaperone, as opposed to a decameric chaperone, that regulates the condensation state of sperm and somatic chromatin by removing linker histones and specific nuclear basic proteins and depositing histone H2A-H2B dimers on the distal face of the NPM2. / Graduate

Temperature is a critical environmental signal, which exerts powerful control over the development and virulence of diverse microbial pathogens. Fungi, along with other microbial species, exploit a diversity of mechanisms to sense and respond to temperature fluctuations that may be encountered in the host or under other conditions of temperature stress. For Candida albicans, the leading fungal pathogen of humans, temperature influences mating, phenotypic switching, resistance to antifungal drugs, and the morphogenetic transition from yeast to filamentous growth. C. albicans morphogenesis is strongly influenced by temperature, and most filament inducing cues depend on a concurrent increase of temperature to 37˚C before morphogenesis can occur. Further elevated temperature of 39˚C to 42˚C can serve as an independent filament-inducing cue, although the molecular mechanisms underpinning this temperature-dependent morphogenetic transition remain largely uncharacterized. Here, I provide the first comprehensive investigation of the molecular mechanisms mediating temperature-dependent morphogenesis in C. albicans. I establish that the thermally responsive molecular chaperone Hsp90 orchestrates temperature-dependent morphogenesis, and that Hsp90 functions as a key temperature sensor, such that elevated temperature is required to relieve Hsp90-mediated repression of the morphogenetic program. Further, I demonstrate that Hsp90 controls morphogenesis via at least two distinct cellular signaling cascades. First, Hsp90 and its co-chaperone Sgt1 physically interact, and together regulate protein kinase A (PKA) signaling via an interaction with the adenylyl cyclase of the PKA cascade, Cyr1, such that genetic depletion of either Hsp90 or Sgt1 activates PKA signaling and induces filamentation. Second, Hsp90 controls temperature-dependent morphogenesis via previously uncharacterized cellular circuitry comprised of the cyclin-dependent kinase Pho85, the cyclin Pcl1, and the transcriptional regulator Hms1. Together, this research illuminates the central role of Hsp90 in coupling temperature sensing and morphogenesis in the human fungal pathogen C. albicans.

Temperature is a critical environmental signal, which exerts powerful control over the development and virulence of diverse microbial pathogens. Fungi, along with other microbial species, exploit a diversity of mechanisms to sense and respond to temperature fluctuations that may be encountered in the host or under other conditions of temperature stress. For Candida albicans, the leading fungal pathogen of humans, temperature influences mating, phenotypic switching, resistance to antifungal drugs, and the morphogenetic transition from yeast to filamentous growth. C. albicans morphogenesis is strongly influenced by temperature, and most filament inducing cues depend on a concurrent increase of temperature to 37˚C before morphogenesis can occur. Further elevated temperature of 39˚C to 42˚C can serve as an independent filament-inducing cue, although the molecular mechanisms underpinning this temperature-dependent morphogenetic transition remain largely uncharacterized. Here, I provide the first comprehensive investigation of the molecular mechanisms mediating temperature-dependent morphogenesis in C. albicans. I establish that the thermally responsive molecular chaperone Hsp90 orchestrates temperature-dependent morphogenesis, and that Hsp90 functions as a key temperature sensor, such that elevated temperature is required to relieve Hsp90-mediated repression of the morphogenetic program. Further, I demonstrate that Hsp90 controls morphogenesis via at least two distinct cellular signaling cascades. First, Hsp90 and its co-chaperone Sgt1 physically interact, and together regulate protein kinase A (PKA) signaling via an interaction with the adenylyl cyclase of the PKA cascade, Cyr1, such that genetic depletion of either Hsp90 or Sgt1 activates PKA signaling and induces filamentation. Second, Hsp90 controls temperature-dependent morphogenesis via previously uncharacterized cellular circuitry comprised of the cyclin-dependent kinase Pho85, the cyclin Pcl1, and the transcriptional regulator Hms1. Together, this research illuminates the central role of Hsp90 in coupling temperature sensing and morphogenesis in the human fungal pathogen C. albicans.

<b>Background:</b> Aryl Hydrocarbon Receptor Interacting Protein-Like 1 (AIPL1) interacts with NUB1 and restricts the entry of NUB1 protein into the nucleus. The interferon-induced NEDD8 ultimate buster (NUB1) protein causes degradation of neddylated and FAT10ylated proteins through the ubiquitin proteasome system. We observed AIPL1 were frequently down-regulated in various cancers compared to normal tissues. The mechanistic roles of AIPL1 and NUB1 protein in cancer cell cycle regulation remain unexplored. <b>Results:</b> Meta-analysis of cancer databases revealed that expression transcripts of chaperones, including AIPL1, were down-regulated in lung, pancreatic cancer and breast cancer relative to the adjacent normal tissues. Opposite levels of both AIPL1 and NUB1 transcripts were observed in the breast cancer. So it triggers the in vitro experiments using breast cancer cells. METABRIC breast cancer clinical cohort highlighted that patients with low NUB1 transcripts had poor survival in the ER-negative subgroup (but not in ER-positive) of breast cancer patients: hazard ratio (HR)=0.66, 95% confidence interval (CI)=0.5-0.87, p=0.003 and triple negative subgroup of breast cancer patients: HR=0.67, 95% CI=0.47-0.96, p=0.028. NUB1 silencing significantly inhibits in vitro cell growth in MDA-MB-231 and MCF7 under hypoxia. AIPL1 protein forms multimers in cancer cells. NUB1 protein moved into the nucleus in hypoxia (0.1% O2 48hrs) with final confluency at 80-90%. p21 (marker of senescence) &amp; p27 (marker of cell cycle arrest) accumulated in NUB1-silent MDA-MB-231 and RCC4 cells. It suggested that low NUB1 nuclear localisation in hypoxia cause cancer cell cycle arrest. In MDA-MB-231 cell, upon hypoxia, neddylation inhibitor (MLN4924) treated and siNUB1 transfected cells showed decreased CUL1 and further accumulated p21 &amp; p27. The evidence suggested lower neddylated CUL1 and reduced NUB1 cooperatively stabilise p21 and p27 as the substrate of CUL1-ubiquitin ligase. The neddylation inhibitor MLN4924 treated and NUB1 knockdown group exhibited more cells in sub-G1 stage as compared to the control group. In connection to higher p21/p27, it is associated with prolonged arrested cellular aging with depletion. After silencing of NUB1, the increases in cell death of cancer cells upon hypoxia happen through the neddylation-dependent CUL1-p27-p21 and CUL2-VHL axis. We then demonstrated that HIF1&alpha; protein could be both neddylated and FAT10ylated upon reoxygenation. In a tissue microarray study of breast cancer, lower cytoplasmic expression (n=57) had worse overall survival than higher cytoplasmic expression (n=57): HR=1.779, 95% CI=1.006-3.346, p=0.048. <b>Conclusions:</b> AIPL1 and NUB1 proteins exert a role in cell cycle regulation in breast cancer. Low cytoplasmic NUB1 levels are observed in the G₁-S transition of cancer cells. NUB1 depletion causes G₀/G₁ phase arrest due to CUL1 and CUL2 ubiquitin E3 ligase-dependent pathways.

No description available.

Molecular chaperones and proteases help maintain protein homeostasis in the cell. While chaperones assist in the folding of polypeptide chains to their native state, proteases degrade misfolded or unfolded proteins and also help regulate protein levels. While mapping chaperone interaction networks, we found that tig (trigger factor chaperone gene), clpP and clpX genes co-localize next to each other on the genome of most examined bacteria. This led us to hypothesize that trigger factor (TF) chaperone and ClpXP protease might interact functionally. TF is a ribosome-associated chaperone that co-translationally folds polypeptide chains. ClpXP is a proteolytic complex that degrades a wide range of substrate proteins. We observed that TF enhanced the rate of the ClpXP degradation of the λO phage protein in vitro and in vivo. TF was also found to enhance the degradation of ribosome-stalled λO thus suggesting the existence of co-translational protein degradation in E. coli.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.