Doctor of Philosophy / Graduate Biochemistry Group / Michal Zolkiewski / ClpB, a bacterial chaperone that belongs to the AAA+ protein family, cooperates with the Hsp70/40 system (DnaK, DnaJ and GrpE in E.coli) in the reactivation of aggregated substrates by translocating them through the central channel of its hexameric form. ClpB is essential for survival of bacteria under heat shock and plays an important role in the infectivity of pathogenic microorganisms. However the detailed mechanism of ClpB disaggregation activity is still not clear. ClpB is a multi-domain protein, which consists of two nucleotide binding domains (NBD1 and NBD2) connected by the middle domain (M domain), and the N-terminal domain connected to the rest of the protein by a flexible linker. In this work, mutations were introduced into the linker region to modify the mobility of the N-terminal domain. It was found that without altering the proper folding and oligomerization of ClpB, all the mutants had deficiencies in aggregate reactivation, possibly due to the weaker binding to aggregated substrates in the initial step of disaggregation. This led to the conclusion that the flexible attachment of the N-terminal domain supports substrate binding and controls the disaggregation by ClpB. Moreover, partial inhibition of the ClpB chaperone activity was observed for all the linker variants, suggesting that the linker sequence might have been optimized by selective pressure to maintain the optimal efficiency of aggregate reactivation. To study the substrate translocation of ClpB, a BAP (ClpB-ClpA P-loop) variant that binds to the protease ClpP was constructed. A FRET-based experiment was designed and the fluorescently-labeled ClpB substrates were produced. This work sets the stage for further studies on the mechanism of aggregate recognition by ClpB. ClpB also plays important roles in pathogenic bacteria invasion and virulence. Recombinant ClpB from Ehrlichia chaffeensis, a pathogenic bacterium that causes human monocytic ehrlichiosis, was purified to study its biochemical properties. Ehrlichia ClpB (Eh_B) and E.coli ClpB (Ec_B) sequences are highly conserved in the nucleotide binding region and poorly conserved in the N-terminal and M domain. The oligomerization, ATPase activity, chaperone activity and substrate binding of the recombinant Eh_B were tested. Recombinant Eh_B was able to reactivate aggregated proteins in the presence of HSP70 from E.coli with equal efficiency as Ec_B. However, the mechanism of Eh_B interactions with substrates and/or substrate specificity may be different from that of E. coli ClpB.

A leishmaniose é uma doença tropical negligenciada que afeta milhares de pessoas podendo até levar a óbito em sua forma visceral. Durante o seu ciclo de vida, o parasita passa por diversas mudanças ambientais como mudança de temperatura e pH, principalmente quando da transfecção do inseto vetor para o hospedeiro mamífero. Tais mudanças geram estresse celular que pode levar proteínas ao enovelamento incorreto assim como a processos agregativos, sendo necessários sistemas de controle de qualidade proteico para manter a homeostase celular, do qual fazem parte as chaperonas moleculares. Chaperonas como a Hsp100, ajudam a manter a homeostase celular e a adaptação desempenhando um papel importante para protozoários como a Leishmania braziliensis, causador da leishmaniose. A Hsp100 tem papel desagregase, atuando com outras chaperonas moleculares para a extração de polipeptídios de agregados proteicos possibilitando seu desenovelamento e posterior reenovelamento, evitando seu efeito tóxico sobre a célula. A Hsp100 parece ser essencial para esses microrganismos, no entanto não há muito dados disponíveis para Hsp100 em Leishmania sp. e Plasmodium sp. Neste trabalho está descrito o protocolo para expressão e purificação da Hsp100 recombinante de L. braziliensis (rLbHsp100), assim como sua caracterização estrutural e funcional inicial in vitro. A proteína foi analisada por espectropolarimetria de dicroísmo circular, apresentando estrutura típica de proteínas ricas em hélices α, a fluorescência estática de triptofano demonstrou que a proteína possui estrutura terciária local com seus triptofanos parcialmente expostos ao solvente. Por cromatografia de exclusão molecular analítica, observou-se que a LbHsp100 se comporta como um oligômero cujo estado é influenciado tanto pela concentração proteica como pela presença de nucleotídeos adenosina. Análises por ultracentrifugação analítica evidenciaram que a rLbHsp100 em solução apresenta um equilíbrio de diversas espécies havendo deslocamento para um hexâmero de maneira concentração dependente. Análises de SAXS confirmaram a estrutura hexamérica e proporcionaram a obtenção de um modelo ab initio da proteína. Através de microscopia eletrônica de transmissão pode-se observar a forma toróide e a dispersividade do sistema. Por fim, atestou-se que a proteína foi obtida funcional com fraca atividade ATPásica, apresentando também interações com nucleotídeos adenosina (ATP e ADP) assim como com a suramina. / Leishmaniasis is a neglected tropical disease that affects thousands of people and may even lead to death in its visceral form. During its life cycle, the parasite undergoes several environmental changes such as temperature and pH changes, especially when transfecting from the vector insect into the mammalian host. Such changes generate a cellular stress that can lead to misfolding as well as to aggregative processes, therefore a protein quality control system is necessary to maintain cell homeostasis, which includes molecular chaperones. Chaperones such as Hsp100 can help maintain cellular homeostasis and adaptation playing an important role for protozoa such as Leishmania braziliensis, which causes leishmaniasis. The Hsp100 has a disaggregase action, acting with other proteins of the chaperone system to extract polypeptides from protein aggregates, allowing their unfolding and subsequent refolding, avoiding their toxic effect on the cell. Hsp100 appears to be essential for these microorganisms, however there is not much data available for Hsp100 in Leishmania sp. and Plasmodium sp. This work describes the protocol for expression and purification of the recombinant Hsp100 of Leishmania braziliensis (rLbHsp100), as well as its initial in vitro characterization. The protein was analyzed by circular dichroism spectropolarimetry, presenting a typical structure of ?-helix rich protein as well as a concentration-dependent structure gain, static fluorescence of tryptophan demonstrated that the protein has local tertiary structure with its tryptophans partially exposed to the solvent. Analytical size exclusion chromatography showed that LbHsp100 behaves as an oligomer whose state is influenced by both the concentration and the presence of adenosine nucleotides. Analysis by analytical ultracentrifugation has shown that the rLbHsp100 in solution exhibits an equilibrium of several species shifting towards a hexamer in a concentration dependent manner. SAXS analyzes confirm the hexameric structure and had provide an ab initio model for the protein. Transmission electron microscopy shows the toroidal form and dispersivity of the system. Finally, the obtained protein had showed catalytic function, and also interacted with adenosine nucleotides (ATP and ADP) as well as suramine.

TRiC (TCP-1 ring complex), a type II chaperonin, facilitates protein folding, and we previously showed that TRiC crosslinks to ribosome-bound actin and luciferase nascent chains. Here, it was found that actin and luciferase nascent chains were adjacent to more than one TRiC subunit at different stages of translation. Six and seven out of the eight TRiC subunits were photocrosslinked to the luciferase and actin nascent chains, respectively. Actin nascent chains with widely-spaced, site-specific probe locations were adjacent to the same three TRiC subunits (a, b and e) at different stages of translation. The exposure of other TRiC subunits to nascent chains varied with the length and identity of the nascent chain. In addition, the presence or absence of ATP influences the photocrosslinking yields. This suggests that ATP alters the conformation of the subunits and/or their affinity for the nascent chain. Photocrosslinking also revealed that TRiC is in close proximity to the exit site of the ribosomal tunnel, presumably to create a protected folding environment for the nascent chain. Immunoprecipitations under native conditions revealed that prefoldin photocrosslinks to the actin nascent chain and that these prefoldin-containing photoadducts are coimmunoprecipitated with antibodies specific for the TRiC a subunit. This result suggests that prefoldin and TRiC bind simultaneously to the same actin nascent chain. Photocrosslinking studies with probes at position 68 in the actin nascent chain revealed that prefoldin binds to the nascent chain subsequently to TRiC binding. An unknown protein with an apparent molecular mass of 105 kDa was shown to photocrosslink to the luciferase nascent chain in a length-dependent manner at specific probe locations close to the N-terminus of the nascent chain. Thus, the nascent chain sees a variety of proteins in its immediate environment as it emerges from the ribosomal tunnel and undergoes its chaperonin-assisted folding.

Cancer immunotherapy aims to use and manipulate the host’s immune system to fight against cancer. The objective of this strategy is to induce specific and persistent immune responses leading to tumor eradication. Heat shock proteins (HSP) purified from cancer tissues have been identified as unique mediators of specific anti-tumor immunity. In our laboratory, we have developed an original vaccine, termed CRCL (Chaperone-Rich Cell Lysate) that consists of multiple HSP complexes enriched from tumor lysates. CRCL immunization leads to an efficient protection against a wide variety of murine cancers by inducing a strong, long-lasting, and specific T and NK-cell dependent immune responses against the tumor from which it has been generated. Tumor-derived CRCL has been shown to be more efficient in triggering DC activation than individual purified HSP or tumor lysates. The immunostimulatory effects of CRCL arise from its superior ability to provide a wide variety of tumor antigens to the immune system and by providing potent adjuvant effects. However, CD4⁺CD25⁺ regulatory T lymphocytes (Treg) critically contribute to the mechanisms of cancer-induced suppression. Data from independent groups including ours suggests they may also restrain the function of antigen presenting cells. The current study was designed to elucidate the molecular signaling events triggered by the tumor-derived CRCL vaccine in antigen presenting cells and evaluate whether CRCL may overcome the inhibitory effects of Treg modulation of DC and macrophage activation. Our results indicate CRCL activates DC and macrophages by inducing proinflammatory cytokine chemokine secretion. CRCL induces iNOS expression and NO production in macrophages. CRCL activation of DC and macrophages results in transcription factor NF-κB activation in vitro and in vivo, and this includes the activation of additional signaling molecules upstream of NF-κB. Following CRCL treatment the phenotypic maturation of DC, the production of DC and macrophage pro-inflammatory cytokines, and the activation of the transcription factor NF-κB are not affected by Treg. Additionally, CRCL induced activation of DC is not diminished by the immunosuppressive cytokine TGF-β 1. Our results indicate tumor-derived CRCL-treated DC and macrophages are refractory to Treg inhibition. These results are important for advancing CRCL-based vaccines in Phase I clinical trials.

The family of Nucleoplasmin (NPM) proteins play an important role in a number of chromatin remodelling processes. The first NPM protein discovered in the eggs and oocytes of Xenopus laevis was NPM2, a tissue specific histone chaperone. In Xenopus, NPM2 has been linked to paternal chromatin decondensation following fertilization through the removal of sperm proteins, nucleosome assembly through the storage and addition of H2A-H2B dimers and apoptosis. In mammals, NPM2 correlates strongly with nucleolus-like bodies, and has been suggested by various groups to differ in its roles when compared to the X. laevis homologue. However, the exact roles of NPM2 in mammals remain to be fully elucidated. In this dissertation, attempts are made to determine the physical interaction sites between mouse NPM2 and core histone proteins, H2A, H2B, H3 and H4, as well as physical interactions between mouse NPM2 and protamines (sperm proteins) P1 and P2. Interaction sites between mouse NPM2 and various chromosomal proteins were investigated using a number of different techniques. First, NPM2: chromosomal protein binding assays were attempted to determine the ratio of NPM2 to both core histones and protamines. When visualized through 12% Native gels, NPM2 was determined to interact with histone octamers at a molar ratio of 1-1.5 mol NPM2/mol histone octamer. Mouse sperm protamines were determined to form complexes with mouse NPM2 at a molar ratio of 2.5 mol protamine/mol NPM2 (or mol protamine/0.4 mol NPM2). Analytical Ultracentrifuge (AUC) analysis was conducted on NPM2 and chromosomal proteins separately and in complex formation. Although determining that isolated, full length mouse NPM2 exists in a pentamer form, attempts with AUC were unsuccessful in determining specific NPM2:chromosomal protein binding affinity and complex formation. Specific physical interaction sites between NPM2 and chromosomal proteins were investigated using Cross Linking Mass Spectrometry. Here, a number of new interaction sites as well as sites previously identified by other groups were determined. In combination, our results present likely interaction sites between NPM2 and chromosomal proteins and represent an interesting point of reference for future work. / Graduate

abstract: Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver cancer which is among the top five of the most common cancers. Therefore, vaccines against HCV are under intense study in order to prevent HCV from harming people's health. The envelope protein 2 (E2) of HCV is thought to be a promising vaccine candidate because it can directly bind to a human cell receptor and plays a role in viral entry. However, the E2 protein production in cells is inefficient due to its complicated matured structure. Folding of E2 in the endoplasmic reticulum (ER) is often error-prone, resulting in production of aggregates and misfolded proteins. These incorrect forms of E2 are not functional because they are not able to bind to human cells and stimulate antibody response to inhibit this binding. This study is aimed to overcome the difficulties of HCV E2 production in plant system. Protein folding in the ER requires great assistance from molecular chaperones. Thus, in this study, two molecular chaperones in the ER, calreticulin and calnexin, were transiently overexpressed in plant leaves in order to facilitate E2 folding and production. Both of them showed benefits in increasing the yield of E2 and improving the quality of E2. In addition, poorly folded E2 accumulated in the ER may cause stress in the ER and trigger transcriptional activation of ER molecular chaperones. Therefore, a transcription factor involved in this pathway, named bZIP60, was also overexpressed in plant leaves, aiming at up-regulating a major family of molecular chaperones called BiP to assist protein folding. However, our results showed that BiP mRNA levels were not up-regulated by bZIP60, but they increased in response to E2 expression. The Western blot analysis also showed that overexpression of bZIP60 had a small effect on promoting E2 folding. Overall, this study suggested that increasing the level of specific ER molecular chaperones was an effective way to promote HCV E2 protein production and maturation. / Dissertation/Thesis / M.S. Biological Design 2011

A leishmaniose é uma doença tropical negligenciada que afeta milhares de pessoas podendo até levar a óbito em sua forma visceral. Durante o seu ciclo de vida, o parasita passa por diversas mudanças ambientais como mudança de temperatura e pH, principalmente quando da transfecção do inseto vetor para o hospedeiro mamífero. Tais mudanças geram estresse celular que pode levar proteínas ao enovelamento incorreto assim como a processos agregativos, sendo necessários sistemas de controle de qualidade proteico para manter a homeostase celular, do qual fazem parte as chaperonas moleculares. Chaperonas como a Hsp100, ajudam a manter a homeostase celular e a adaptação desempenhando um papel importante para protozoários como a Leishmania braziliensis, causador da leishmaniose. A Hsp100 tem papel desagregase, atuando com outras chaperonas moleculares para a extração de polipeptídios de agregados proteicos possibilitando seu desenovelamento e posterior reenovelamento, evitando seu efeito tóxico sobre a célula. A Hsp100 parece ser essencial para esses microrganismos, no entanto não há muito dados disponíveis para Hsp100 em Leishmania sp. e Plasmodium sp. Neste trabalho está descrito o protocolo para expressão e purificação da Hsp100 recombinante de L. braziliensis (rLbHsp100), assim como sua caracterização estrutural e funcional inicial in vitro. A proteína foi analisada por espectropolarimetria de dicroísmo circular, apresentando estrutura típica de proteínas ricas em hélices α, a fluorescência estática de triptofano demonstrou que a proteína possui estrutura terciária local com seus triptofanos parcialmente expostos ao solvente. Por cromatografia de exclusão molecular analítica, observou-se que a LbHsp100 se comporta como um oligômero cujo estado é influenciado tanto pela concentração proteica como pela presença de nucleotídeos adenosina. Análises por ultracentrifugação analítica evidenciaram que a rLbHsp100 em solução apresenta um equilíbrio de diversas espécies havendo deslocamento para um hexâmero de maneira concentração dependente. Análises de SAXS confirmaram a estrutura hexamérica e proporcionaram a obtenção de um modelo ab initio da proteína. Através de microscopia eletrônica de transmissão pode-se observar a forma toróide e a dispersividade do sistema. Por fim, atestou-se que a proteína foi obtida funcional com fraca atividade ATPásica, apresentando também interações com nucleotídeos adenosina (ATP e ADP) assim como com a suramina. / Leishmaniasis is a neglected tropical disease that affects thousands of people and may even lead to death in its visceral form. During its life cycle, the parasite undergoes several environmental changes such as temperature and pH changes, especially when transfecting from the vector insect into the mammalian host. Such changes generate a cellular stress that can lead to misfolding as well as to aggregative processes, therefore a protein quality control system is necessary to maintain cell homeostasis, which includes molecular chaperones. Chaperones such as Hsp100 can help maintain cellular homeostasis and adaptation playing an important role for protozoa such as Leishmania braziliensis, which causes leishmaniasis. The Hsp100 has a disaggregase action, acting with other proteins of the chaperone system to extract polypeptides from protein aggregates, allowing their unfolding and subsequent refolding, avoiding their toxic effect on the cell. Hsp100 appears to be essential for these microorganisms, however there is not much data available for Hsp100 in Leishmania sp. and Plasmodium sp. This work describes the protocol for expression and purification of the recombinant Hsp100 of Leishmania braziliensis (rLbHsp100), as well as its initial in vitro characterization. The protein was analyzed by circular dichroism spectropolarimetry, presenting a typical structure of ?-helix rich protein as well as a concentration-dependent structure gain, static fluorescence of tryptophan demonstrated that the protein has local tertiary structure with its tryptophans partially exposed to the solvent. Analytical size exclusion chromatography showed that LbHsp100 behaves as an oligomer whose state is influenced by both the concentration and the presence of adenosine nucleotides. Analysis by analytical ultracentrifugation has shown that the rLbHsp100 in solution exhibits an equilibrium of several species shifting towards a hexamer in a concentration dependent manner. SAXS analyzes confirm the hexameric structure and had provide an ab initio model for the protein. Transmission electron microscopy shows the toroidal form and dispersivity of the system. Finally, the obtained protein had showed catalytic function, and also interacted with adenosine nucleotides (ATP and ADP) as well as suramine.

No description available.

No description available.

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.