Trypanosomes are protozoans, of which many are parasitic, and possess complex lifecycles which alternate between mammalian and arthropod hosts. As is the case with most organisms, molecular chaperones and heat shock proteins are encoded within the genomes of these protozoans. These proteins are an integral part of maintaining the structural integrity of proteins during normal and stress conditions. Heat shock protein 40 (Hsp40) is a co-chaperone of heat shock protein 70 (Hsp70) and in some cases can act as a chaperone. These proteins work together to bind non-native polypeptide structures to prevent unfolded protein aggregrate formation in times of stress, translocate proteins across organelle membranes, and transport unsalvageable proteins to proteolytic degradation by the cellular proteasome. Hsp40s are divided into four types based on their domain structure. Analysis of the nuclear genomes of eight trypanosomatid species revealed that less than 10 of the approximate 70 Hsp40 sequences per genome were Type 1 Hsp40s, many of which contained putative orthologues in the other seven trypanosomatid genomes. One of these Type 1 Hsp40s from T b. brucei, Trypanosoma brucei DnaJ 2 (Tbj2), was functionally characterised in T brucei brucei. RNA interference knockdown of expression in T brucei brucei showed that cells deficient in Tbj2 displayed a severe inhibition of the growth of the cell population. The levels of the Tbj2 protein population in T brucei brucei cells increases after exposure to 42°c and the protein was found to have a generalized cytoplasmic subcellular localization at 37°c. These findings provide evidence that Tbj2 is an orthologue of Yeast DnaJ 1 (Y dj l), an essential S. cerevisiae protein. Hsp40s interact with their partner Hsp70s through their J-domain. The amino acids of the J-domain important for a functional interaction with Hsp70 were examined in Trypanosoma cruzi DnaJ 2 (Tcj2) (the orthologue of Tbj2) and T cruzi DnaJ protein 3 (Tcj3) by testing their ability to substitute for Y dj l in Saccharomyces cerevisae and for DnaJ in Escherichia coli. In both systems, the positively charged amino acids of Helix II and III of the J-domain disrupted the functional interaction of these Hsp40s with their partner Hsp70s. Substitutions in Helix I and IV of the J-domains of Tcj2 and Tcj3 produced varied results in the two different systems, possibly suggesting that these helices serve to define with which Hsp70s a given Hsp40 can interact. The inability of an Hsp40 and an Hsp70 to interact functionally does not necessarily mean a total absence of physical interaction between these proteins. The amino acid substitution of the histidine in the HPD motif (H34Q) of the J-domain of Tcj2 and Tcj3 removed the ability of these proteins to interact functionally with S. cerevisiae Hsp70 (Ssal) in vivo. However, preliminary binding studies using the quartz crystal microbalance with dissipation monitoring (QCM-D) show that Tcj2 and Tcj2(H34Q) both physically interact with M sativa Hsp70 in vitro. This study is the first report to provide evidence that certain trypanosoma! Type 1 Hsp40s are essential proteins. Futhermore, the interaction of these Hsp40s with Hsp70 identified important features of the functional interface of this chaperone machinery.

Current dogma suggests that the Heat Shock Protein (Hsp) molecular chaperones and associated co-chaperones function primarily within the cell, although growing evidence suggests a role for these proteins on the plasma membrane of cancer cells. Hsp90 does not function independently in vivo, but instead functions with a variety of partner chaperones and co-chaperones, that include Hsp70 and Hsp90/Hsp70 organising protein (Hop), which are thought to regulate ATP hydrolysis and the binding of Hsp90 to its client proteins. Hsp90 on the plasma membrane appears to have distinct roles in pathways leading to cell motility, invasion and metastasis. We hypothesised that Hsp90 on the plasma membrane is present as part of a multi-chaperone complex that participates in the chaperone-assisted folding of client membrane proteins in a manner analogous to the intracellular chaperone complex. This study characterised the membrane expression of Hsp90, Hsp70 and Hop in different cell models of different adhesive and migratory capacity, namely MDA-MB-231 (metastatic adherent breast cancer cell line), MCF-7 (non-metastatic adherent breast cancer cell line), U937 and THP1 (monocytic leukemia suspension cell lines). Membrane expression of the Hsps was analysed using a combination of subcellular fractionation, biotin-streptavidin affinity purification and immunofluorescence. This study provided evidence to suggest that Hsp90, Hsp70 and Hop are membrane associated in MDA-MB-231 and MCF-7 breast cancer cells. Hsp90, Hsp70 and Hop associated with the plasma membrane such that at least part of the protein is located extracellularly. Immunofluorescence analysis showed that Hsp90, Hsp70 and Hop at the leading edge may localize to membrane ruffles in MDA-MB-231 cells, in accordance with the published role of Hsp90 in migration. An increase in this response was seen in cells stimulated to migrate with SDF-1. By immunoprecipitation, we isolated a putative extracellular membrane associated complex containing Hsp90, Hsp70 and Hop. Using soluble Hsp90 and antibodies against membrane associated Hsp90, we suggested roles for soluble extracellular Hsp90 in mediating migration by wound healing assays and inducing actin reorganisation and vinculin-based focal adhesion formation. The effects of extracellular Hsp90 are mediated by signalling through an ERK1/2 dependent pathway. An anti-Hsp90 antibody against an N-terminal epitope in Hsp90 appeared to be able to overcome the death inducing effects of a combination of SDF-1 and AMD3100, while soluble Hsp90 could not overcome this effect. We propose that this study provides preliminary evidence that extracellular Hsp90 functions as part of a multi-chaperone complex that includes Hsp70 and Hop. The extracellular Hsp90 chaperone complex may mediate cell processes such as migration by modulating the conformation of cell surface receptors, leading to downstream signalling.

To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.

No description available.

No description available.

A HP1076 null mutant has been constructed to provide a better understanding of the biological significance of HP1076 in H. pylori . The DeltaHP1076 mutant displays impaired motility and resistance to the antibiotic drug metronidazole. Using a proteomic study, an overall of 40 differentially expressing proteins involved in metabolism and pH homeostasis for bacterial survival, adhesion for colonization, virulence factor to gastric epithelial cells and antigenic proteins have been identified. The virulence factor, Cag pathogenicity island protein (Cag 26) and urease UreA and UreB are confirmed to have enhanced and reduced expression in null mutants. These findings may provide new insight into the infection of H. pylori. / FliS is an export chaperone that binds to flagellin molecules in cytosol in order to prevent pre-mature polymerization. Disruption of FliS would result in formation of shorter flagella and impaired adhesion ability to epithelial cells. Previous yeast two-hybrid study has identified various FliS associated proteins in H. pylori, but with no known implications. Here, we have demonstrated the interaction of FliS and a hypothetical protein HP1076 by biochemical and biophysical methods. Moreover, HP1076 possesses anti-aggregation ability on insoluble FliS-mutants and chaperone activity. Thus, HP1076 is proposed to be a co-chaperone that promotes the folding and chaperone activity of FliS. FliS is demonstrated to have a broad range of substrate specificity that binds to flagellin and flagellar related proteins which may play a key role in flagellar export system different from other flagellated bacteria. / Helicobacter pylori is a pathogenic bacterium and adheres to the gastric mucosal cells. Chronic infection would lead to gastritis or peptic ulceration and is one of the leading causes of gastric cancer. Formation of functional flagella is essential for infection, that it aids in motility of bacteria and colonization on gastric epithelial cells. The process is complex and involves more than 50 proteins in assembly of structural proteins, regulatory proteins, an export apparatus, a motor and a sensory system. Cytosolic chaperones are required to bind to exported proteins in order to facilitate the export or prevent the aggregation of proteins in cytosol. Divergence is found in flagellar system H. pylori that may account for survival inside gastric environment. / The crystal structures of FliS, HP1076 fragment and FliS/HP1076 complex are determined at 2.7A, 1.8A and 2.7A resolution respectively to provide better understanding of their molecular interactions. FliS consists of four helices and HP1076 consists of helical rich bundle structure with three helices and three beta strands that share similar fold to that of a flagellin homologue, hook-associated protein and FliS, suggesting HP1076 is involved in flagellar system. The FliS/HP1076 complex reveals an extensive electrostatic and hydrophobic binding interface which is distinct from the flagellin binding pocket on FliS. HP1076 stabilizes two alpha helices of FliS and therefore the overall bundle structure. Our findings provide new insights into the flagellar export chaperones and other secretion chaperones in Type III secretion system. / Lam, Wai Ling. / Adviser: An Wing-Ngor. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 223-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 103-117). Also issued on the Internet.

The Arabidopsis HSP101 protein belongs to the Hsp100/ClpB family of AAA+ proteins (ATPases Associated with various cellular Activities). This family of proteins, in collaboration with the Hsp70 chaperone system, has the remarkable ability to solubilize protein aggregates and refold proteins back to their native forms. Thus, their chaperone activity is necessary for acquired thermotolerance in organisms as diverse as bacteria and plants. My dissertation project focused on understanding the mechanism of HSP101 action using Arabidopsis thaliana as a model system. The first approach used genetics to screen for suppressors of a specific missense mutant allele of HSP101, hot1-4, in order to find interacting cofactor proteins or key substrates of HSP101, or other processes involved in thermotolerance. Four extragenic suppressors that can overcome the heat-hypersensitive phenotype caused by the hot1-4 mutation were isolated and one of them (shot1) was identified as a mutation in a mitochondrion-targeted protein. Although shot1 mutations do not directly interfere with HSP101 function, they reveal independent mechanisms required for thermotolerance, which involve reduced oxidative stress. The second approach used to investigate HSP101 function was to affinity-purify HSP101 and identify associated proteins. For this purpose, transgenic Arabidopsis plants were generated expressing affinity-tagged wild-type and mutant variants of HSP101. As predicted, cytosolic Hsp70s were identified as an interacting partner of HSP101. Surprisingly, 26S proteasome regulatory subunits were also identified, suggesting a possible link between the protein degradation and reactivation pathways. Further experiments were also undertaken to define the importance of different domains of HSP101, as well as the localization of HSP101. Transgenic Arabidopsis plants expressing N- or C-terminally truncated HSP101 indicate that the N-terminal domain of HSP101 is required for full activity in protecting plants from heat stress. However, in contrast to the yeast ortholog, Hsp104, the C-terminal extension of HSP101 was found to be completely dispensable for thermotolerance of Arabidopsis. Additional transgenic plants expressing an HSP101-GFP were also characterized. Initial microscopic analysis confirms nuclear/cytoplasmic localization as has been reported previously for yeast Hsp104. However, the dynamics of subcellular redistribution upon heat stress need to be further investigated to fully understand the potential significance of the observed localization.

As chaperonas moleculares atuam no enovelamento de proteínas, montagem de complexos, prevenção/recuperação de proteínas de agregados e encaminhamento de proteínas mal enoveladas para depuração. As Hsp90 são chaperonas moleculares que atuam estabilizando proteínas relacionadas a vias de sinalização, crescimento celular, processos transcricionais e traducionais, estabilidade do genoma, entre outras, sendo essencial para a viabilidade celular. Em protozoários do gênero Leishmania, as Hsp90 são imprescindíveis no desenvolvimento, adaptação e transformação celular. Estes fatores fazem das Hsp90 alvos potenciais para o tratamento de patologias, como a leishmaniose, uma doença tropical negligenciada. As Hsp90 são homodímeros flexíveis onde cada protômero é dividido em três domínios denominados N, M e C. As Hsp90 possuem um ciclo conformacional associado ao seu ciclo funcional e sua baixa atividade ATPásica, o qual é direcionado e regulado por proteínas auxiliares, as co-chaperonas. A co-chaperona Aha1 atua estimulando a atividade ATPásica da Hsp90, participando da maturação de proteínas quinase e receptores de hormônios. O objetivo deste trabalho foi caracterizar estruturalmente a proteína Aha1 de L. braziliensis (LbAha1) e seu mecanismo de interação com a Hsp90 desse organismo (LbHsp90). A LbAha1 é formada por dois domínios, LbAha1N e LbAha1C, conectados entre si por um linker flexível. Experimentos de identificação in vivo mostraram que a LbAha1 e LbHsp90 são proteínas cognatas. A LbAha1 e as construções de seus domínios (LbAha1N e LbAha1C) recombinantes foram obtidas puras e enoveladas. A LbAha1 é estruturada em dois domínios com diferentes estabilidades, que não interagem entre si e se enovelam independentemente, porém influenciam-se reciprocamente. Em solução, a LbAha1 se comporta como um monômero alongado e possui notável flexibilidade, com dimensão suficiente para interagir com os domínios N e M da LbHsp90. A análise da interação entre a LbAha1 e LbHsp90 revelou que a associação destas proteínas é dirigida entalpicamente, ocorrendo através de interações eletrostáticas e com estequiometria de 2 moléculas de LbAha1 por dímero de LbHsp90. O mapeamento de regiões envolvidas na interação indicou que o domínio LbAha1N e o domínio M da LbHsp90 compõem o cerne da interação e somente a LbAha1 íntegra é capaz de encaminhar a LbHsp90 para um estado fechado. Experimentos de cinética enzimática mostraram que somente a LbAha1 íntegra estimula a atividade ATPásica da LbHsp90 por meio de um mecanismo cooperativo positivo. Assim, é proposto que a conexão entre os domínios da LbAha1, via linker, é essencial para o direcionamento da LbHsp90 para um estado conformacional fechado e competente na hidrólise de ATP. / Molecular chaperones play a role in protein folding, complex assembly, prevention/recover of proteins from aggregates and targeting misfolded proteins to depuration. Hsp90 molecular chaperones work stabilizing proteins related to signaling pathways, cell growth, transcription and translation processes, genome stability, among others, and are essential to cell viability. In protozoa of the genus Leishmania, Hsp90s are indispensable for cell developing, adaptation and transformation. These factors make Hsp90s potential targets for pathologies treatment, such as leishmaniasis, a neglected tropical disease. Hsp90s are flexible homodimers and each protomer is divided into three domains named N, M and C. Hsp90s have a conformational cycle associated to its functional cycle and low ATPase activity, which is directed and regulated by auxiliary proteins, so-called cochaperones. Aha1 co-chaperone stimulates Hsp90 ATPase activity, participating on protein kinase and hormone receptors maturation. This work aimed to characterize the structure of the Aha1 from L. braziliensis (LbAha1) and its mechanism of interaction with the Hsp90 from the same organism (LbHsp90). LbAha1 is formed by two domains, LbAha1N and LbAha1C, connected to each other by a flexible linker. In vivo experiments identified LbAha1 and LbHsp90 as cognate proteins. Recombinant LbAha1 and its domains construct (LbAha1N and LbAha1C) were obtained pure and folded. LbAha1 is divided into two domains with dissimilar stabilities and they do not interact to each other. In spite of this they fold independently and influence each other reciprocally. LbAha1 behaves as an elongated monomer in solution and has a remarkable flexibility, with sufficient dimension to interact to LbHsp90 N and M domains. The analysis of the LbAha1-LbHsp90 interaction revealed that the association between these two proteins is enthalpically driven, occurring through electrostatic interactions in a stoichiometry of 2 LbAha1 molecules per LbHsp90 dimer. Domain mapping experiments indicated that LbAha1N and LbHsp90 M domains compose the core of the interaction and only full length LbAha1 is able to direct LbHsp90 toward a closed state. Enzyme kinetics experiments showed that only full length LbAha1 stimulates LbHsp90 ATPase activity through a positive cooperative mechanism. Thus, it is proposed that the connection between the LbAha1 domains, via linker, is essential to direct the LbHsp90 toward a closed and ATPase-competent conformational state.