The Heat Shock Protein 90 (Hsp90) is an essential and highly conserved chaperone that facilitates the maturation of a wide array of client proteins, including many kinases. These clients in turn regulate a wide array of cellular processes, such as signal transduction, and transcriptional reprogramming. As a result, the activity of Hsp90 has the potential to influence physiology, which in turn may influence the ability to adapt to new environments. Previous studies using a deep mutational scanning approach, (EMPIRIC) identified multiple substitutions within a 9 amino acid substrate-binding loop of yeast Hsp90 that provides a growth advantage for yeast under elevated salinity conditions and costs of adaptation under alternate environments. These results demonstrate that genetic alterations to a small region of Hsp90 can contribute to evolutionary change and promote adaptation to specific environments. However, because Hsp90 is a large, highly dynamic and multi-functional protein the adaptive potential and evolutionary constraints of Hsp90 across diverse environments requires further investigation. In this dissertation I used a modified version of EMPIRIC to examine the impact of environmental stress on the adaptive potential, costs and evolutionary constraints for a 118 amino acid functional region of the middle domain of yeast Hsp90 under endogenous expression levels and the entire Hsp90 protein sequence under low expression levels. Endogenous Hsp90 expression levels were used to observe how environment may affect Hsp90 mutant fitness effects in nature, while low expression levels were used as a sensitive readout of Hsp90 function and fitness. In general, I found that mutations within the middle domain of Hsp90 have similar fitness effects across many environments, whereas, under low Hsp90 expression I found that the fitness effects of Hsp90 mutants differed between environments. Under individual conditions multiple variants provided a growth advantage, however these variants exhibited growth defects in other environments, indicating costs of adaptation. When comparing experimental results to 261 extant eukaryotic sequences I find that natural variants of Hsp90 support growth in all environments. I identified protein regions that are enriched in beneficial, deleterious and costly mutations that coincides with residues involved in co-chaperone-client-binding interactions, stabilization of Hsp90 client-binding interfaces, stabilization of Hsp90 interdomains and ATPase chaperone activity. In summary, this thesis uncovers the adaptive potential, costs of adaptation and evolutionary constraints of Hsp90 mutations across several environments. These results complement and extend known structural and functional information, highlighting potential adaptive mechanisms. Furthermore, this work elucidates the impact environment can have on shaping Hsp90 evolution and suggests that fluctuating environments may have played a role in the long-term evolution of Hsp90.

No description available.

The deposition of β-amyloid peptides and of α-synuclein proteins is a neuropathological hallmark in the brains of Alzheimer’s disease (AD) and Parkinson’s disease (PD) subjects, respectively. However, there is accumulative evidence that both proteins are not exclusive for their clinical entity but instead co-exist and interact with each other. Here, we investigated the presence of a newly identified, pyroglutamate79-modified α-synuclein variant (pGlu79-aSyn)—along with the enzyme matrix metalloproteinase-3 (MMP-3) and glutaminyl cyclase (QC) implicated in its formation—in AD and in the transgenic Tg2576 AD mouse model. In the human brain, pGlu79-aSyn was detected in cortical pyramidal neurons, with more distinct labeling in AD compared to control brain tissue. Using immunohistochemical double and triple labelings and confocal laser scanning microscopy, we demonstrate an association of pGlu79-aSyn, MMP-3 and QC with β-amyloid plaques. In addition, pGlu79-aSyn and QC were present in amyloid plaque-associated reactive astrocytes that were also immunoreactive for the chaperone heat shock protein 27 (HSP27). Our data are consistent for the transgenic mouse model and the human clinical condition. We conclude that pGlu79-aSyn can be generated extracellularly or within reactive astrocytes, accumulates in proximity to β-amyloid plaques and induces an astrocytic protein unfolding mechanism involving HSP27.

Molecular chaperones are proteins that interact with and aid in stabilization and activation of other proteins. Chaperones help proteins attain their three dimensional conformation, without forming a part of the final structure. Many of the chaperones are stress proteins known as Heat shock proteins (Hsps). Their expression is upregulated in response to various kinds of stress such as heat stress, oxidative stress etc., which threaten the protein homeostasis, by structurally destabilizing cellular proteins, and increasing the concentration of aggregation-prone folding intermediates. The Hsps are classified according to their molecular weight into Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, and the small Hsp families. Some of them are constitutively expressed and play a fundamental role in de novo protein folding. They further aid in proteome maintenance by assisting in oligomeric assembly, protein trafficking, refolding of stress denatured protein, preventing protein aggregation and protein degradation. Heat shock protein 90 (Hsp90) are one of the important representatives of this class of proteins. Hsp90 are highly conserved class of molecular chaperones. They are found in bacteria, eukaryotes, but not in archaea. In contrast to the eukaryotes which require a functional cytoplasmic Hsp90 for viability, the bacterial counterpart (HtpG) is typically nonessential. Hsp90 is an ATP dependent chaperone. Hsp90 form dimers, with each protomer consisting of three functional domains: N- terminal, ATP binding domain, Middle domain and C-terminal domain. Hsp90 is a dynamic protein, and undergoes an elaborate conformational cycle during its ATPase cycle, which is essential for its chaperoning activity. The Hsp90 chaperone cycle is regulated by interaction with diverse cochaperones. Hsp90 interacts with specific set of substrate proteins. Many of these substrate proteins function at the heart of several cellular processes like signalling, cell cycle, apoptosis. Studies from protozoans like Leishmania, Plasmodium, Trypanosoma etc. have also implicated the role of Hsp90 in their growth and stage transitions. Thus, selective inhibition of Hsp90 has been explored as an intervention strategy against important human diseases such as cancer, malaria and other protozoan diseases. The ATP binding N-terminal domain (NTD), has been explored as the target domain for inhibition of Hsp90 using competitive inhibitors of ATP. Several chemical classes of Hsp90 inhibitors are known, including ansamycins, macrolides, purines, pyrazoles, and coumarin antibiotics. However, many inhibitors are observed to be toxic, less soluble and unstable. Hence, there is a requirement for new approach to design inhibitors which are more soluble and less toxic and serve as effective therapeutic drugs.inhibitors are observed to be toxic, less soluble and unstable. Hence, there is a requirement for new approach to design inhibitors which are more soluble and less toxic and serve as effective therapeutic drugs. The work presented in this thesis mainly concerns with the structural studies and biochemical and biophysical characterization of Hsp90 from two different sources viz. Dictyostelium discoideum, a cellular slime mould and a plant source Oryza sativa (rice). The structural analyses of these two proteins have been carried out by X-ray crystallography. Though yeast has been explored extensively as a model system to understand the different roles of Hsp90, it lacks the various signalling pathways essential for growth and development present in case of higher eukaryotes. D. discoideum has been employed as a model system to understand multicellular development, which occurs in response to starvation induced stress. D. discoideum has the advantages due to its ease of manipulation. The organism's genome also shows many signalling pathway for growth and differentiation that are conserved between D. discoideum and mammals. With this motivation, we have studied several structural aspects of the cytosolic isoform of Hsp90 from D. discoideum called HspD. HspD was also observed to play a role in the multicellular development of D. discoideum. It has been demonstrated that the treatment of D. discoideum with inhibitors like Geldanamycin or Radicicol causes an arrest in the multicellular development at the mound stage, and the few which escaped this arrest gave rise to abnormal fruiting bodies. A subset of the proteins involved in this mound arrest phenotype, were observed to have homologs in humans, which are clients of Hsp90. Therefore, a structural perspective of HspD can aid in better understanding of the role of this protein in the organism, as well as, elucidate any structural differences observed as compared to other species, which may have an impact on its activity. Studies on the physiological role of Hsp90 in plants began much later as compared to fungi and humans. In plants Hsp90 are involved in various abiotic stress responses. In addition, their roles have also been implicated in plant growth and development, innate immune response and buffering genetic variations. However, the molecular mechanisms of these various actions are not clearly understood. Also, the structural aspects of plant Hsp90 are yet to be explored. The structure of the NTD of Hsp90 from barley is the only one available from a plant source till now. We have initiated the studies on rice Hsp90 with the objective to understand the mechanism of Hsp90 in plants, which may aid in improving stress tolerance in plants. The thesis has been divided into five chapters. The first chapter introduces the various aspects of Hsp90 protein. The chapter starts with a general overview of concept of molecular chaperones and describes briefly the different classes of molecular chaperones. This is followed by a detailed description of different aspects of Hsp90 with main emphasis on the structure and its conformational flexibility. The chapter describes the association of Hsp90 with other accessory proteins like cochaperones and its interaction with its substrate proteins and explains the functional significance of Hsp90 as a drug target and the need for the development of new class of inhibitors, followed by the significance of the study of Hsp90 in the two model systems (D. discoideum and rice) chosen to be studied. The second chapter gives a brief overview of the principles behind the different experimental methods employed during the course of this research, which includes the tools of X-ray crystallography and other biochemical and biophysical techniques employed for the characterization of the protein. Chapter 3 describes the crystal structure of NTD of Hsp90 from D. discoideum. The structure of NTD was solved in two different native (ligand-free) forms viz. monoclinic and hexagonal. The two forms differed in local structural rearrangement of a segment of NTD known as the lid region. The lid region in the hexagonal form showed a shift in its position as compared to the other solved structures of NTD. The structure of NTD was also solved in complex with various ligands which include ADP, substrate analogs and an inhibitor molecule. A comparison of all the structures showed that the overall structure is well-conserved. One of the crystal structures of NTD showed a heptapeptide (part of the vector) bound at the active site. The peptide was observed to make several complementary interactions with the residues of the ATP binding pocket and retain several interactions which the nucleotide makes with the NTD. The NTD showed subtle conformational differences when compared with the NTD of Hsp90 from yeast. Chapter 4 details the structural and functional characteristics of full length Hsp90 protein from D. discoideum. Due to the large size and flexibility, the full length protein did not crystallize in spite of several attempts. Hence, HspD was studied using different solution studies like Small Angle X-ray Scattering (SAXS) and Dynamic Light Scattering (DLS). Both the studies showed the presence of higher oligomers. The SAXS data showed the presence of tetramers and hexamers while, the addition of the ligand shifts the protein from a dimer to a higher oligomer as observed from DLS studies. The chapter also describes the study of interaction of HspD with a cochaperone protein p23. The interactions were studied using ITC, which showed a strong binding. The ATPase activity was also evaluated in the presence of increasing concentrations of p23, which was observed to decline with increasing concentrations of p23. In chapter 5, we describe the biochemical characterization of Hsp90 from Oryza sativa (rice) and the crystallographic analysis of its NTD. Binding of the rice Hsp90 to ATP and an inhibitor were studied by fluorescence. The ATPase activity of rice Hsp90 was checked by radioactive assay and the protein was observed to be active. The NTD of rice Hsp90 crystallized as a monomer in complex with a substrate analog AMPPCP and the structure was determined.

The topic of the presented thesis is the regulation of gene expression level of the three HSP70 genes in mononuclear cells. We investigated the dependence of expression regulation (induction) abiliy on selected point mutations, so-called SNPs (single nucleotide polymorphism) in the observed genes. The mononuclear cells were obtained from peripheral blood samples of healthy individuals. In order to analyze their gene expression, we selected individuals who were homozygous for at least one of the monitored point mutations. Taking into account the chosen criteria for healthy individuals we based on interviews with these individuals and their personal history. We determined the polymorphisms observed in two cell stress-inducible HSP70-1 (HSPA1A) and HSP70-2 (HSPA1B) genes and in one constitutively expressed gene HSP70-Hom (HSPA1L). Further, we have analyzed HSP70s gene expression regulation and the relation between the expression regulation and studied polymorphisms. We determined the degree of regulation of a gene expression in the studied genes in relation to two SNPs -110A/C (rs1008438), +190G /C (rs1043618) gene HSP70-1, and two SNPs +1267A/G (rs1061581), +2074G /C (rs539689 ) of the HSP70-2 gene, and the mutation of one five-nucleotide (rs9281590) HSP70-2 gene, and one SNP +2437T/C (rs2227956) of...

Macrófagos infectados com Leishmania são protegidos de apoptose, entretanto não se conhece o mecanismo de transdução de sinal intracelular que interfere neste processo de morte. Neste trabalho, células J 774 em cultura, com privação de nutrientes, sofrem apoptose, a qual aumenta na presença dos indutores camptotecina (CPT) ou fator de necrose tumoral recombinante (rTNF). Estas células quando infectadas com amastigotas ou promastigotas de Leishmania (L.) chagasi (5 parasitos/uma célula) são protegidas de apoptose. Avaliando as possíveis vias intracelulares envolvidas nesse processo, observamos que a privação de nutrientes altera o potencial de membrana da mitocôndria, havendo reversão com a infecção tanto com promastigotas e amastigotas, entretanto a reversão da alteração do potencial de membrana induzida por rTNF só foi observada com infecção com promastigotas. Tanto a atividade de caspase 3, como a detecção de caspase 3 clivada induzidas por H202 são revertidas com a infecção com promastigotas ou amastigotas. Quando analisamos a expressão de poli (ADP ribose) polimerase (PARP), em relação às células sem indução, a indução por CPT não levou ao aumento da PARP de 116 kDa, mas, aumento da banda de 24 kDA. Por outro lado, a infecção por amastigota de Leishmania (L.) chagasi em células J774 levou à diminuição da expressão de PARP de 116 kDa, mas aumento da de 24 kDa. Nas células infectadas por promastigotas de Leishmania (L.) chagasi, observamos uma diminuição da banda de 116 kDa, aparecimento de uma molécula de 89kDa e diminuição da expressão da de 24 kDa. Nas células sob indução por CPT, a infecção levou a resultados similares, exceto a diminuição da molécula de 24 kDa quando infectado por amastigota. Avaliando-se a influência da proteína do choque térmico de 83 kDa de Leishmania infantum, como possível fator que interferiria no processo de apoptose, observamos que a fagocitose de bactérias Escherichia coli (M15) contendo plasmídio com gene de HSP83 expressando essa proteína, leva a diminuição da apoptose nessas células, mesmo quando induzidas por CPT ou rTNF. Nossos dados mostram que a infecção de macrófagos J774 in vitro por Leishmania (L.) chagasi, interfere no processo de apoptose afetando diversas vias de sinalização intracelular de apoptose, tanto extrínsecas quanto intrínsecas, sendo que promastigota é mais efetiva em inibir apoptose nesta linhagem macrofágica. / Macrophages infected by Leishmania are protected from apoptosis, however the mechanism of intracellular signal transduction that interferes in this death process remains unknown. In this work, J774 cells in culture, under nutrient deprivation undergo apoptosis, which is increased in the presence of inducers: camptothecin (CPT) or recombinant tumoral necrosis factor (rTNF). These cells infected by amastigotes or promastigotes of Leishmania (L.) chagasi (5 parasites per cell) are protected from apoptosis. Evaluating the possible intracellular pathways involved in this process, we observed nutrient deprivation alters the mitochondrial membrane potential, reversed by both amastigote and promastigote infection, in contrast, mitochondrial membrane potential was altered by rTNF and it was reversed only by promastigotes. Both caspase 3 activity and caspase 3 cleavage detection induced by H2O2 are reversed with amastigote or promastigote infection. When we analysed the expression of poly (ADP-ribose) polymerase, related to no induced cells . CPT induction didnLt increase 116 kDa PARP, but increased a 24 kDa fragment. Otherwise, Leishmania (L.) chagasi amastigote infection in J774 cells decreased 116 kDa PARP, but increased a 24 kDa fragment. Incells infected by Leishmania (L.) chagasi , we observed a decrease of 116 kDa fragment, appearance of a 89 kDa fragment and a decreasing of a 24 kDa fragment. In the cells under CPT induction similar results were found, except a decreasing of a 24 kDa when infected by amastigote. Evaluating the Leishmania (L.) infantum Heat Shock Protein of 83 kDa, as a possible factor that interferes in the apoptosis process, we observed that a phagocytosis of Escherichia coli (M15) bacteria with a HSP83 gene within a plasmid expressing this protein induced by isopropyl β - D- tiogalactopiranosideo (IPTG), considerably diminished apoptosis in these cells even when induced by CPT or rTNF. Our data show that Leishmania (L.) chagasi infection in J774 macrophages in vitro notoriously interferes in the apoptosis process affecting several intracellular pathways involved in both extrinsic and intrinsic pathways, more prominently with promastigote in this macrophage cell lineage.

The citrulline-nitric oxide (NO) cycle, comprised of the enzymes argininosuccinate synthase (AS), argininosuccinate lyase (AL) and endothelial nitric oxide synthase (eNOS), is responsible for the regulated production of endothelial NO. Although most studies have focused on eNOS to uncover important regulatory mechanisms, we and others have determined that AS is an essential and regulated step in endothelial NO production. AS is rate limiting for endothelial NO production and is the primary source of arginine, the substrate for eNOS-mediated NO production, despite saturating intracellular levels of arginine and available arginine transport systems. AS is essential for endothelial cell viability and its expression is regulated coordinately with eNOS by TNF and thiazolidenediones with concomitant effects on NO production. Given the importance of AS for endothelial health, we explored three independent regulatory mechanisms. In Chapter One, the functional consequences of altered AS expression due to overexpression, insulin, VEGF and ceramide were studied. We demonstrated that overexpression of AS leads to enhanced NO production and that insulin, VEGF and ceramide coordinately regulate the expression of AS and eNOS. In Chapter Two, the first post-translational modifications of AS in the endothelium were characterized. We determined that AS is an endogenous phosphoprotein in the endothelium, described several levels of biological significance of AS phosphorylation, identified 7 sites of AS phosphorylation and began to uncover the direct impact of phosphorylation on AS function. Finally, in Chapter Three, endothelial AS subcellular localization was defined and important protein interactions were identified including caveolin-1 and HSP90. The work presented in this dissertation demonstrates that multiple mechanisms regulate the function of AS, often coordinately with eNOS, and have a direct impact on nitric oxide production. Our findings suggest that the global understanding of the citrulline-NO cycle as a metabolic unit will unravel new paradigms that will re-define our understanding of the regulation of vascular function by NO.

The heat shock response (HSR) is a highly conserved mechanism that enables organisms to survive environmental and pathophysiological stress. In Drosophila, the HSR is regulated by a single transcription factor, heat shock factor (HSF). During stress, HSF trimerizes and binds to over 200 loci on Drosophila polytene chromosomes with only nine mapping to major heat shock (HS) inducible gene loci. The function of HSF binding to the other sites in the genome is currently unknown. Some of these sites may contain yet unidentified “minor” HS genes. Interestingly, the binding of HSF also coincides with puff regression at some sites. Two such sites contain the major developmentally regulated genes Eip74 and Eip75: key regulators in the response to 20-hydroxyecdysone (20E), the main hormone responsible for the temporal co-ordination of post-embryonic development in Drosophila. Previous work in our and other labs indicates that the regression of non-HS puffs during the HSR is dependent on the presence of functional HSF. Using chromatin immunoprecipitation (ChIP) followed by hybridization to genome tiling arrays (Chip), I have identified 434 regions in the Drosophila Kc cell genome that are bound by HSF during HS, and have determined that 57% of these sites are located within the transcribed regions of genes. By examining the transcriptional response to HS in Kc cells and third instar larvae using expression microarrays, I found that only about 10% of all genes within 1250 bp of an HSF binding site are transcriptionally regulated by HS and many genes whose transcript levels change during HS do not appear to be near an HSF binding site. Furthermore, genes with an HSF binding site within their introns are significantly enriched (modified Fisher Exact p-value between 2.0x10-3 and 1.5x10-6) in gene ontology terms related to developmental processes and reproduction. Using expression microarray technology, I characterized the transcriptional response to 20E and its structural analog ponasterone A. I have identified multiple HSF binding sites within Eip74 and Eip75, and show that induction of the HSR correlates with repression of these genes and all other 20E-inducible genes. Taken together, this work provides a basis for further investigation into the role of HSF binding to sites not associated with HS genes and its possible function as a repressor of gene transcription during conditions of stress and as a regulator of developmental genes under stress and non-stress conditions.

Molecular chaperones have emerged in recent years as major players in many aspects of cell biology. Molecular chaperones are also known as heat shock proteins (HSPs) since many were originally discovered due to their increased synthesis in response to heat shock. They were initially identified when Drosophila salivary gland cells were exposed to a heat shock at 37°C for 30 min and then returned to their normal temperature of 25°C for recovery. A “puffing” of genes was found to have occurred in the chromosome of recovering cells, which was later shown to be accompanied by an increase in the synthesis of proteins with molecular masses of 70 and 26 kDa. These proteins were hence named “heat shock proteins”. The first identification of a function for HSPs was the discovery in Escherichia coli that five proteins synthesized in response to heat shock were involved in λ phage growth. The products of the groEL and groES genes were found to be essential for phage head assembly while the dnaK, dnaJ and grpE gene products were essential for λ phage replication. It was later shown that GroEL and GroES are part of a chaperonin system for protein folding in the prokaryotic cytosol while DnaK is a member of the Hsp70 family that works in conjunction with the DnaJ (Hsp40) co-chaperone and the nucleotide exchange factor GrpE to promote phage replication by dissociating the DnaB helicase from the phage-encoded P protein. Since then, a large number of other proteins collectively referred to as HSPs have been discovered. However, heat shock is not the only signal that induces synthesis of heat shock proteins. Stress of any kind, such as nutrient deprivation, chemical treatment and oxidative stress among others causes increased production of HSPs and therefore, they are also known as stress proteins. The term “molecular chaperone” was originally used to describe the function of nucleoplasmin, a Xenopus oocyte protein that promotes nucleosome assembly by binding tightly to histones and donating the bound histone to chromatin. However, since then, chaperones have been defined as “a family of unrelated classes of proteins that mediate the correct assembly of other proteins, but are not themselves components of the final functional structure”. This view of molecular chaperones, though undoubtedly correct, doesn’t capture the multifaceted roles they have since been discovered to play in cellular processes. In recent years, molecular chaperones have been shown to perform other functions in addition to the maintenance of protein homeostasis: translocation of proteins across organelle membranes, quality control in the endoplasmic reticulum, turnover of misfolded proteins as well as signal transduction. As a result, many chaperones are also essential under non-stress conditions and play crucial roles in cell growth and development, cell-cell communication and regulation of gene expression. Heat shock protein 90 (Hsp90) is one of the most abundant and highly conserved molecular chaperones in organisms ranging from bacteria to all branches of eukarya. It has been shown to be essential for cell viability in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster. Although the bacterial homolog HtpG is dispensable under normal conditions, it is important for cell survival during heat shock. In addition to its role as general chaperone in protein folding following stress, Hsp90 has a more specialized role as a chaperone for several protein kinases and transcription factors. Many Hsp90 client proteins are signaling proteins involved in regulation of cell growth and survival. These proteins are critically dependent on Hsp90 for their maturation and conformational maintenance resulting in a key role for Hsp90 in these processes. Recent reports have also highlighted a role for Hsp90 in linking the expression of genetic and epigenetic variation in response to environmental stress with morphological development in Drosophila melanogaster and Arabidopsis thaliana. In Candida albicans, Hsp90 augments the development of drug resistance, implicating a role for Hsp90 in the evolution of infectious diseases. The malarial parasite, Plasmodium falciparum, is the causative agent of the most lethal form of human malaria. The parasite life cycle involves two hosts: an invertebrate mosquito vector and a vertebrate human host. As the parasite moves from the mosquito to the human body, it experiences an increase in temperature resulting in a severe heat shock. The mechanisms by which the parasite adapts to changes in temperature have not been deciphered. Our laboratory has been interested in investigating the role of heat shock proteins during acclimatization of the parasite to such temperature fluctuations. Heat shock proteins of the Hsp40, Hsp60, Hsp70 and Hsp90 families have been characterized in the parasite and are being examined in our laboratory. This thesis pertains to understanding the functional role of Plasmodium falciparum Hsp90 (PfHsp90) during adaptation of the parasite to fluctuations in environmental temperature. The parasite expresses a single gene for cytosolic Hsp90 on chromosome 7 (PlasmoDB accession no.: PF07_0029) coding for a protein of 745 amino acids with a pI of 4.94 and Mw of 86 kDa. Eukaryotic Hsp90 regulates several protein kinases and transcription factors involved in cell growth and differentiation pathways resulting in a crucial role for Hsp90 in developmental processes. A role for PfHsp90 in parasite development, therefore, seems likely. Indeed, PfHsp90 has previously been implicated in parasite development from the ring stage to the trophozoite stage during the intra-erythrocytic cycle. Pharmacological inhibition of PfHsp90 function using geldanamycin (GA), a specific inhibitor of Hsp90 activity, abrogates stage progression. These experiments suggest that PfHsp90 may play a critical role in parasite development. This is further substantiated by the fact that several pathogenic protozoan parasites such as Leishmania donovani, Trypanosoma cruzi, Toxoplasma gondii and Eimeria tenella depend on Hsp90 function during different stages of their life cycles. It appears, therefore, that a principal role of Hsp90 in protozoan parasites may be the regulation of their developmental cycles. However, the precise functions of PfHsp90 during the intra-erythrocytic cycle of the malarial parasite are not clear. In this study we have carried out a functional analysis of PfHsp90 in the malarial parasite. We have examined the role of PfHsp90 in parasite development during repeated exposure to febrile temperatures. We have investigated its involvement in parasite development during a commonly used synchronization protocol involving cyclical changes in temperature. We have examined the interaction of GA with the Hsp90 multi-chaperone complex from P. falciparum as well as the human host. Finally, we have carried out a systems level analysis of chaperone networks in the malarial parasite as well as its human host using an in silico approach. We have analyzed the protein-protein interactions of PfHsp90 in the chaperone network and predicted putative cellular processes likely to be regulated by parasite chaperones, particularly PfHsp90.

No description available.