Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 199-227).

The heat shock or stress response is a transient response to stressful stimuli that protects vital cellular proteins from damage and irreversible aggregation. Heat shock proteins (Hsps) are molecular chaperones that bind to unfolded protein and inhibit their aggregation, thereby maintaining their solubility until they can be refolded to their native conformation. Hsp47 is an endoplasmic reticulum (ER)-resident protein that serves as a molecular chaperone during collagen production. Collagen is the major class of insoluble fibrous protein found in the extracellular matrix and in connective tissues. It is the single most abundant protein of the animal kingdom; at least 14 different forms exist, each with distinct structures and binding properties. The various types of collagen all possess protein regions with the distinct triple helical conformation. This complex physical structure requires very organized assembly and HSP47 has been established as an integral component of this process for collagen types I-V. Most of the previous studies examining the expression and function of hsp47 have been conducted with mammalian cultured cells. The present study represented the first investigation of the expression of hsp47 in the poikilothermic vertebrate, <i>Xenopus laevis</i>. Full-length <i>Xenopus</i> hsp47 nucleotide and amino acid sequences were obtained from Genbank and compared with hsp47 from chicken, mouse, rat, human and zebrafish. <i>Xenopus</i> HSP47 protein had an identity of approximately 77% with chicken, 73% with mouse, 72% with rat and human, and 70% with zebrafish. Most of the sequence identity between HSP47 from all investigated organisms occurred centrally in the amino acid sequence and in several carboxyl terminal regions. Three key features were conserved between HSP47 proteins from most species investigated: a hydrophobic leader sequence, two potential glycosylation sites and the ER-retention signal, RDEL. A partial cDNA clone encoding <i>Xenopus</i> hsp47 was obtained from the American Type Culture Collection (ATCC) and used to generate hsp47 antisense riboprobe for the purpose of investigating hsp47 mRNA accumulation in <i>Xenopus</i> A6 kidney epithelial cells and embryos. Northern blot analysis detected hsp47 mRNA constitutively in A6 cells. The expression pattern for hsp47 mRNA was compared with two other <i>Xenopus</i> heat shock proteins that have been previously characterized in our laboratory: hsp70, a cystolic/nuclear hsp and BiP, an ER-resident hsp. The results of hsp47 mRNA accumulation in A6 cells suggested that the expression pattern for <i>Xenopus</i> hsp47 was unique but, with respect to some stressors, resembled that of a cytosolic hsp rather than an ER-resident hsp. HSP47 protein levels were also examined in A6 cells. Heat shock, sodium arsenite and b-aminopropionitrile fumerate treatments enhanced hsp47 accumulation. In some experiments, western blot analysis revealed the presence of two closely sized protein bands. It is possible that minor differences in HSP47 protein size may be due to post-translational modification, namely phosphorylation or glycosylation. The present study also examined the accumulation and spatial pattern of hsp47 mRNA accumulation during <i>Xenopus laevis</i> early development. Hsp47 was constitutively expressed throughout <i>Xenopus</i> early development. Constitutive levels of hsp47 mRNA in unfertilized eggs, fertilized eggs and cleavage stage embryos indicated that these transcripts were maternally inherited. Constitutive hsp47 mRNA accumulation was enhanced in neurula and tailbud embryos compared to earlier stages. This finding may be explained by the shift towards organogenesis during these stages. Whole mount <i>in situ</i> hybridization revealed hsp47 message along the dorsal region of the embryo, in the notochord and somites, as well as in the head region including the eye vesicle. Hsp47 mRNA induction in <i>Xenopus</i> embryos was also examined in response to heat shock. Hsp47 mRNA accumulated in response to heat shock immediately following the midblastula transition (MBT). In tailbud stages, hsp47 mRNA accumulated in the notochord, somites and head region. Northern blot analysis and whole mount <i>in situ</i> hybridization results revealed an expression pattern that coincided well with the development of collagen-rich tissues thereby substantiating the proposed role of HSP47 as a procollagen molecular chaperone.

Cadmium is a highly toxic chemical and has been classified by the International Agency for Research on Cancer as a human carcinogen. Cadmium is abundant in the environment, at specific work places, and in food and water. Toxicological responses to cadmium exposure include respiratory diseases, neurological disorders and kidney damage. The present study examined the effects of cadmium on heat shock protein (HSP) accumulation in Xenopus laevis A6 kidney epithelial cells. HSPs are molecular chaperones involved in protein folding and translocation. In response to environmental stress these proteins bind to unfolded protein and inhibit their aggregation. Stress-inducible hsp gene transcription is mediated by the heat shock promoter element (HSE), which interacts with heat shock transcription factor (HSF). In the present study, hsp30 and hsp70 mRNA and protein were induced by heat shock, as determined by northern and western blot analysis. Exposure of A6 cells to cadmium chloride also induced the expression of hsp genes. For example, northern and western blot analysis revealed that exposure of A6 cells to cadmium chloride induced the accumulation of hsp30 and hsp70 mRNA and their respective proteins. Western blot analysis also revealed that A6 cells recovering from a cadmium chloride treatment retained relatively high levels of HSP30 and HSP70 protein accumulation over 24 h after the removal of the stress. Treatments combining a mild heat shock and cadmium chloride resulted in a synergistic increase in hsp30 and hsp70 gene expression at mRNA and protein levels. Further experiments in which two stressors were combined revealed that synergistic effects occurred with varying cadmium concentrations and different temperatures. Immunocytochemistry and confocal microscopy were used to confirm the results attained from western blot analysis. Further, this technique allowed the determination of intracellular localization of HSP30 in A6 cells and the examination of cellular morphology and cytoskeletal structure during cadmium chloride treatments. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the cytoplasm, whereas a 2 h heat shock at 35°C resulted in some HSP30 accumulation in the peripheral region of the nucleus. This is in contrast to cells treated with cadmium chloride, where HSP30 accumulation was restricted to the cytoplasm. A 14 h 50 μM cadmium chloride treatment resulted in the accumulation of HSP30 in approximately 10% of cells. The proportion of cells displaying HSP30 accumulation increased to 80% and 95% in cells treated with 100 μM and 200 μM, respectively. HSP30 accumulation frequently occurred in large granular structures. High concentrations of cadmium chloride resulted in cell membrane ruffling at areas of cell-cell contact, as well as actin disorganization. This study characterized the pattern of hsp gene expression, accumulation and localization under various cadmium chloride conditions. These results suggest that hsp30 and hsp70 gene expression can be used as potential biomolecular markers for cadmium exposure.

Stress-induced heat shock protein (HSP) gene transcription is controlled primarily by the transcription factor heat shock factor 1 (HSF1). HSF1 activation involves trimerization, heat shock element (HSE)-binding, and transactivation. During prolonged stress or upon removal of stress HSF1 activity attenuates. The mechanism(s) regulating HSF1 activity are unknown. Some reports have suggested that HSF1 may be regulated in some manner by the HSP90 chaperone (Nadeau, K., 'et.al.', 1993; Nair, S., 'et.al.', 1996). Utilizing the 'Xenopus' oocyte model system I tested the hypothesis that the HSP90 chaperone machine, known to function in the folding and maturation of molecules such as steroid receptors, might also participate in HSF1 regulation. Characterization experiments illustrated that the 'Xenopus' oocyte was capable of responding to some but not all forms of stress at the level of HSF1-HSE binding illustrating that certain stress pathways may be absent or inactive in the oocyte. Through transcriptional assaysit was also shown that HSF1-DNA binding and transactivation are regulated by independent mechanisms in the oocyte. HSP90 was shown to interact with and regulate the activity of HSF1 in oocytes. HSP90-HSF1 associations were illustrated ' in vivo' and 'in vitro' by co-immunoprecipitation and gel supershift assays. Immunotargeting HSP90 caused activation of HSF1 under control conditions and delayed deactivation during recovery. These data support a role for HSP90 in the oligomeric changes associated with HSF1 activation/deactivation. Immunotargeting HSP90 also inhibited HSF1 dependent transcription, supporting a role for HSP90 in mediating HSF1 transcriptional activity. HSP90 does not regulate HSF1 alone. Gel supershift analysis showed that p23, HSP90 and FKBP52 exist in a complex with activated HSF1. Furthermore, elevating the levels of various co-chaperones through injection of protein or mRNA had various effects on HSF1 during recovery from stress. Immunotargeting HSP90 or p23 induced HSF1-DNA binding in the absence of stress indicating these proteins may act together to repress HSF1 'in vivo'. Furthermore, injection of HSP90, Hip, Hop, p23, FKBP51, and FKBP52 antibodies significantly delayed HSF1 deactivation supporting a role for these proteins in trimer disassembly. Therefore multiple components of the HSP90 chaperone complex function to regulate HSF1 during its activation and/or deactivation cycle.

In this study, possible involvement of the small heat shock proteins (sHsps) from a thermoacidophilic archaeon, Thermoplasma (Tp) volcanium in the stress response was investigated. Our results showed that heterologous, high level expression of TVN0775/sHsp gene in E.coli increased its thermotolerance at 53&deg / C for two hours. But, the second sHsp of the Tp. volcanium, TVN0984/sHsp was not effective in improvement of the thermal resistance of the mesophilic bacterium (i. e., E.coli). The expression of the TVN0775/sHsp and TVN0984/sHsp genes increased about 3 fold after heat-shock at 65&deg / C, as revealed by Real-Time PCR analysis. Although expression of the both genes was induced at 70&deg / C, TVN0984/sHsp gene expression was increased higher (about 5 fold) than that of the TVN0775/sHsp gene expression (about 1.5 fold). Tp. volcanium cells were exposed to high pH (pH: 3.5, pH: 4.0, pH: 4.5, pH: 5.0), and the change in the sHsp genes&rsquo / expression profile were analyzed. The results showed that TVN0775/sHsp gene expression was more sensitive to increased pH than TVN0984/sHsp gene expression. The TVN0775/sHsp gene transcription induced at most 2.5 fold at pH 4.0 and the gene expression either reduced or did not change at higher pH values (i.e., pH 4.5 and 5.0). On the other hand, TVN0984/sHsp gene expression did not change at pH 4.0 but significantly reduced at higher pH values. The effect of oxidative stress on the expression of TVN0775 and TVN0984 genes was investigated by treatment of Tp. volcanium cells with 0.01 mM, 0.02 mM, 0,03 mM and 0.05 mM H2O2. For both sHsp genes, transcription was induced at lower concentrations of H2O2 (0.01 mM and 0.02 mM). At higher concentrations of H2O2 expression of both genes&rsquo / transcription either did not changed or down regulated. Lastly, in this study we have purified the recombinant TVN0775/sHsp, as an Nterminal 6x his-tag fusion to homogeneity on Ni-NTA affinity column. Purified protein samples were used in the chaperone activity assays using bovine glutamate dehydrogenase enzyme (boGDH) as substrate. We have found that the recovery of glutamate dehydrogenase activity at 45&deg / C, 50&deg / C and 53&deg / C in the presence of the Tp. volcanium sHsps was higher than that of spontaneous refolding. Also, TVN0775/sHsp increased the recovery of the boGDH enzyme that was denatured at 2.5 M GdnHCl concentrations for 30 min.

Hest shock protein 70 (Hsp70) class of chaperones is highly conserved and present ubiquitously in all cellular organisms They play important role in folding of nascent polypeptides and translocation of precursor proteins to endoplasmic reticulum, mitochondria and chloroplast Hsp70 assists in assembly of proteins complexes as well as in disassembly e g uncoatmg of clathrin coated vesicles Chaperone function of Hsp70 is modulated by cochaperones of DnaJ class, Hip, Hop etc Hsp70 is a component of multi chaperone complex with Hsp90 and helps in maturation of kinases or transcription factors. Plasmodium falciparum is responsible for most severe form of human malaria Plasmodmm in its intraerythrocytic cycle presents an example of a cell with multiple, complex membrane bound structures both inside the parasite as well as m the infected erythrocyte cytosol Parasite deploys proteins in host erythrocyte cytosol, at erythrocyte plasma membrane or traffics them for secretion outside the infected cell in addition to trafficking of proteins to its own organelles like mitochondria, apicoplast, food vacuole, ER etc It is of interest to malaria biologists to understand these trafficking events and role of chaperones in regulating them This study was aimed at understanding the function(s) of Hsp70 in Plasmodium infected erythrocyte in protein maturation and trafficking events We have attempted to study Hsp70 chaperone present in Plasmodium infected erythrocytes We have largely focused on the cytosohc Hsp70, PfHsp70, in the parasite and systematically analyzed its expression, localization, abundance and complexes in the intraerythrocytic cycle To gain insight into its function, we have identified a subset of PfHsp70 interacting proteins, parasite Hsp90, Hsp70-3, Hsp60 and beta tubulin by coimmunoprecipitation experiments in conjunction with proteomic tools like 2DGE and mass spectrometry Parasite Hsp60 is a mitochondria-targeted protein and we have examined the involvement of PfHsp70 in translocation of Hsp60 precursor protein to parasite mitochondrion PfHsp70 and PfHsp90 were found to be present in a complex Geldanarnycm, a drug that affects Hsp70-Hsp90 complex, was used to investigate the role of PfHsp70 in parasite protein trafficking Since there are no known parasite derived chaperones in erythrocyte cytosol compartment, we have examined the possible "involvement of host Hsp70 in supporting transport and assembly of parasite proteins in erythrocyte cytosol Hsp70 in Plasmodium falciparum intraerythrocytic cycle P. falciparum genome codes for five Hsp70 homologs Two of these, pfHsp70-l and PfBiP are expressed in intraerythrocytic stage and have been localized to nucleocytoplasmic and endoplasmic reticulum fraction of the parasite respectively We have focused this study on PfHsp70 of the parasite We show that PfHsp70 is an abundant protein in the cytosol constituting about 2% of the total soluble pool It gets further induced during stress like heat shock and translocates to nuclear fraction indicating that PfHsp70 may be involved in protective function in the parasite nucleus during stress Nuclear translocation of mammalian Hsp70 during stress has been linked to its phosphorylation at Tyr524 We found PfHsp70 to be phosphorylated by in vivo phosphate labeling m the parasite Analysis of PfHsp70 by 2-dimensional gel electrophoresis on narrow gradient IPG strips indicated that it exists in four forms differing in their isoelectnc points (pi) Phosphatase treatment combined with analysis using a phosphorylation prediction tool,Proteomod (http //www biochem use ernet in/proteomod html) suggested that PfHsp70 is phosphorylated at three residues in the parasite The extent of phosphorylation of PfHsp70 may determine substrate specificity or subcellular localization or both Using 2DGE and mass spectrometry approach, we also identified chaperones like Hsp909 BiP, Hsp60, and protein disulphide isomerase (PDI) m P falciparum proteome In summary, PfHsp70 appears to be a highly abundant, cytosohc chaperone It is inducible by stress and multiply phosphorylated and is likely to participate in multiple processes in the parasite. PfHsp70 complexes and interacting proteins in the parasite To gam insight into the functions of Hsp70, we looked for PfHsp70 interacting proteins in the parasite We used gel filtration chromatography to resolve and enrich PfHsp70 complexes and also employed coimmunoprecipitation approach to identify interacting proteins We found parasite Hsp90, Hsp70-3, Hsp60 and beta-tubulin interact with PfHsp70 Fractionation of parasite lysate indicated that PfHsp70 is present in two major complexes of 200 kDa and 450 kDa We find that PfHsp90 interacts with PfHsp70 and both are present in 450 kDa complex Our analysis indicated that 450-kDa complex is like Hsp70-Hsp90 multichaperone complex described in mammalian cells while 200 kDa complex is likely to be an Hsp70-cochaperone complex Smaller complex appears to be a precursor for multichaperone complex Use of an Hsp90 inhibitor, geldanamycin (GA), to study the function of this multi chaperone showed that GA inhibits parasite growth Maturation of four phosphoproteins interacting with PfHsp70 was affected by GA implicating them in regulation of parasite growth GA appeared to mediate its effects by inhibiting H§p^0 phosphorylation Amongst the other three interacting proteins, PfHsp70-3 is amoveJ/Hsp70 homolog that was found at the protein level for the first time in this study PfHsp60 is mitochondria-targeted protein in the parasite and it is likely that cytoshc PfHsp70 helps in translocation of PfHsp60 to mitochondria from cytosol Tubuhn is a cytoskeletal protein and its interaction with PfHsp70 suggests possible role of PfHsp70 in cytoskeleton organization during invasion, growth or cell division In all, we find that Hsp70 in the parasite exist in a multi chaperone complex with Hsp90 which might be responsible for maturation of signaling molecules important for growth The smaller complex of PfHsp70 is a precursor of multi chaperone complex and is likely to be an Hsp70- co chaperone complex Role of Hsp70 in protein translocation and trafficking Cytosolic Hsp70 aids in translocation of precursor proteins from cytosol to mitochondria (or chloroplast) We found a mitochondnal chaperone, PfHsp60, interact with PfHsp70 and we examined the possibility that PfHsp60 translocation is assisted by cytosolic PfHsp70 We found that PfHsp60 had a cleavable, N-thermal targeting sequence Examination of PfHsp60 forms present in mitochondnal and cytosolic fraction of the parasite showed that mitochondnal form was more acidic in pi than cytosolic form as expected after targeting sequence cleavage Cytosolic PfHsp60 interacted with both PfHsp70 and PfHsp90 Interestingly, while mitochondnal PfHsp60 appeared to be in a chaperonm like complex, as expected, cytosolic form was present in smaller ohgomeric complex of about 450 kDa This suggested that PfHsp60 precursor form could be bound to multichperone complex All these experiments together strongly indicated that PfHsp60 precursor interacts with cytosolic Hsp70 and Hsp90 before former's translocation into mitochondria This interaction might be required to keep the precursor in the transport competent state P falciparum lives inside a vacuole in the infected cells but it deploys a number of proteins to host cell cytosol and to the plasma membrane To examine the involvement of multichaperone complex in trafficking, we studied the effect of GA on targeting of two parasite proteins, knob associated histidme-rich protein (KAHRP) and glycogen synthase kinase (GSK) KAHRP is indispensable for the formation of cytoadherence complexes called knobs at erythrocyte plasma membrane We found that KAHRP transport to erythrocyte plasma membrane was blocked in GA-treated parasites and it appeared all over the infected cell Further analysis showed that GA caused block in KAHRP transport at some step beyond its exit from parasite ER The targeting of GSK to membranous inclusions in the infected RBC cytosol was not severely affected m the GA-treated parasites suggesting that GSK transport may not be regulated by multi chaperone complex It also indicated that parasite may be using different pathways for trafficking of proteins to the host compartment In summary, PfHsp70 and PfHsp90 interact with PfHsp60 precursor in the cytosol They probably help keep the precursor in transport competent form before arrival at the translocase complex of mitochondria The multi chaperone complex may also be important for trafficking of at least one parasite protein, KAHRP, to the host cell compartment Analysis of erythrocyte Hsp70 in Plasmodium falciparum infected cells The remodeled plasma membrane of parasite-infected erythrocytes is important for the cytoadherence property of the infected cells Knobs, supramolecular complexes on the infected cell surface, formed by parasite proteins, PfEMPl, KAHRP, and PfEMP3 are responsible for cytoadherence of infected cells to vascular endothehum or placenta KAHRP transport is BFA-sensitive inside the parasite while PfEMP proteins undergo vesicle mediated trafficking in the erythrocyte cytosol The involvement of molecular chaperones has been implicated in the trafficking and assembly of knob components in the erythrocyte cytosol There is no evidence for the presence of bona fide parasite derived chaperones in the host compartment The chaperones of the erythrocyte origin, Hsp70, Hsp90, Hip and Hop were readily detected in the host cytosol, on the other hand By analyzing localization, abundance and biochemical characteristics of the host chaperones of erythrocyte origin, we examined if host chaperones are being utilized by the parasite for its functions Localization experiment showed that while PfHsp70, PfHsp90 and PfBiP were present in the parasite compartment, host-Hsp70 was present in erythrocyte cytosol fraction Host~Hsp70 was about 60% as abundant as PfHsp70 and was potentially capable of facilitating chaperone function in the erythrocyte cytosol Though host-Hsp70 was soluble in unmfected cells, it was present in membrane bound, triton-insoluble complexes, containing KAHRP, in infected cells Since knobs are triton-insoluble complexes at the erythrocyte plasma membrane, we isolated erythrocyte ghost (plasma membrane) fraction and could detect both Hsp70 and KAHRP Hsp70 association with erythrocyte plasma membrane was specific as it could be crosshnked to KAHRP in ghost fraction of infected cells Host-hsp70 was present in purified cytoskeleton fraction containing knobs from infected cells along with cochaperone Hop All these evidences suggest that parasite may be exploiting host-Hsp70 in erythrocyte cytosol compartment Summary This study gives insight into some functions performed by PfHsp70 in mtraerythrocytic cycle of malarial parasite PfHsp70 is an abundant cytosohc chaperone in the parasite It gets induced during stress and translocates to the nucleus It is also phosphorylated at three sites Analysis of Pfhsp70 complexes shows that it is present in bimodal complexes (450 kDa and 200 kDa), which are in equilibrium PfHsp70 and PfHsp90 interact and are part of 450 kDa multichaperone complex This multichaperone complex appears to regulate trafficking of one parasite protein to host cytosol compartment In addition, PfHsp70 and PfHsp90 are also bound to mitochondria-targeted PfHsp60 precursor in the cytosol probably keeping them m a transport competent state In addition to PfHsp90 and PfHsp60, PfHsp70 interacts with a novel Hsp70 homolog of the parasite, PfHsp70-3, and cytoskeletal protein, beta-tubuhn Examination of chaperones available in erythrocyte cytosol, showed that parasite chaperones were absent while host chaperone (Hsp70) was present and exhibited altered properties during parasite infection It was associated with membrane-bound, triton-insoluble complexes on the infected cell plasma membrane suggesting that host-Hsp70 might be involved in trafficking and/or assembly of parasite proteins In all, PfHsp70, as part of multichaperone complex, appears to be regulating translocation and trafficking of parasite proteins to organellar locations or outside the parasite Host-Hsp70, in erythrocyte cytosol, might also be engaged in specific chaperone function upon infection

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 2, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Department of Organismal Biology and Anatomy, June 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 99-121). Also available in electronic version. Access restricted to campus users.