RNA editing had been considered as a rare exception to the central dogma of molecular biology in which the mRNA truthfully carries genetic code from nucleus to the ribosome for translation. However, researches in the last five years have revealed numerous, widespread RNA A-to-I editing sites in human genome. Although the effects of these editing events require further study, this finding strongly suggests RNA editing occurs frequently, and affects large number of genes. By selectively modifying a few sequences of a gene, RNA editing allows a cell to produce a population of proteins with different properties from a single gene. The major question of this thesis study is whether such editing event is actually dynamically regulated when the cellular physiological processes have to be adjusted in response to changing environment. A previous study screening for Drosophila mutants defected in hypoxia and heat tolerance discovered a hypnos-2 mutant strain which was later found to be defective in dADAR, the drosophila gene encoding the A-to-I editing enzyme, supporting the hypothesis that cells/organisms response to stressful environment by dADAR-mediated RNA editing. Two directions are used to approach how Drosophila uses A-to-I editing to adapt ¡§heat¡¨ environment stress. First, whether the expression pattern of dADAR changes after heat shock was investegated. The result showed the dADAR gene exon 7 self-editing frequency was decreased by heat shock, thus possibly enhances dADAR activity after heat shock processing. Moreover it is worth noting that the isoform without -1 exon transcript were obviously up-regulated, and transcript with -1 sequence is relatively down-regulated. On the other hand, no significant changes in the dADAR mRNA expression levels and in the degrees of two dADAR promoters activity were observed. Second, the changes of editing frequency of 30 known A-to-I editing sites were investigated. Generally the editing frequency of majority editing sites changed after heat shock. Therefore, the dADAR activity, the dADAR gene transcript expression alternations, and A-to-I editing frequency of dADAR target genes did change after heat shock, supporting the notion that change of RNA editing pattern is a mechanism for organism to adapt to drastic environmental change. However, how the edited protein isoforms contribute to heat resistance requires further investigation.

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.

The heat shock response (HSR) is the cell's molecular reaction to protein damaging stress and is critical in the management of denatured proteins. Activation of HSF1, the master transcriptional regulator of the HSR, results in the induction of molecular chaperones called heat shock proteins (HSPs). Transcription of hsp genes is promoted by the hyperphosphorylation of HSF1, while the attenuation of the HSR is regulated by a dual mechanism involving negative feedback inhibition from HSPs and acetylation at a critical lysine residue within the DNA binding domain of HSF1, which results in a loss of affinity for DNA. SIRT1 is a NAD+-dependent histone deacetylase that has been reported to deacetylate HSF1, thus promoting stress-induced HSF1 DNA binding ability and increasing HSP expression (Westerheide, Anckar et al. 2009). While an abundance of research is aimed to investigate SIRT1 substrate regulation, the mechanism in which SIRT1 itself is regulated is less understood (Haigis and Sinclair 2010). Positive and negative modulators of SIRT1 include AROS and DBC1, respectively, and have yet to be investigated in relation to SIRT1-dependent regulation of the HSR. In addition, metabolic stress such as caloric restriction has been shown to modulate SIRT1 activity in yeast (Rahat, Maoz et al. 2011), but the effect of caloric restriction on the HSR is unknown. Using cell-based assays, we have investigated how the HSR may be controlled by factors influencing SIRT1 activity. We found that heat shock results in an increase in the cellular NAD+/NADH ratio and an increase in recruitment of SIRT1 to the hsp70 promoter. Furthermore, we found that the SIRT1 modulators, AROS and DBC1, impact hsp70 transcription, HSF1 acetylation status, and HSF1 recruitment to the hsp70 promoter. The nematode Caenorhabditis elegans is a useful model organism for testing the relationship between the HSR and metabolism, as these animals can easily be calorically-restricted via bacterial limitation and possess the mammalian SIRT1 homolog, Sir2.1. Using C. elegans, we demonstrate that caloric restriction and heat shock have a synergistic effect on the HSR in a sir2.1-dependent manner. We show that caloric restriction increases the ability of heat shock to promote thermotolerance and fitness in wild-type animals and to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1. These studies provide insight into SIRT1-dependent regulation of the HSR and the impact of metabolism on this response. We highlight the SIRT1 modulators AROS and DBC1 as two new targets available for therapeutic regulation of the HSR and add caloric restriction as another HSR activator that can synergize with heat shock.

Apoptosis is a conserved program of cell death that promotes organism homeostasis in all stages of life. Two main pathways activate caspases, which are cysteinyl-aspartate proteases that execute apoptosis. The extrinsic pathway is initiated by cell surface death receptors, while the intrinsic pathway is initiated by intracellular signals that cause permeabilization of the outer mitochondrial membrane (MOMP). The Bcl-2 protein family regulates MOMP, which causes the release of several pro-apoptotic proteins (such as cytochrome c, Smac) into the cytosol. Bcl-2 proteins share homology in up to four "BH" domains and are subdivided into three subgroups. Pro-apoptotic Bax and Bak catalyze pore formation in the mitochondria, while anti-apoptotic members (Bcl-2, Mcl-1) inhibit MOMP. The third subgroup, termed BH3-only, promotes MOMP by either antagonizing Bcl-2 proteins or by directly activating Bax/Bak, and initiate apoptosis in response to various stressors, including heat shock (HS). Hyperthermia or acute HS reportedly induces apoptosis through caspase-2-mediated cleavage of BID, engaging the intrinsic pathway. However, additional evidence suggests that this pathway could represent an amplification loop. Thus we hypothesized that during HS, another BH3-only protein such as BIM, that does not require cleavage, could engage MOMP. Herein, we report that BIM mediates an alternative HS-induced apoptosis pathway. Cells lacking BIM are resistant to HS and exhibit better short and long-term survival than either Bid[superscript -/-] or Bax[superscript -/-]Bak[superscript -/-]. Moreover, caspase-2 induces apoptosis in Bim[superscript -/-] but not Bid[superscript -/-] cells, implying that caspase-2 kills exclusively through BID. Interestingly, Bim[superscript -/-] and Bax[superscript -/-]Bak[superscript -/-] cells are entirely resistant to MOMP, but the Bax[superscript -/-]Bak[superscript -/-] cells still undergo caspase-3 activation and remain partially sensitive to HS, indicating that BIM triggers caspase-3 activation upstream of mitochondria. Thus, BIM plays an important role in HS-induced apoptosis. Hyperthermia has clinical applications for the treatment of solid tumors. Unfortunately, a practical limitation is the development of thermotolerance, which confers resistance not only to subsequent HS but also to radiotherapy and chemotherapy. Therefore, a better understanding of the molecular mechanisms involved both in heat-induced apoptosis and thermotolerance could lead to new therapeutic interventions. Here we also show evidence for a putative role for the stress kinase JNK signaling pathway in the regulation of thermotolerance. / text

<p>Proteotoxic stress is implicated in numerous human diseases including neurodegeneration, cancer, and diabetes. Unfortunately, our mechanistic understanding of the cellular response to proteotoxic stress is limited. A critical feature of the cellular stress response is the activation of Heat Shock Transcription Factors (HSFs) that regulate the expression of numerous genes involved in protein folding, protein degradation, and cellular survival. The studies presented here utilize a diverse array of techniques including yeast genetics, recombinant protein expression and purification, biochemical analysis of protein-DNA interactions, x-ray crystallography, in vitro post-translational modification, and mammalian cell culture to illuminate novel aspects of HSF biology. Critical findings include understanding key principles of HSF-DNA interactions, identification of a novel negative regulator of HSF activity, and identification of structural features of HSF paralogs that enable precise combinatorial regulation. These unique insights lay the foundation for a greater understanding of HSF in specific cellular contexts and disease states.</p> / Dissertation

<p>Inducible Heat shock protein (Hsp70i) is a protein chaperone that is utilized during tumorigenesis and viral infections for efficient propagation. Overexpression of Hsp70i is observed in a wide spectrum of human tumors, and this overexpression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Hsp70i aids in cancer cell propagation through regulation of anti-apoptotic and cell survival pathways. Furthermore, Hsp70i is induced following infection for several viruses and aids viral propagation, in part through regulation of anti-apoptotic pathways as well as promoting the folding of newly synthesized proteins. Due to the parallel role of Hsp70i in both cancer and viral pathogenesis, identification of small-molecule inhibitors selective for Hsp70i could provide tools for the development of novel therapeutics and further elucidate the role of Hsp70i in both cancer and viral infections.</p><p>To date, few Hsp70 inhibitors have been identified and characterized, and their efficacy in clinical settings is unknown. Through the fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, an allosteric inhibitor selective for Hsp70i was identified, called HS-72. We show that HS-72 is highly selective for Hsp70i, over the broader purinome and other Hsp70 family members, in particular the closely related constitutively active Hsp70 family member, Hsc70. Additionally, HS-72 acts as an allosteric inhibitor to induce a conformational change and inhibit Hsp70i activity. HS-72 displays hallmarks of Hsp70i inhibition in vitro by promoting Hsp70i substrate protein degradation, protein aggregation, and selective growth inhibition of cancer cells. In wild type mice HS-72 is well tolerated and a limited PK study shows HS-72 is bioavailable. Furthermore, in a MMTV-neu breast cancer mouse model, HS-72 shows efficacy to inhibit tumor growth and promote survival.</p><p>Due to the similar utilization of Hsp70i in cancer and viral pathogenesis, this suggests the potential for HS-72 as an antiviral agent. Dengue virus (DENV) is of great public health importance due to estimates of up to 400 million infections per year, coupled with the geographic distribution of the virus, which is now endemic in over 100 countries worldwide. There is also a pressing need for DENV interventions, owing to the lack of approved vaccines or antiviral therapies. DENV is reliant on host factors throughout the viral life cycle and Hsp70i has been implicated as a host factor in DENV pathogenesis. Additionally, the complete role of Hsp70i in DENV pathogenesis remains to be elucidated, highlighting a unique opportunity to use HS-72 as a tool to specifically probe Hsp70i function. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Furthermore, inducing Hsp70i expression prior to infection, correlates with an increase in DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained, through inhibiting the entry stage of the viral life cycle. Following infection, Hsp70i localizes to the cell surface and interacts with the DENV receptor complex to mediate viral entry. While, HS-72 treatment results in a disruption of the interaction of Hsp70i with the DENV receptor complex, yielding a reduction in infected cells. </p><p>Collectively this work further supports Hsp70i as an anticancer and anti-dengue virus target, and identifies HS-72, a chemical scaffold that is amenable to resynthesis and iteration, as an ideal starting point for a new generation of therapeutics targeting Hsp70i.</p> / Dissertation