Yes / Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality.

No description available.

Amyotrophic Lateral Sclerosis (ALS) is an always lethal motor neuron disease with unknown pathogenesis. Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) have limited neuroprotection in some models of motor neuron degeneration. However the direct effect of Hsp90 inhibition on motor neurons is unknown. Here we show that Hsp90 inhibition induced motor neuron death through activation of the P2X7 receptor. Motor neuron death required phosphatase and tensein homolog (PTEN)-mediated inhibition of the PI3K/AKT pathway leading to Fas receptor activation and caspase dependent death. The relevance of Hsp90 for motor neuron survival was investigated in mutant Cu/Zn superoxide dismutase (SOD) transgenic animal models for ALS. Nitrated Hsp90, a posttranslational modification known to induce cell death (Franco, Ye et al. 2013), was present in motor neurons after intracellular release of zinc deficient (Zn, D83S) and the SOD in which copper binding site was genetically ablated (Q) but not after copper deficient (Cu) wild type SOD. Zn deficient and Q mutant SOD induced motor neuron death in a peroxynitrite mediated and copper dependent mechanism. Nitrated Hsp90 was not detected in the spinal cord of transgenic animals for ALS-mutant SOD animal models until disease onset. Increased nitrated Hsp90 concentrations correlated with disease progression. Addition of Zn or Q SOD to nontransgenic brain homogenate treated with peroxynitrite led to an increase level of nitrotyrosine in comparison to wild type controls. However, in the same samples there was a 2 to 10 time increase in Hsp90 nitration as compared to nitrotyrosine. The selective increase is likely due to the binding of Hsp90 to Zn deficient and Q SOD as oppose to wild type SOD. These results suggest that Hsp90 nitration facilitated by mutant SOD may cause motor neuron degeneration in ALS. Targeted inhibition of nitrated Hsp90 may be a novel therapeutic approach for ALS. An alternative therapeutic strategy is to target the production of survival factors by glial cells. Riluzole is the only FDA approved drug for the treatment of ALS and it shows a small but significant increase in patient lifespan. Our results show that acute riluzole treatment stimulated trophic factor production by astrocytes and Schwann cells. However long-term exposure reversed and even inhibited the production of trophic factors, an observation that may explain the modest increase in patient survival in clinical trials. Discontinuous riluzole treatment can maintain elevated trophic factor levels and prevent trophic factor reduction in spinal cords of nontransgenic animals. These results suggest that discontinuous riluzole administration may improve ALS patient survival. In summary, we demonstrated that Hsp90 has an essential function in the regulation of motor neuron survival. We have also shown that Hsp90 was nitrated in the presence of mutant SOD and was present during symptom onset and increases as disease progresses, which may explain the toxic gain of function of mutant SOD. Finally we demonstrate a biphasic effect of riluzole on trophic factor production and propose changes in administration to improve effects in ALS patients.

No description available.

Cells invest a lot of energy in order to get their proteins to fold correctly and attain functionality. It is the functional proteome of a cell that defines the ‘life of a cell’. Cells have therefore employed dedicated machinery called chaperones to enable protein folding. One class of these chaperones is heat shock proteins named so because they were initially discovered to be heat inducible and particularly important during heat stress. However the role of heat shock proteins has now been extended from merely being important for stress tolerance. Heat shock proteins are prominently involved in maintaining the correct folding and conformation of proteins and are vital in regulating the stability between protein synthesis and degradation. One of the heat shock proteins, Hsp90, is an evolutionarily conserved molecular chaperone essential in all known eukaryotes examined so far. Unlike other chaperones, Hsp90 is unique in binding to substrate proteins, which are at a late stage of folding, poised for activation by either ligand binding or interaction with other cellular factors. The most common clients of Hsp90 are signaling proteins, the classic example being steroid hormone receptors and signaling kinases. Several other proteins including transcription factors, proteins involved in cell division and development have also been shown to rely on Hsp90 functioning for their maturation. Hsp90 has emerged as an important molecular chaperone due to the large number of proteins that depend on the activity of Hsp90 for their functionality. Hsp90 plays a central role in multiple cellular processes. Since knock-out of hsp90 is lethal to most eukaryotes, inhibitors of Hsp90 have been widely used to study its function. The most widely used inhibitor is geldanamycin (GA). GA binds to the N-terminal/ATP binding site of Hsp90 which results in the degradation of client proteins. Hsp90 clients have been shown to be proteins important for diverse cellular processes such as protein trafficking, signal transduction, cell-cycle, cellular motility and development in eukaryotes. Exploring new Hsp90 clients gives an insight into more pathways that Hsp90 regulates. Intriguingly, many proteins interact with Hsp90 in a context dependent manner, i.e., under certain environmental cue, or in a particular tissue, or only under certain diseased states. It is therefore essential to study Hsp90 functioning and examine Hsp90-client interactions in more than one model organism. Dictyostelium discoideum: a model organism to study the role of Hsp90 in development The eukaryote, Saccharomyces cerevisiae that has been explored extensively for studying the diverse clientele of Hsp90, lacks various signaling pathways important for growth and differentiation as prevalent in higher eukaryotes. It is desirable to develop a model system that would combine the advantages of a lower eukaryote, in terms of its ease of manipulation and retain the complexities of higher eukaryotes. With this motivation, the social slime mold D. discoideum was explored to examine potential roles of cytoplasmic Hsp90 in growth and development. D. discoideum is ideal for studying signaling pathways important for growth and differentiation and to understand how these pathways control cellular responses to external stimuli. Multicellular development in D. discoideum occurs in response to starvation induced stress. As in case of many other protozoans, we conjectured that Hsp90 may participate in regulating developmental transition from unicellular to multicellular stages in Dictyostelium as well. My initial study attempts, to address the role of Hsp90 (HspD), in development of D. discoideum. Towards this two approaches were taken: through genetic interference of HspD, and the other, through its pharmacological inhibition. An antisense HspD plasmid was designed which upon transfection in D. discoideum, showed a very slow growth phenotype, and the cells did not survive beyond few generations. Therefore to further study the functions of HspD, I resorted to pharmacological inhibition by using the specific, well characterized inhibitor, GA. As a first step towards this I examined whether GA was capable of binding to HspD from D. discoideum cell lysate. Towards this, GA was immobilized to NHS-sepharose beads, and bound proteins were examined. Western blot of the bound fraction, using antibody specific to HspD, identified it as a predominant protein being pulled down. This was further confirmed by mass spectrometry. To be able to compare Hsp90 from D. discoideum with Hsp90s from other model organisms, HspD was cloned, purified and biochemically characterized. Comparison of ATPase activities of HspD with Hsp90’s from other systems indicates HspD to possess a relatively low ATPase activity with a Kcat of 1.6 x 10-3 min-1. The dissociation constant of GA for HspD was found to be 0.8 µM, which was in the range similar to Hsp90s from other systems. In addition, we have now obtained structural data on HspD in collaboration with crystallography groups. The N-terminal domain of HspD has been crystallized, both in -free and ligand-bound forms. Crystal structure comparison of HspD with Hsp90 from S. cerevisiae shows overall fold similarity yet some important differences in side chain orientations of specific residues in the ATP binding domain. Interestingly, on treating D. discoideum cells with GA or another Hsp90 N-terminal inhibitor, Radicicol, it was found that, while control cells progressed to develop into fruiting bodies, GA/Radicicol treated cells resulted in delayed development, and were finally arrested at the ‘mound’ stage. This suggested potential involvement of HspD in developmental progression beyond the mound stage. In order to identify the pathways that are probably affected by HspD in D. discoideum development, cells were treated with/without GA and subjected to comparative proteomics using mass spectrometric analysis. Amongst other differences, there was an obvious absence of peptides corresponding to the protein paxillin in GA treated cells. The results were verified by Western blot analysis, using a specific antibody against paxillin, wherein a drastic decrease in paxillin levels were observed in cells treated with GA. Paxillin is a key player in focal adhesion sites that functions as an adaptor protein to recruit diverse cytoskeletal and signaling proteins into a complex, and is essential for cellular proliferation and cell-substrate adhesion. My studies suggest that one of the pathways through which HspD regulates development is through cellular motility as Hsp90 was involved in regulating proteins necessary for motility and cytoskeletal organization at focal adhesion points during development in D. discoideum. Hsp90 as a target for Trypanosoma evansi infections In addition to examining the role of Hsp90 in differentiation in D. discoideum, I have also looked at the potential of Hsp90 under diseased conditions. Towards this, I explored the protozoan parasite, T. evansi, which causes a fatal disease ‘surra’. Surra is a neglected disease that mainly affects domestic and wild animals including equines, camels, cattle and buffaloes. The parasite causes significant economic losses to livestock industry. While this infection is mainly restricted to domestic (camels, equines, cattle, buffaloes, goats, sheep, pigs, dogs etc.) and wild animals, recent reports indicate their ability to infect humans. There are no reliable sensitive and specific diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable. With a view to identifying potential diagnostic markers and drug targets I have studied the clinical proteome of T. evansi infection using mass spectrometry. I have been able to identify almost 166 proteins of T. evansi, which also included potential drug and vaccine targets. Due to absence of any genome sequence information from T. evansi, most of the peptides obtained matched to its related species, T. brucei, T. cruzi and also few from Leishmania major. Importantly, I was also able to identify peptides from Hsp90. Hsp90 from T. evansi was cloned and its sequence was also obtained. To investigate the possibility of exploring Hsp90 as a target against Surra infections, TeHsp90 protein was purified by expressing it in bacterial cells, and its drug (GA) binding ability was examined in-vitro. The dissociation constant of GA for HspD was found to be 1.4 µM, which was in the range similar to Hsp90s from other systems. The ability of 17AAG (a derivative of GA) was examined in inhibiting T. evansi infection at pre-clinical level. Towards this, swiss female mice were infected with purified parasites and then the drug was injected either immediately, in one group of mice, and in another group of mice the parasites were challenged with the drug only after the onset of infection. Interestingly, both groups of mice were found to get cured using Hsp90 inhibitor. The pre-clinical results suggested that Hsp90 was an interesting drug target and its inhibitor could indeed be used against ‘surra’ infections. Hsp90 from Giardia lamblia: An unusual case Hsp90 was also examined from another pathogenic protozoan, Giardia lamblia, one of the leading causes of diarrhea in the world. Previous studies from our lab have shown Gardial Hsp90 to be coded by two different ORFs, spliced together in trans. This is indeed the only example of trans-splicing in Hsp90 known so far. My study further characterizes this finding through analysis of transcription levels of the individual ORFs, using Northern blot analysis. Importantly, I was able to detect transcripts of all three forms of Hsp90; full-length, N terminus as well as C terminus, suggesting that these are expressed and may have biological significance. To understand the significance of these independent transcripts, I have examined relative levels of expression of all three forms by Real-time PCR analysis wherein there was almost 90 fold and 5 fold lesser transcript level of N terminus and C terminus Hsp90 observed, respectively as compared to the full-length GlHsp90 expression. Previous reports have shown Hsp90 from all known organisms, to get up regulated during heat shock. Thus it was important to examine the effect of heat stress on the expression of these independent transcripts. Interestingly, different domains were found to get independently induced during heat stress. The transcript level of HspC was seen to be almost similar to that of full-length upon heat shock. There was also a significant up regulation observed in HspN transcript upon heat shock. Taking together all these observations, these results suggest a possible role for the independent domains, HspN and HspC during heat stress in G. lamblia. Furthermore, I have cloned and purified one of the individually expressed domains, HspN and characterized it biochemically. HspN was found to be able to bind to ATP, however lacked ATPase activity. Taking together all these observations, it suggests a possible role for the independent domains, HspN and HspC which needs to be investigated further. Summary Altogether, my studies establish the importance of alternate model systems in understanding the biology of Hsp90. The importance of Hsp90 was first established in growth and development of a nonpathogenic protozoan D. discoideum. My results provide significant insights into the additional pathways that Hsp90 regulates during D. discoideum development. One such important pathway was delineated to be cellular locomotion and motility. Further, I have also studied the importance of Hsp90 in neglected infectious diseases. In addition to providing a glimpse into the pathways operational during disease manifestation in T. evansi, we have shown Hsp90 to be effective in pre-clinical trials against T. evansi infections. Hsp90 from another pathogenic protozoan, G. lamblia, has also been studied. This is by far the only organism, in which there is an independent expression of the N-and C-terminal domain of Hsp90. The rare gene organization, coupled with independent expression of domains of Hsp90, makes this organism important to examine novel functions of this chaperone.

Heat shock protein 90 (HSP90) is a molecular chaperone protein that protects proteins from degradation, repairs damaged proteins, and assists proteins in carrying out their functions. HSP90 has hundreds of clients, many of which are inflammatory signaling kinases. The mechanism by which HSP90 enables inflammatory pathways is an active area of investigation. The HSP90 inhibitors such as geldanamycin (GA) and its derivative 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) have been shown to reduce inflammation. It was hypothesized that inhibiting HSP90 would reduce inflammatory signal cascade levels. To test this, J774 mouse macrophage cells were treated with 17-DMAG and immune-stimulated with lipopolysaccharide (LPS). 17-DMAG treatment reduced nitric oxide (NO) production and the expression of pro-inflammatory cytokines interleukin (IL)-6, IL-12, and TNF-α. Inhibition of HSP90 also prevented nuclear translocation of NF-κB. To investigate the anti-inflammatory effects of HSP90 inhibition in vivo, MRL/lpr lupus mice were administered 5 mg/kg 17-DMAG for six weeks via intraperitoneal injection. Mice treated with 17-DMAG were found to have reduced proteinuria and reduced splenomegaly. Flow cytometric analysis of splenocytes showed that 17-DMAG decreased double negative T (DNT) cells. Renal expression of HSP90 was also measured and found to be increased in MRL/lpr mice that did not receive 17-DMAG. The mechanistic interactions between HSP90 and the pro-inflammatory nuclear factor-κB (NF-κB) pathway were studied and a computational model was developed. The model predicts cellular response of inhibitor of κB kinase (IKK) activation and NF-κB activation to LPS stimulation. Model parameters were fit to IKK activation data. Parameter sensitivity was assessed through simulation studies and showed a strong dependence on IKK-HSP90 binding. The model also accounts for the effect of a general HSP90 inhibitor to disrupt the IKK-HSP90 interaction for reduced activation of NF-κB. Model simulations were validated with experimental data. In conclusion, HSP90 facilitates inflammation through multiple signal pathways including Akt and IKK. Inhibition of HSP90 by 17-DMAG reduced disease in the MRL/lpr lupus mouse model. A computational model supported the hypothesis that HSP90 is required for IKK to activate the NF-κB pathway. Taken together, HSP90 is a prime target for therapeutic regulation of many inflammatory processes and warrants further study to understand its mechanism of regulating cell signaling cascades. / Ph. D.

The major evolutionary transition from single-celled to multicellular life is believed to have occurred independently of the main metazoan lineages in the cellular slime moulds, of which Dictyostelium discoideum is the best-studied species. Unusually, in this case multicellular development is a facultative trait and part of an asexual life cycle. It is triggered by starvation and involves aggregation of hitherto independent and possibly unrelated free-living cells. The consequences of multicellularity in D.discoideum are strongly influenced by the environment and meaningful external perturbations are easily carried out. This makes the organism ideally suited to a study of epigenetic factors that regulate development. In an attempt to understand how conserved epigenetic pathways are integrated within the developmental framework, two likely players were chosen for investigation - heat shock protein 90 (Hsp90) and histone deacetylases (HDACs). Hsp90 has been implicated in diverse biological processes such as protein folding, cell cycle control, signal transduction, and morphological evolution. The role of Hsp90 in D.discoideum life cycle was studied using a specific inhibitor, geldanamycin. Inhibition of Hsp90 function in D.discoideum caused a delay in aggregation and an arrest of development at the ‘mound’ stage. A reduction in Hsp90activity in starving cells of D.discoideum resulted in the generation of a range of phenotypes. The study suggests that Hsp90 is required for a specific developmental transition of the social amoeba and is important in generating a reliable outcome of the developmental process. Histone acetylation regulates gene expression and leads to the establishment and maintenance of cellular phenotypes during development of plants and animals. To study the roles of HDACs in D.discoideum, biochemical, pharmacological and genetic approaches were employed. The inhibition of HDAC activity by trichostatin A resulted in histone hyperacetylation and a delay in cell aggregation and differentiation. Cyclic AMP oscillations were normal in starved amoebae treated with trichostatin A but the expression of a subset of cAMP-regulated genes was delayed. Bioinformatic analysis indicated that there are four genes encoding putative HDACs in D.discoideum. One of these four genes, hdaB, was found to be dispensable for growth and development under laboratory conditions; but formed spores with lower efficiency than the wild type in chimeras. The work shows that HDAC activity is important for regulating two aspects of multicellular development: (a) heterochrony, namely the relative timing of developmental events, and (b) modulating the behaviour of single cells in a manner that is sensitive to their social environment.

Abstract Calcific aortic valve disease (CAVD) is the most common valvular heart disease in the Western world. Although it shares mainly the same risk factors as coronary heart disease (CHD), i.e. similar initial events in both diseases but with time, they lead to different clinical outcomes. Thus, when it affects the coronary arteries, the disease leads to an obstructive or rupture-prone plaque whereas in the aortic valve, it causes massive calcification and ossification. This obstructs the blood flow from the left cardiac ventricle, causing myocardial hypertrophy, and if left untreated, heart failure and death. Many of the pathobiological differences between CAVD and CHD remain unknown. Currently, there are no effective lifestyle- or pharmacologic treatments for CAVD and the only therapy is a valve replacement operation. In this thesis, several studies utilizing large-scale methods were undertaken to profile the molecular events leading to CAVD. Surgically removed valves from patients in different stages of the disease were obtained and gene transcripts, microRNA-molecules and several proteins were identified as being differentially expressed. Several of these were investigated further, including two pro-inflammatory CC-type chemokine ligands 3 and 4 (CCL3 and CCL4), microRNA-125b, several granzyme-proteins and heat-shock protein 90. The results of this thesis provide a large dataset of hundreds of molecular changes associated with CAVD. It is proposed that they can be used as a basis for the generation of new hypotheses and assist in the design of experiments to clarify the mechanisms driving CAVD. / Tiivistelmä Aorttaläpän kalkkeutuva ahtauma on länsimaiden yleisin sydänläppäsairaus. Riskitekijät ovat pääosin samat kuin sepelvaltimotaudissa, ja molemmat saavat alkunsa samalla tavalla. Ajan myötä ne kuitenkin johtavat varsin erilaisiin kliinisiin ilmenemismuotoihin: sepelvaltimoihin kasvaa ahtauttavia ja repeytymisherkkiä plakkeja, kun taas aorttaläppään muodostuu runsaasti kalkkia ja luuta. Se haittaa verenvirtausta sydämen vasemmasta kammiosta aorttaan, mikä aiheuttaa sydänlihaksen paksuuntumista. Hoitamattomana tauti johtaa lopulta sydämen vajaatoimintaan ja kuolemaan. Monet syyt eroihin sepelvaltimotaudin ja aorttaläpän ahtauman välillä ovat edelleen tuntemattomia. Tällä hetkellä aorttaläpän ahtaumaan ei ole olemassa tehokasta elintapa- tai lääkehoitoa, ja ainoa hoitomuoto onkin vioittuneen aorttaläpän korvaaminen proteesilla. Tässä väitöskirjatyössä tehtiin useita laaja-alaisia molekyylitason profilointitutkimuksia, joilla selvitettiin aorttaläpän ahtaumaan mahdollisesti johtavia mekanismeja. Aineistona oli leikkauksessa potilailta poistettuja, erilaisissa taudin vaiheissa olevia aorttaläppiä. Niistä kerättiin tietoja kaikkien geenien ilmentymisestä, mikroRNA-molekyyleistä sekä koko proteomitason muutoksista. Useat tunnistetuista molekyyleistä valittiin jatkotutkimuksiin niiden tarkempien ominaisuuksien selvittämiseksi. Näitä olivat tulehdusta välittävät kemokiinit CCL3 ja CCL4, mikroRNA-125b, useat grantsyymiproteiinit sekä lämpöshokkiproteiini 90. Väitöskirjatyön tuloksista voidaan muodostaa ainutlaatuinen aineisto sadoista erilaisista aorttaläpän ahtaumaan johtavista molekyylitason muutoksista. Sitä voidaan hyödyntää uusien tutkimushypoteesien muodostamisessa sekä aorttaläpän ahtauman tarkempien mekanismien selvittämiseen tähtäävien kokeellisten tutkimusten suunnittelussa.

Molecular chaperones are a group of proteins which maintain cellular homeostasis by assisting de novo protein folding and their refolding to native state after destabilization due to external stress. They are also known as heat shock proteins as they were first discovered as a response to heat stress. It is now well established that the function of this group of proteins is not only restricted to protein homeostasis but also extends to diverse cellular processes such signal transduction, development and differentiation. Heat shock protein 90 (Hsp90) is one of the most abundant molecular chaperones that is highly conserved from prokaryotes to eukaryotes. Hsp90 is an essential chaperone and is required for the viability of all eukaryotes examined so far including yeast, Drosophila and Caenorhabditis elegans. Hsp90 has emerged as an important regulator of cellular activities by virtue of its ability to interact with a diverse set of client proteins many of which include transcription factors, protein kinases and signaling molecules. Through interaction with these proteins it is involved in regulating cellular processes including growth, cell cycle, endocrine functions, apoptosis, differentiation and development. Further in Drosophila and plants, Hsp90 is thought to function as a capacitor for morphological evolution and phenotypic variation. Recently, it has also been implicated in the emergence of drug resistance in Candida albicans. Furthermore, the importance of Hsp90 in disease states, particularly in cancer, is strongly evident, where chaperoning of mutated and oncogenic proteins is critical for continuous proliferation of cells. This has led to the development of Hsp90 inhibitors as an anti-cancer drug. Geldanamycin (GA), a benzoquinone ansamycin was the first molecule shown to inhibit Hsp90 activity by binding to its ATP binding domain. A derivative of GA, 17-allylamino-17-demethoxygeldanamycin (17AAG), has shown promise in clinical studies and has entered Phase III clinical trials. Hsp90 has been shown to be important for growth and development of many protozoan parasites. Inhibition of Hsp90 function in Leishmania, Emiera, Toxoplasma, Trypanosoma as well as Plasmodium causes a block in their developmental cycle. Previous studies from our laboratory have shown that inhibition of Hsp90 function prevents growth of malaria parasite in human erythrocytes in vitro. P. falciparum Hsp90 (PfHsp90) has also been shown to regulate parasite growth during the febrile episodes that are characteristic of malaria. While most of the studies highlighting the importance of PfHsp90 have relied on its pharmacological inhibition, its biochemical characterization and quantitative measurement of its interaction with GA in isolated system has not been explored. It was also not understood whether the in vitro model of Hsp90 inhibition could translate into inhibition of the parasite growth in an animal model of malaria. Since Hsp90 is a split ATPase requiring proper co-ordination between the residues on its N-terminal and middle domains, it would be desirable to biochemically characterize full length PfHsp90 to gain insights into its potential as an anti-malarial target. The present study was initiated with an objective of understanding the biochemical properties of Hsp90 from P. falciparum in terms of ATP binding, ATP hydrolysis and its GA binding ability. We have also examined the potential of PfHsp90 to serve as a chemotherapeutic target using its clinically well-established inhibitor, 17AAG, in a preclinical mice model. Apart from using in vitro and in vivo models of malaria, we have also explored the efficacy of 17AAG in the P. falciparum samples collected from malaria patients. Additionally, we have examined the relevance of chaperones, in particular PfHsp90 in the samples collected from malaria patients. Finally, we have attempted to understand the unexplored biology of another malaria parasite P. vivax by a high throughput proteomics approach. Biochemical characterization of PfHsp90 and its comparison with host Hsp90 Hsp90 belongs to GHKL (gyrase, Hsp90, histidine kinase, MutL) protein family having a characteristic novel ATP-binding Bergerat fold. The ATP binding pocket of GHKL family differs from the conventional nucleotide binding fold in the formation of a cone shaped pocket made up of four anti-parallel β-sheets and three α helices as opposed to parallel βsheets surrounded by α-helices in the latter. The most distinctive feature of Bergerat fold is the presence of ATP lid. Further, even within the GHKL family members the composition and the conformation of this ATP-lid differs, leading to different solvent exposure of the bound ATP. All Hsp90s from different organisms, characterized so far, have been shown to posses ATP binding and hydrolysis activity but so far PfHsp90 ATPase activity has not been characterized. Using intrinsic tryptophan fluorescence measurements, we found PfHsp90 to bind ATP with about 30% higher affinity than human Hsp90 (hHsp90). We further, 32 determined the ATPase activity of PfHsp90 by monitoring the direct conversion of (γ-P) 32-2 ATP to Pi. PfHsp90 bound and hydrolyzed ATP with a Km of 611 µM and kcat of 9.9 x 10 -1m . Interestingly, PfHsp90 showed six times higher ATPase activity as compared to its human homologue and more intriguingly the ATPase activity exhibited by PfHsp90 was highest among all the Hsp90s studied so far. Previous studies from our laboratory have provided sufficient evidence for inhibitory action of GA on Plasmodium growth inside the infected erythrocytes. GA is known to exert its inhibitory effect by binding to the ATP binding domain of Hsp90 thus inhibiting its chaperone activity. Earlier reports have shown that despite a high similarity between the ATP/GA binding region in Hsp90 from different organisms, there is a difference in their ability to bind GA. For example, in spite of all the hallmarks of ATP-binding pocket of Hsp90 family C. elegans Hsp90 does not bind GA. We have employed fluorescence spectroscopy to examine whether PfHsp90 can bind to GA. In parallel, we have also determined the binding affinity of human Hsp90 (hHsp90) to GA. We observed small but reproducible differences in the binding affinity of GA to Hsp90s from human host and P. falciparum with latter having fourfold higher affinity. A sequence analysis of the GA binding domain of Hsp90s from P. falciparum and human host showed a homologous substitution of K112 of hHsp90 to R98 in PfHsp90. In order to examine the effect of this substitution, if any, on the observed difference in GA binding abilities, we mutated R98 to K in PfHsp90. However, we did not find any difference in the binding ability of R98K PfHsp90 to GA, suggesting that this homologous substitution has minimal or no effect on drug protein interaction in vitro. However, in view of this phylogenetically conserved substitution, we cannot rule out its role in vivo. The chaperone function of Hsp90 is dependent on its ATPase activity which is susceptible to GA mediated inhibition. We next examined the extent of inhibition of GA on the ATPase activity of Hsp90s from P. falciparum and human host. Interestingly, we found the PfHsp90-ATPase activity to be three times more sensitive than hHsp90-ATPase activity to GA mediated inhibition suggesting that the malaria parasite, P. falciparum is likely to be more sensitive to GA when compared to human host. This result is in accordance with a recent study, which has shown that yeast expressing PfHsp90 in lieu of native yeast Hsp90 was more sensitive to GA than yeast expressing either yeast Hsp90 or human Hsp90. Acetylation of Plasmodium falciparum Hsp90 Post-translational modification of Hsp90 such as acetylation has been shown to affect its binding with GA. We first examined whether, PfHsp90 can be acetylated. With the use of various purified Histone acetyl transferases (HATs) of human origin, we have shown PfHsp90 to undergo acetylation in vitro. We found that among different HATs (pCAF, Gcn5 and p300) used, only p300 was able to acetylate PfHsp90 suggesting a role for it in PfHsp90 in vivo acetylation as well. We next examined the in vivo acetylation status of PfHsp90 from parasite lysate. To enrich the acetylated fraction of PfHsp90, we have used Histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). Immunoprecipitation of PfHsp90 followed by immunoblotting with an acetyl-lysine antibody confirmed that PfHsp90 undergoes acetylation in vivo. In order to identify the lysine residues which underwent acetylation we subjected the acetylation enriched fraction of PfHsp90 to in-gel trypsin digestion followed by mass spectrometry. Analysis of trypsin digested PfHsp90 from the parasites identified three sites of acetylation, one of which overlapped with PfHsp90 cochaperone (Aha1 and p23) binding residue, suggesting that acetylation could play a potential role in modulating PfHsp90 multi-chaperone complex assembly. Indeed, treatment of P. falciparum cultures with a HDAC-inhibitor resulted in partial dissociation of PfHsp90 complex as observed from size-exclusion chromatography. Adding to this observation, we also found that co-treatment of TSA and GA showed a synergistic and additive effect in inhibiting parasite growth in vitro. The above results suggest the possibility of using Hsp90 inhibitor in combination with HDAC inhibitor to arrest Plasmodium growth and development. Clinically tested GA-analogue 17AAG inhibits Plasmodium growth in vitro and in vivo The specificity of GA inside the cell has been a matter of debate since the discovery of its medicinal importance. In the past, Hsp90 has been implicated as a target of GA by carrying out immunoblotting of GA pull-down fraction with an anti-Hsp90 antibody. Crystal structure of GA with yeast Hsp90 has shown it to bind within the well conserved ATP-binding pocket of Hsp90. However, the specificity of GA inside the cell is still a conjecture. We have performed GA pull down assays from the parasite lysate followed by Coomassie Blue staining, which gave a single band corresponding to 86 kDa PfHsp90. The identity of PfHsp90 was further confirmed by immunoblotting with antibody specific to PfHsp90. This result indicates that inside the cells, inhibitory effect of GA is mediated by and large through its interaction with Hsp90. However, we cannot rule out the presence of other minor, less significant, interactors of GA. Earlier work from our laboratory has shown that GA inhibits Plasmodium growth inside the infected erythrocytes. However, issues related to GA toxicity have excluded its development as a therapeutic. Nevertheless, interest in this class of molecule has led to the generation of a large number of less toxic derivatives of GA. One classical example is 17AAG which has gained clinical importance over the years and has entered in phase III trial. Intrigued by the clinical success of 17AAG, we were interested in determining its ability to modulate parasite growth. Indeed, 17AAG was able to inhibit parasite growth in a manner similar to that of GA. We further extended our study to parasites isolated from patient samples. Here too, we found 17AAG to be effective in inhibiting growth of the parasite. Finally, we examined the efficacy of 17AAG at a pre-clinical level using a mouse model of malaria. Using Peters’ four-day test we found 17AAG, to be effective in attenuating parasite growth and prolonging the survival of parasite infected mice (n=4, p=0.00692; n=10, p=0.001). Clinical relevance of heat shock proteins of Plasmodium falciparum A recent study using in vivo expression profiles of parasites derived from blood samples of infected patients has revealed previously unknown physiological diversity in the biology of malaria parasites. According to gene expression profiles, parasites were clustered into three different physiological states – starvation, glycolysis dependent active growth and environmental stress response. In order to examine the clinical relevance of molecular chaperones in malaria, we reanalyzed the previously published gene expression data of clinical parasites from 46 patients. Our analysis of this data showed that organellar chaperones were up-regulated upon starvation (cluster1) while cytosolic chaperones such as Hsp90 were up-regulated in active growth conditions (cluster2) indicating up-regulation of distinct group of Hsps in response to different environmental cues. Interestingly, Hsp90 and its co-chaperones, previously implicated as drug targets in malaria, clustered in the same group. Further, some patients of cluster 3 (environmental stress response) showed higher expression of Hsp90 while others showed lower expression. In general, cluster 3 group of patients were heterogeneous in terms of expression of chaperones. Using non-negative matrix factorization (NMF), cluster 3 was sub-clustered into two groups 3a and 3b. Cluster 3b showed up-regulation of cytosolic chaperones similar to cluster 2 indicating these two clusters are inter-related. Most of the Hsp90 dependent pathways such as trafficking, signaling, anti apoptotic and pro-survival found to be most active in cluster 2 indicating the dependence of this group of parasites on Hsp90. The two main outcomes of our chaperone analysis are (1) the up-regulation of molecular chaperones in parasites are not a general response to hostile conditions as perceived previously, but is largely determined by the host factors and may differ from one host to another (2) the disease specific pathways may exist in natural condition by the up-regulation of specific chaperone and its interactors as a response to different host environment. Clinical proteomics of human malarial parasites Much of our understanding about the life cycle of parasites and importance of parasite proteins have been gleaned from the studies in laboratory strain or with the laboratory adapted clinical parasites. Although, these studies provide us first hand information about the functionality and the importance of these proteins, but they often fail to mimic the actual disease environment. In the patient, parasites are exposed to host factors such as hormones, metabolites, inflammatory mediators which can influence the expression of proteins and thus parasite biology. Further, instead of parasite exposure to 37°C temperature throughout the erythrocytic cycle in vitro, it is exposed to several rounds of febrile episodes inside human, which can also influence the parasite life cycle. Furthermore, clinical analysis is important to validate the presence and expression of drug targets in actual disease environment. Therefore, analysis of malaria parasite from clinical settings has become an important component in our laboratory and this thesis. Proteomic analysis of clinical samples has emerged as an important tool to understand the proteins dynamicity as response to disease environment. We have initiated clinical proteomic study of P. falciparum, the cause of most common and fatal malaria in humans and extended it further to the neglected malaria parasite P. vivax. The study of P. vivax has largely been over-shadowed by the enormous attention devoted to P. falciparum. Notably, the drugs which have been discovered against P. falciparum are not as effective against P. vivax. Further several unique features of P. vivax such as dormant hyponozoites, reticulocyte host preference and formation of specialized caveolae vesicle complex structure distinguish its biology from P. falciparum and warrant concerted effort directed at this parasite. A major limitation in studying this parasite is the absence of a long-term culturing system. Therefore, research on this parasite requires samples obtained directly from patients. In spite of the inherent difficulty in obtaining such samples, this method provides us an opportunity to study this parasite in its real environment which has a huge effect on the expression as well as function of parasites and host proteins. Our current knowledge about the life cycle of this parasite has been gained from the recently published transcriptome study. Even though transcriptome analyses provide useful understanding at the level of gene expression, they do not reflect the active protein component of a cell. In other words, most of disease outcome is a result of interaction of the protein component with the environment. We therefore attempted to understand the protein component of this parasite in the disease environment to shed light on its pathogenicity. Despite facing several challenges in the way of proteomic analysis of this parasite such as availability of samples, low parasitemia, contamination of parasite proteins with abundant host proteins etc, we were able to identify 154 P. vivax proteins abundantly expressed in clinical environment using mass-spectrometry based approach. We found many proteins unique to this parasite along with known drug targets. This study is the first of its kind and could prove to be a very important step towards gaining insights into the physiology of this parasite.This study serves as a proof-of-principle method which in future is likely to help in identifying many more potential drug targets, vaccine candidates and diagnostic markers from clinically relevant samples as opposed to cultured samples. Summary Despite the importance of PfHsp90 in malaria biology, it has not been characterized in terms of its biochemical properties and its interaction with the inhibitor. In this study, we have successfully cloned, expressed, purified and characterized full length PfHsp90. We found that PfHsp90 exhibits a hyper-ATPase activity and is more sensitive to GA mediated inhibition as compared to human Hsp90. We have also shown that its sensitivity towards GA is dependent on its acetylation status as treatment of infected erythrocytes with HDAC inhibitors increases its sensitivity to GA. Using a pull-down assay, we have determined, unequivocally, that GA specifically binds to Hsp90. Most importantly, we have demonstrated that 17AAG, a clinically well-established inhibitor of Hsp90, inhibits parasite growth in a laboratory strain, field isolates and an in vivo mouse model of malaria. Overall, our biochemical characterization and drug interaction studies underscore the importance of PfHsp90 as a potent drug target and its inhibitors as a candidate drugs for the treatment of malaria, one of the deadly human infectious diseases. Our efforts to understand the importance of molecular chaperones in parasites isolated directly from patient samples (clinical setting) has revealed conspicuous association of Hsps with previously defined parasite physiological states. In particular, parasites obtained from a specific group of patients exhibited heightened dependence on Hsp90-dependent pro-survival pathways, indicating an increased response to host stressors in this group of parasites. Thus, parasite encoded chaperones, in particular PfHsp90, play a major role in defining the pathogenesis of malaria. A disease is an outcome of interaction between pathogens and its host, therefore it is important to study parasite in its real environment to understand disease pathogenesis. Our lab has previously reported the first ever proteomic analysis of P. falciparum from malaria patients. In this study, we have made an attempt to understand the unexplored biology of another important malaria parasite P. vivax. We have used a mass-spectrometry based approach to identify the protein content of this parasite. This technically challenging attempt has enabled us to identify many proteins. This study is an important step towards understanding the biology of this parasite in dearth of any information available on the proteins involved in this parasite’s pathogenicity.

Heat shock proteins (Hsps) are a class of molecular chaperones which were first discovered as proteins up-regulated in response to heat stress in Drosophila. Later, it was found that these set of proteins get up-regulated as a general stress response associated with destabilization of native protein structures. Over a period of time, intricate involvement of Hsps in various biological processes has been well established. Heat shock protein 90 (Hsp90) is one of the important representative of this class of proteins. Hsp90 is an essential molecular chaperone which is evolutionarily conserved. It has a selective set of proteins to chaperone called as clients, which majorly include transcription factors and protein kinases. Through its interaction with its clients it modulates cell cycle, signal transduction, differentiation, development and evolution. Previous studies from Candida, Leishmania and Plasmodium have implicated Hsp90 to be involved in stage transition and growth. It is also critically involved in regulating growth of other protozoans such as Dictyostelium, Entamoeba and Trypanosoma. Thus, selective inhibition of Hsp90 has been explored as an intervention strategy against important human diseases such as cancer, malaria and other protozoan diseases. In Plasmodium falciparum, Hsp90 plays a critical role in stage transition. The parasite inside the human RBC develops from ring to trophozoite to schizont stage and inhibition of Hsp90 using specific pharmacological inhibitor arrests the growth of parasite at ring stage. In Dictyostelium, it has been observed that Hsp90 function is required for development. Inhibition of Hsp90 causes mound arrest and stops the cells from entering to its next developmental stage, fruiting bodies. In parallel, Hsp90 in Candida has been shown to be involved in morphogenesis. In nature Candida exists as a single cell yeast form and upon entry into the human host these yeast forms undergo morphogenesis to form virulent filamentous fungi. Inhibition of Hsp90 mimics temperature mediated morphogenesis. All together, these studies suggest that Hsp90 functions in a context dependent manner and each biological system explored has given new insights into the Hsp90 biology. Giardia lamblia, a protozoan parasite of humans and animals, is an important cause of diarrheal disease causing significant morbidity and also mortality in tropical countries. In the present study we focus on the biology of Hsp90 from Giardia lamblia. Giardia has a biphasic life cycle with infective cyst stage and pathogenic trophozoite stage. These cysts are present in the environment and enter mammalian host through oral route. They undergo a process called as excystation in the intestine giving rise to trophozoites. The trophozoites so formed colonize the upper part of the small intestine which causes the symptoms of giardiasis. Some of the trophozoites escape from the nutrition rich milieu of the upper part of small intestine to the lower part. In this region, trophozoites undergo a process called as encystation, wherein each trophozoite forms a cyst which escapes through faeces back into the environment. As seen in the life cycle of Giardia there are two major biological transitions, excystation and encystation; and till date no definitive player or pathway is known to regulate these processes. With the knowledge of Hsp90 playing an important role in similar biological transitions in other organisms we were encouraged to study role of Hsp90 in Giardia lamblia. Trans-splicing based generation of a full length Hsp90 in Giardia lamblia To understand the role of Hsp90, we first carried out sequence alignment of Hsp90 predicted ORFs in Giardia genome with yeast Hsp90. On alignment we observed that Hsp90 in Giardia is discontinuous and is annotated to be encoded by two different ORFs. Hsp90 in most organisms is coded by a single ORF with none to many cis-spliced introns. In a relatively intron poor organism G. lamblia, cytosolic Hsp90 is coded by two different ORFs separated by 777 kb in the genome. On multiple sequence alignment, we noticed that these two ORFs correspond to two independent regions of the Hsp90 protein. The ORFs are designated as hspN and hspC, containing the N-terminal and the C-terminal region of the protein respectively. We began our study by sequencing whole genome of Giardia lamblia clinical strain. Our genome sequencing confirmed the split nature of hsp90 and showed high ‘synteny’ between the other sequenced isolates. Using PCR based approach we have ruled out the possibility of having a full length gene in the genome. In contradiction to the genome result, we have observed a higher molecular weight protein in the lysate on proteomic analysis which was further confirmed by western blotting. The protein was observed to have a molecular weight of 80 kDa which could be a resultant of combination of two ORFs, suggesting the presence of a full length mRNA for Hsp90. PCR amplification using primers against both the fragments resulted in amplification of 2.1 kb product from the RNA pool of Giardia. Sequencing of this product showed that hspN and hspC were stitched together to form a mature messenger for full length Hsp90. In total our results suggest a post transcriptional process, trans-splicing, to be involved in the construction of Hsp90. The transition marked by this fusion coincides with the canonical GU¬AG splice site transitions as observed in other eukaryotes. Interestingly, a 26 nt near-complementary region was observed inside and upstream of hspN and hspC ORFs respectively. Put together these results suggest that the 26 nt complementary region acts as the positioning element to bring these two precursors in spatial proximity. With efficient spliceosomal activity these two precursor forms are trans-spliced to generate a full length cytosolic Hsp90 in Giardia. There are only four genes which have cis-spliced introns in the Giardia genome and the core components of the spliceosomal machinery are also present. The presence of canonical splice site in both the transcripts suggests that these transcripts are fused together by the spliceosomal machinery by the phenomenon of trans-splicing. The formation of full length Hsp90 RNA by its fragmented gene is the first example of trans-splicing in Giardia. To understand, are there any other genes which are also similarly trans-spliced we have carried out shotgun proteomic analysis of the total cell lysate obtained from Giardia trophozoites. Using Hsp90 as template, in our proteomic datasets, we have designed an algorithm for identification of additional trans-spliced gene products at the protein level. We have identified a total of 476 proteins of which hypothetical proteins constitute the major class followed by metabolic enzymes. We have compared the theoretical molecular weights for the identified proteins with the experimentally determined mass. Any discrepancy in the molecular mass was further analyzed and we assigned a gene to be potentially trans-spliced based on three criteria: if they were encoded by two or more different ORFs (loci), absence of a single full length counterpart and presence of splice sites with branch point and positional elements. Using this algorithm we were able to identify dynein as a potential candidate of trans-splicing reaction which was confirmed by the nucleotide sequence analysis of the predicted ORFs. Interestingly, dynein gene fragments were observed to be scattered on different chromosomes with minor splice sites unlike hsp90 genes. In vivo Expression of Hsp90 sub-fragments, HspN and HspC In the mature Hsp90 mRNA formed upon trans-splicing, 33 additional codons are present right between hspN and hspC sequences and they were acquired from the upstream region of hspC ORF. The 33 codons encode for an important region of Hsp90 which harbours the conserved catalytic “Arg” residue; suggesting that the full length Giardia Hsp90 (GlHsp90) formed could be an active ATPase. To confirm the same we have carried out in vitro characterization of trans-spliced Hsp90. Towards this, we have cloned, expressed and purified His tag-GlHsp90. As a first step, highly purified protein was used to assess its efficiency in binding to it cognate ligand, ATP, and the known inhibitors. Our binding studies show that GlHsp90 binds to ATP with a dissociation constant of 628 M and to its inhibitors, GA and 17AAG with 1.5 μM and 17.5 μM respectively. The bound ATP will be subsequently cleaved by Hsp90 which is an essential step in the chaperone cycle. As determined in our ATPase assay we observed that GlHsp90 hydrolyzes bound ATP with the catalytic efficiency of 4.4 × 10-5μM-1.min-1which confirms that Hsp90 generated upon trans-splicing is an active ATPase. The uniqueness of the hsp90 gene arrangement in Giardia posed a new question. Do these gene fragments also get translated? Our results suggest that HspN and HspC are poly¬adenylated. In order to determine the levels of these transcripts we performed qRT-PCR using primers specific to HspN, HspC and GlHsp90. We have observed that, in comparison with HspN transcript level, HspC and GlHsp90 transcripts are 15 and 75 folds higher respectively. To check for the presence of translation products of these transcripts, we have re-analyzed our proteomic datasets wherein we could identify peptides corresponding to HspN and HspC in their respective molecular weight region, 45 to 35 kDa. To confirm the proteomic data, western blot analysis was performed for trophozoite lysate on both 1D and 2D gels using anti-HspN antibody. Two specific bands (1D) / spots (2D) corresponding to the full length Hsp90 and HspN were identified. Gel filtration analysis revealed that HspN co¬eluted with full length Hsp90 thereby suggesting that both the proteins are in a same complex. With the background that HspN and HspC are present at the protein level, we asked if these fragments in combination can hydrolyse ATP. We reconstituted recombinant HspN and HspC in equimolar amounts and scored for the hydrolysis of ATP. However, no Pi release was observed. To determine whether HspN and HspC could modulate Hsp90 function, ATPase activity was monitored in the presence of HspN or HspC, in vitro. It was observed that ATPase activity was inhibited by both the fragments thus suggesting that HspN and HspC negatively regulate Hsp90 ATPase activity. Role of Hsp90 in Giardia encystation Giardia has a biphasic life cycle with proliferative trophozoites and latent cyst stage. In Giardia, in vitro encystation was established nearly two decades back by modulating the medium conditions. However, the mechanism and triggers underlying this transition are not well characterized. To understand whether Hsp90 has any role in this transition, in vitro conversion of trophozoites to cysts was achieved. The cysts obtained showed all the characteristic features of mature Giardia cyst with cyst wall protein 1 (CWP1) on the cyst wall and four nuclei as determined by immunofluorescence analysis. Further, the levels of Hsp90 in trophozoites were compared with mature cysts at both transcript and protein levels and it was found that cysts show more than 50% reduction in the level of Hsp90 in comparison with normal trophozoites. In accordance, exogenous inhibition of Hsp90 using 17AAG promoted the formation of cysts in vitro by 60 folds in a dose dependent manner; however, the window period of Hsp90 function compromise plays an important role in this process. Higher numbers of cysts were obtained from the cells treated with inhibitors during pre-encystation condition but inhibition of Hsp90 during encystation did not affect the formation of cysts, suggesting that Hsp90 down-regulation plays an important role during commitment towards encystation. To further show that cyst formation is a specific response to Hsp90 inhibition we have carried out encystation in the presence of metranidazole and from heat shocked cells; however, in both the conditions we did not observe any significant change in cyst formation, thus confirming that Hsp90 plays an important role during encystation in Giardia lamblia. Summary In Conclusion, Our study throws light on a unique aspect of Hsp90 biology in Giardia Lamblia, wherein the formation of the full length protein is dependent on a unique trans splicing reaction of its gene components representing different domains. We have also shown that HsP90 fragments, HspN and HspC, are also expressed in Trophozoites. Our in vitro data suggests that these fragments possibly regulate the function of Hsp90. Furthermore, the full length of Hsp90 plays an important role in stage transition in Giardia wherein inhibition of Hsp90 induces encystations. The study has opened many new avenues for research. Understanding the exact role of HspN and HspC in vivo will provide better appreciation for the evolution of such a complex biogenesis of an essential protein.