http://hdl.handle.net/2005/7 Sat, 04 May 2013 18:36:04 GMT 2013-05-04T18:36:04Z http://hdl.handle.net/2005/1960 Title: Functional Characterization And Regulation Of UvrD Helicases From Haemophilus Influenzae And Helicobacter Pylori, And Recj Exonuclease Fron Haemophilus Influenzae Authors: Sharma, Ruchika Abstract: DNA repair processes are crucial for mutation avoidance and the maintenance of genetic integrity in all organisms. Organisms rely on repair processes to combat genotoxic stress imposed by hostile host environment, and sometimes by therapeutic agents. Most pathogens rapidly generate genetic variability to acquire increased virulence and evade host immune response. Therefore, there needs to exist a fine balance between mutation avoidance and fixation, which is perhaps regulated by repair processes. Haemophilus influenzae and Helicobacter pylori contribute significantly to morbidity and mortality caused by bacteria worldwide. H. influenzae is an obligate commensal of upper respiratory tract with the potential to cause a variety of diseases in humans like meningitis and respiratory infections. H. pylori, which inhabits the human stomach, is associated with gastric and duodenal ulcers and cancerous gastric lesions. One of the striking differences between these two genetically diverse bacterial species is the absence of recognized DNA mismatch repair (MMR) pathway homologs in H. pylori. MMR is a highly conserved post-replicative process, which corrects base pairing mismatches and small loops arising during DNA replication and recombination due to misincorporated nucleotides, insertions, and deletions. Defective MMR results in increased mutation frequency that can alter the pathogenic potential and antibiotic resistance of pathogens. MMR has been extensively studied in Escherichia coli, and requires an orchestrated function of different proteins like MutS, MutL, MutH, UvrD, SSB, RecJ, ExoVII, ExoI, ExoX, beta-clamp, DNA polymerase III and DNA ligase. A growing body of evidence suggests that bacteria other than the well-characterized E. coli paradigm differ in basic DNA repair machinery. MMR proteins involved in mismatch recognition and strand discrimination like MutS, MutL and MutH from H. influenzae have been characterized, but other downstream repair genes like UvrD helicase and exonucleases like RecJ have not been studied functionally in detail. H. pylori harbors a UvrD homolog, which shares limited homology with other UvrD proteins (29% identity with E. coli UvrD and 31 % with H. influenzae UvrD) and its cellular functions are not clear. Moreover, it is not well-understood how the activities of UvrD and RecJ proteins are regulated within these pathogens. It was, therefore, envisaged that biochemical characterization of UvrD and RecJ would lead to a better understanding of the mechanistic aspects of repair processes within these pathogens. The following sections summarize the results presented in this investigation. Functional characterization of UvrD from H. influenzae UvrD or DNA helicase II is a member of superfamily I of DNA helicases with well-documented roles in nucleotide excision repair (NER) and MMR, in addition to roles in replication and recombination. The 727-amino acid H. influenzae Rd KW20 UvrD (HiUvrD) protein was purified as an N-terminal (His)6-tagged protein to near homogeneity, and its authenticity was confirmed by peptide mass fingerprint analysis. HiUvrD displayed robust binding with single-stranded (ss) DNA as compared to double-stranded (ds) DNA. HiUvrD was found exhibit ~ 1000-fold higher affinity for ssDNA as compared to dsDNA as determined by surface plasmon resonance (SPR). In addition, to gain insights into the role of HiUvrD in replication, repair, recombination and transcription, the ability of HiUvrD to bind different DNA structures resembling intermediates of these processes was investigated using electrophoretic mobility shift assays. HiUvrD exhibited relatively high affinities for a number of branched DNA substrates and the order of affinity observed was; splayed-duplex ≥3’-flap ≥ ssDNA > 3’-overhang > four-way junction > three-way junction > nicked duplex > looped duplex ≥ duplex. Concurrent with its high affinity for ssDNA, HiUvrD exhibited a robust ssDNA-specific and Mg2+ - dependent ATPase activity. HiUvrD was able to unwind different DNA structures with varying efficiencies (3’ flap ≥ 3’-overhang > three-way junction > splayed-duplex > four-way junction > nicked > loop = duplex >>> 5’-overhang) and with a 3’-5’ polarity, which underpins its role in replication fork reversal, recombination and different DNA repair pathways. Multiple sequence alignment of HiUvrD with other helicases showed the presence highly conserved helicase motifs of which motif I and II are essential for ATP binding and hydrolysis. Mutation of an invariant glutamate residue (E226Q) in motif II of HiUvrD resulted in a dominant negative growth phenotype since, it was not possible to recover transformants when wild-type E. coli expression strains BL21(DE3)plysS or BL21(DE3)plysE were transformed with expression vector carrying hiuvrDE226Q. Mutation of a conserved arginine residue to alanine (R288A) in motif IV resulted in approximately 80 % reduction in ATP hydrolysis, and abrogation of helicase activity as compared to the wild-type protein. This can be attributed to ~ 70 % reduced ATP binding by HiUvrDR288A as determined by UV-crosslinking of radioactive ATP without change in affinity for ssDNA. HiUvrD was found to exist predominantly as a monomer with small amounts (~ 2-3 %) of higher oligomers like dimers and tetramers in solution. Deletion of 48 amino acid residues from distal C-terminus of HiUvrD resulted in abrogation of the oligomeric species implicating C-terminus to be involved in protein oligomerization. Interplay of UvrD with MutL and MutS in H. influenzae, and its modulation by ATP To investigate the effects of H. influenzae MutS (HiMutS) and MutL (HiMutL) on the helicase activity of HiUvrD, two different nicked DNA substrates were generated- a homoduplex and a heteroduplex DNA with a GT mismatch. HiMutL and HiMutS did not exhibit any helicase activity on either homoduplex or heteroduplex DNA, and unwinding of these substrates was observed only in presence of HiUvrD. In the presence of HiMutL the helicase activity of HiUvrD was stimulated on both homoduplex and heteroduplex nicked substrates whereas no significant modulation of HiUvrD ATPase activity in presence of HiMutL was observed. A much higher stimulation of unwinding of heteroduplex DNA was obtained, in presence of increasing concentrations of HiMutS. With increasing concentrations of HiMutL a progressive increase in HiUvrD mediated unwinding of the radiolabeled DNA strand was observed, which was ~ 15-fold higher than unwinding by HiUvrD alone. To investigate the effect of ATP in the stimulation of HiUvrD by HiMutL, two mutants of HiMutL–E29A (E29 is involved in ATP hydrolysis in E. coli UvrD), and D58A (D58 is essential for ATP binding in E. coli UvrD) were generated. HiMutLE29A retained only ~ 30 % of the wild-type ATPase activity, which was completely abolished in HiMutLD58A. Similar to wild-type protein, HiMutLE29A was able to stimulate HiUvrD helicase activity whereas HiMutLD58A failed to stimulate this activity. This indicated that ATP-bound form of MutL was essential for stimulation and perhaps interaction with UvrD. SPR analysis was carried out to validate and quantitate the direct protein-protein interaction between HiUvrD and HiMutL in absence or in presence of ATP, AMPPNP, and ADP. In the presence of ATP as well as AMPPNP, almost ~ 10,000-fold increase in the affinity between HiMutL and HiUvrD was observed but the same was not the case in presence of ADP. This clearly suggested that ATP binding rather than its hydrolysis promotes the interaction of MutL with UvrD. The effect of HiMutS on MutL-stimulated DNA unwinding by HiUvrD was determined using a heteroduplex nicked DNA with a GT mismatch. Interestingly, in the presence of HiMutS ~ 20-fold activation of DNA unwinding was observed, which is higher than the stimulation by HiMutL alone. The role of ATP-hydrolysis by MutS in regulation of UvrD helicase was studied by replacing wild-type protein with HiMutSE696A in the helicase assays. HiMutSE696A failed to hydrolyze ATP but was able to bind ATP with the same affinity as the wild-type protein and interacted with heteroduplex DNA with ~ 8-fold reduced affinity as compared to wild-type MutS. Intriguingly, increasing concentrations of HiMutSE696A failed to stimulate HiUvrD helicase activity in presence of HiMutL indicating that ATP hydrolysis by HiMutS is essential for stimulation of HiUvrD helicase activity post MutH-nicking during MMR. SSB, an essential component of all DNA metabolism pathways, possibly functions to stabilize the ssDNA tract generated by UvrD and exonucleases during MMR. ATPase and helicase activities of HiUvrD were inhibited by the cognate SSB protein. This inhibition could be overcome by increasing the concentration of HiUvrD helicases thus, pointing out the fact that SSB and UvrD perhaps compete with each other for ssDNA substrate. Noticeably, MutL and MutS proteins could alleviate the inhibition of HiUvrD by HiSSB. Functional characterization of UvrD from H. pylori In H. pylori, UvrD has been reported to limit homologous recombination and DNA-damage induced genomic recombinations but the protein has not been functionally studied. UvrD from H. pylori strain 26695 (HpUvrD) was over-expressed and purified as an N-terminal (His)6-tagged protein, and its authenticity was confirmed by peptide mass fingerprint analysis. HpUvrD exhibited high affinity for ssDNA as compared to dsDNA as determined by electrophoretic mobility shift assays and SPR. In addition, HpUvrD was able to bind a number of branched DNA structures (splayed duplex > ssDNA > 3’-flap > 3’overhang > three-way junction = four-way junction > loop >>> nicked ≥ duplex) suggesting its role in different DNA processing pathways. HpUvrD exhibited a Mg2+ - dependent ssDNA-specific ATPase activity, and a 3’-5’ helicase activity. HpUvrD was able to unwind different branched DNA structures with 3’-ssDNA regions like splayed duplex, 3’-overhang and 3’-flap. Blunt-ended duplex, duplexes with nick and loop as well as three-way and four-way junctions were unwound with less efficiency. Interestingly, the helicase activity of HpUvrD was supported by GTP and dGTP to almost the same level as ATP and dATP, which is in stark contrast to other characterized UvrD proteins. Moreover, HpUvrD was able to hydrolyze GTP albeit with ~ 1.5-fold reduced rate as compared to ATP. However, motifs associated with GTP binding and hydrolysis were not found in HpUvrD and it is possible that GTP binds in the same site as ATP. To investigate this possibility, helicase assay was done in the presence of ATP together with different concentrations of GMP-PNP, which is a non-hydrolysable analog of GTP, and did not support HpUvrD helicase activity. With increasing concentrations of GMP-PNP, a progressive inhibition of DNA unwinding by HpUvrD was observed suggesting that GMP-PNP could compete with ATP for a common binding site within HpUvrD. Replacement of a highly conserved glutamate residue with gluatamine (E206Q) in Walker B motif of HpUvrD resulted in ~17-fold reduced ATPase activity, and abrogation of helicase activity as compared to the wild-type protein. HpUvrDE206Q was able to bind ssDNA and ATP with comparable affinities as the wild-type protein suggesting the role of E206 in ATP hydrolysis. Like HiUvrD, HpUvrD was found to exist predominantly as a monomer in solution together with the presence of small amounts of higher oligomeric species. However, unlike HiUvrD, deletion of distal C-terminal 63 amino acids in HpUvD did not abrogate the oligomeric species suggesting that additional regions of the protein may be involved in protein oligomerization. The ATPase and helicase activities of HpUvrD were inhibited by the cognate SSB protein, and this inhibition could be overcome by increasing HpUvrD concentrations again suggesting that both UvrD and SSB proteins compete for ssDNA substrate. To investigate the role of UvrD in the physiology of H. pylori, a knock-out of hpuvrD was constructed in H. pylori strain 26695 by insertion of chloramphenicol cassette in its open reading frame. The mutant H. pylori strain 26695 obtained after disruption of hpuvrD was extremely slow growing under the normal microaerophilic conditions compared to the wild-type strain. Growth defect of H. pylori strain 26695ΔhpuvrD highlights the importance of UvrD in H. pylori cellular processes and in vitro fitness. Characterization of H. influenzae RecJ and its interaction with SSB Among the four exonucleases involved in MMR pathway, RecJ is the only known nuclease that degrades single-stranded DNA with 5’ to 3’ polarity. RecJ exonuclease plays additional important roles in base-excision repair, repair of stalled replication forks, and recombination. RecJ exonuclease from H. influenzae (HiRecJ) is a 575 amino acid protein, which harbors the characteristic motifs conserved among RecJ homologs. Due to limited solubility of HiRecJ, the protein was purified as a fusion protein with maltose binding protein (MBP). The purified protein exhibited a Mg2+ or Mn2+- dependent, and a highly processive 5’ to 3’ exonuclease activity, which is specific for ssDNA. MBP did not affect the exonuclease activity of HiRecJ. The processivity of HiRecJ was determined as ~ 700 nucleotides per binding event, using a ssDNA substrate labelled internally with 3H and at its 5’-terminus with 32P. Cd2+ inhibited the Mg2+ - dependent exonuclease activity of RecJ, which could not be overcome by increasing Mg2+ concentration. Site-directed mutagenesis of highly conserved residues in HiRecJ- D77A, D156A and H157A abolished the enzymatic activity. Interestingly, HiRecJD77A was found to interact with ssDNA with a 10-fold higher affinity than wild-type protein suggesting that this conserved aspartate residue may function to coordinate the binding of metal ion or DNA to hydrolysis of DNA. E. coli HU protein inhibited the HiRecJ exonuclease activity in a concentration-dependent manner possibly due to sequestration of ssDNA, thus making it unavailable for HiRecJ. During MMR, ssDNA tracts generated by UvrD helicase activity are most probably stabilized by SSB and hence, the in vivo substrate for RecJ would be SSB-ssDNA complex. The exonuclease activity of HiRecJ was stimulated approximately 3-fold by H. influenzae SSB (HiSSB) protein. HiSSB was able to stimulate HiRecJ exonuclease activity on a ssDNA substrate, which formed either a very strong secondary structure or on a homopolymeric ssDNA substrate, which did not form any secondary structure, suggesting that HiRecJ exonuclease was stimulated independent of the ability to HiSSB to melt secondary structures and stabilize ssDNA. Significantly, steady-state-kinetic analysis clearly showed that HiSSB increases the affinity of HiRecJ for ssDNA. H. influenzae SSBΔC and T4 gene 32 protein, a SSB homolog from bacteriophage T4, failed to enhance the HiRecJ exonuclease activity suggesting a specific functional interaction between HiSSB and HiRecJ mediated by C-terminus tail of HiSSB. More importantly, HiRecJ was found to directly associate with its cognate SSB. The C-terminus of HiSSB protein was found to be essential for this interaction. To delineate the regions of HiRecJ that interact with HiSSB, different truncated forms of HiRecJ were generated in which regions external to conserved motifs required for exonuclease activity were deleted. Different deletion mutants of HiRecJ- RecJ∆N34, RecJ∆C76 and the core catalytic domain (which contains amino acid residues 35-498) were purified as fusion proteins with MBP. HiSSB was found to interact with all the truncated forms of HiRecJ suggesting that its core-catalytic domain harbors a site for interaction with SSB. Taken together, the results presented in this study lead to a better understanding of the structure-function relationships of the UvrD helicase and RecJ exonuclease. Importantly, they provide insights into the interplay between various proteins in DNA MMR pathway. Characterization of repair proteins that are involved in multiple genome fidelity pathways is of fundamental importance to understand repair processes, more so in pathogenic bacteria wherein they regulate mutation rates, which can alter the fitness and virulence of the pathogens. Publication Sharma R., and Rao, D.N. (2009). Orchestration of Haemophilus influenzae RecJ exonuclease by interaction with single-stranded DNA-binding protein. J. Mol. Biol., 385, 1375-1396. Sun, 31 Mar 2013 18:30:00 GMT http://hdl.handle.net/2005/1960 2013-03-31T18:30:00Z http://hdl.handle.net/2005/1965 Title: Understanding Heat Shock Protein 90 Biology And Exploring Its Potential As A Target Against Neglected Protozoan Diseases Authors: Roy, Nainita Abstract: 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. Wed, 03 Apr 2013 18:30:00 GMT http://hdl.handle.net/2005/1965 2013-04-03T18:30:00Z http://hdl.handle.net/2005/1341 Title: Nonstructural Protein, NSs Encoded By Groundnut Bud Necrosis Virus (Tomato) Is A Multifunctional Enzyme Authors: Bhushan, Lokesh Abstract: 1 Viruses are submicroscopic obligate parasites that depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. In the recent times, many new RNA viruses have evolved that possess the potential threat to plants and animals. One among them is Tospovirus (Family Bunyaviridae) which has severely affected the agricultural productivity in India. One of the Tospoviruses GBNV is a major challenge of crop production in south India. Tospoviruses shares several features such as morphology, genome structure and organization with members of other genera in the family Bunyaviridae. Virus particles are 80–120 nm in diameter. The genome includes three RNAs referred to as large (L), medium (M) and small (S). The L RNA is in negative-sense while the M and S RNAs are ambisense. The L RNA codes for the RNA-dependent RNA polymerase (RdRp), and the M RNA for the precursor of two glycoproteins (GN and GC) and a non-structural protein (NSm). The S RNA codes for the N protein and another non-structural protein (NSs). Tospovirus infection is an emerging threat for agricultural productivity in India. Therefore, biochemical and molecular characterization of these viruses is essential for developing various strategies for control of these diseases. 2 Present thesis deals with biochemical characterization of nonstructural protein, NSs of GBNV. 3 A review of literature on Tospovirus genome organization, replication, transcription, translation and assembly is presented in Chapter I. This chapter also includes the recent work on all the proteins encoded by the tospoviruses. 4 The objectives of the present study are as follows; a. Cloning, expression, purification and biophysical characterizations of rNSs. b. Analysis of its NTPase/dATPase activity c. Demonstration of nucleic acid 5’ phosphatase activity d. Characterization of nucleic acid unwinding activity of rNSs 5 The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in bacteria, protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology are described in chapter II 6 NSs of TSWV was shown to be suppressor of gene silencing (PTGS) in 2002. Since then there has been no further work on this protein. Till date neither in vitro nor in vivo study of NSs of any tospovirus has been carried out in detail. To gain insight into the biochemical function of rNSs, the NSS gene was cloned, overexpressed in E.coli and purified. The NSS gene, was cloned into pRSET-C vector. 7. Chapter 3 deals with cloning, overexpression, purification and biophysical characterization of GBNV NSs in terms of secondary structure analysis as well as its interaction with siRNA and ssRNA. The results provide the evidence that rNSs was successfully expressed in E.coli and purified (Fig. 3.1). Molecular mass of purified rNSs was confirmed by MALDI TOF, which gave the molecular mass of expected size 51.5 kDa (Fig. 3.2) Circular dichroism study revealed that rNSs has negative ellipticity peak at 215 and 223 nm typical of a globular protein. The protein had an emission maximum at 340 nm (Fig 3.3 B) when exited at 280 nm, which reflects that rNSs is well folded. Thermal melting study (Fig 3.3 C) showed rNSs had a reasonably high Tm (65°C). So overall, spectral study suggested that purified rNSs was soluble, well folded and thermally stable and could be used for further biochemical assay. The oligomeric status of the protein was determined by size exclusion chromatography to be trimeric (156 kDa, Fig 3.5). Purified rNSs was used to raise the polyclonal antibodies in rabbit. The antiserum could detect rNSs specific band only in IPTG induced sample not in uninduced sample (Fig 3.6). 50% binding was observed at 100 ng/ml of antigen showing that these antibodies were of high affinity (Fig 3.7 B). Further, the 50% binding was observed at 1:34000 dilution of the antiserum, which suggests that high titer antibodies against rNSs were obtained (Fig 3.7 A). 8 Further, the RNA binding property of rNSs was examined. Synthetic 21 bp siRNA and in vitro transcribed 100 nt ssRNA was used to analyze the RNA binding property of rNSs. Indeed rNSs was able to bind with 100 nt ssRNA (Fig 3.8 A) or 21 nt siRNA in a protein concentration dependent manner (Fig 3.8 B). The binding however did not require presence of divalent cation such as Mg 2+ (Fig 3.8 C). In order to understand the biological function of rNSs, its interaction with the structural protein, NP by ELISA was investigated. rNSs could interact with the NP protein (Fig 3.9) . Further 15 amino deletions from C terminus of NP did not affect its interaction with rNSs protein (Fig 3.9), which suggest that the C terminal 15 amino acid residues of NP are not essential for interaction with rNSs in vitro. 9. Sequence analysis of GBNV NSs revealed the presence of Walker motifs A (GxxxxGKT) and B (DExx) in its primary structure (Fig 4.2). The proteins that possess the Walker motifs A and B exhibit ATPase activity. Therefore, the purified rNSs was tested for its ability to hydrolyze ATP in the absence and presence of poly(A) (chapter IV). rNSs could hydrolyze [γ-32P] ATP in a concentration-dependent manner (Fig. 4.3 A). Further, ATPase activity was stimulated in presence of poly(A) (Fig. 4.3 B). Quantitative analysis of reaction product suggested that the reaction was linear in the presence of poly(A) upto 1.6 µg of rNSs (Fig. 4.3 C). 10. The product of ATP hydrolysis by rNSs had the same mobility as the phosphate released by RecoP51 ATPase, a positive control used in the assay. In contrast, another viral protein from the Cotton leaf curl virus, His tagged-AV2, purified in same way as rNSs, did not show the release of phosphate, suggesting that the activity was not due to the histidine tag present at the N-terminus of rNSs. Further, no release of phosphate could be seen when immunodepleted rNSs was used suggesting that the activity was inherent to the protein and was not due to bacterial contamination (Fig 4.3 lane 7). Time course analysis of ATPase activity revealed that the reaction is linear up to 25 mins (Fig 4.4). Further, pH profile was a typical bell shaped curve with a distinct pH optimum at pH 7.0 (Fig 4.5 A) and the temperature optimum was at 25 °C(Fig 4.5 B). Most of the known viral ATPases require the divalent cation for their activity. The rNSs exhibited the optimum ATPase activity between 2-2.5 mM of MgCl2. The reaction was inhibited by increasing concentration of EDTA demonstrating the requirement of Mg2+ for ATP hydrolysis (Fig. 4.7). Further, the ATPase activity of rNSs was inhibited by increasing concentrations of non-hydrolyzable analog of ATP (Fig. 4.8) and was not inhibited by AMP (Fig 4.9) suggesting that rNSs is not a nucleotidyl phosphatase and is a true ATPase. Limited proteolysis of rNSs suggested that core domain was 23 kDa in size and could catalyze ATP hydrolysis (Fig. 21 and 4.22). 11. Interestingly rNSs not only cleaved ATP rather it could hydrolyze all rNTPs as well as dATP (Fig 4.10). Kinetic parameters were determined for its enzymatic activity. Comparison of the kinetic constants of rNSs NTPase activity revealed little variation, suggesting that the rNSs has a broad substrate specificity (Fig 4.10- 4.15 and table 4.1). 12. To assess the role of amino acids in Walker motif A and B (Fig. 4.16) site specific mutants K189A and D159A were generated ( Fig 4.17) confirmed by sequencing, overexpressed in E.coli and purified (Fig. 4.18). Point mutation in Walker motif B (D159A) reduced the ATPase activity (Fig 4.19) where as point mutation in Walker motif A (K189A abolishes the activity (Fig 4.19). 13. Chapter V deals with the nucleic acid 5’ phosphatase activity of rNSs. Experimental evidence presented in this chapter clearly shows that rNSs can cleave the single phosphate from the ssDNA, ssRNA, dsRNA and dsDNA. Nucleic acid 5’ phosphatase activity of rNSs was inhibited by AMP and ATP (Fig 5.2 and Fig 5.3). Interestingly the K189A mutant rNSs was as active as wild type rNSs where as D159A mutant showed slightly reduced activity (Fig 5.7 C). 14. As mentioned earlier, rNSs was shown to possesses the RNA stimulated NTPase/dATPase activity, a hallmark of all known helicases. Therefore, its nucleic acid unwinding activity was examined using dsDNA and dsRNA as a substrate. rNSs was able to unwind the dsDNA as well as dsRNA in a ATP dependent manner (chapter VI, Fig. 6.1 and 6.5 respectively). ATP and Mg2+ are essential cofactors for the unwinding activity (Fig. 6.1). While the unwinding activity could be observed with ATP and to some extent with dATP, all other NTPs and dNTPs failed to support the helicase function of rNSs (Fig 6.2) Further experimental evidence suggested that rNSs is a bidirectional helicase (Fig. 6.3). D159A mutation in Walker motif B resulted in reduced helicase activity where as K189A mutation in walker Motif A completely abolished the DNA as well as RNA helicase activity of rNSs (Fig. 6.6 and Fig 6.7 respectively). Therefore, mutational analysis clearly suggests that helicase activity is an intrinsic property of rNSs. 15. In conclusion rNSs of GBNV is multifunctional enzyme. This is the first report on the demonstration that rNSs is an non canonical ATP dependent helicase in the Bunyaviridae family. In addition to being a suppressor of PTGS, NSs may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. This new research finding on NSs might pave way for further studies on its role in viral replication and transcription. Sun, 07 Aug 2011 18:30:00 GMT http://hdl.handle.net/2005/1341 2011-08-07T18:30:00Z http://hdl.handle.net/2005/1465 Title: Molecular Characterization Of Movement Protein Encoded By ORF-1 Of Sesbania Mosaic Virus (SeMV) Authors: Chowdhury, Soumya Roy Sun, 09 Oct 2011 18:30:00 GMT http://hdl.handle.net/2005/1465 2011-10-09T18:30:00Z