NF-кB/Rel family proteins regulate genes that are critical for many cellular processes including apoptosis, inflammation, immune response, as well as development. NF-кB/Rel proteins function as homodimers or heterodimers, which recognize specific DNA sequences within target promoters. I examined the activity of different Drosophila Rel-related proteins in modulating Drosophila immunity genes by expressing the Rel proteins in stably transfected cell lines. I also compared how different combinations of these transcriptional regulators control the activity of various immunity genes. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. Furthermore, expression of drosomycin and defensin is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively; whereas attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulates target gene expression. Another area of my research is to investigate the function of p38 MAP kinase (mitogen-activated protein kinase) in Drosophila immune response. In vertebrates, one of the responses evoked by the pro-inflammatory cytokines and lipopolysaccharide (LPS) is the initiation of a kinase cascade that leads to the phosphorylation of p38 MAP kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. Two genes that are highly homologous to the mammalian p38 MAP kinases were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of both Drosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression induced by LPS.

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins Polycystin-1 (PKD1) and Polycystin-2 (PKD2). The most proximal effects of polycystin mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The stress-activated mitogen-activated protein kinase (MAPK) pathway c-Jun N-terminal kinase (JNK) promotes proliferation in specific contexts and is activated in acute and chronic kidney disease. Previous work found evidence of JNK activation in cystic tissues (Le et al., 2005) and others showed that JNK signaling is activated by aberrant expression of PKD1 and PKD2 in cell culture (Arnould et al., 1998; Arnould et al., 1999; Parnell et al., 2002; Yu et al., 2010) but the contribution of JNK signaling to cystic disease in vivo has not been investigated. This body of work describes the use of conditional and germline deletion of Pkd2, Jnk1 and Jnk2 to model ADPKD and JNK signaling inhibition in juvenile and adult mice. Immunoblots and histological staining were used to measure JNK activation and evaluate the effect of JNK deletion on cystic disease. Results show that Pkd2 deletion activated JNK signaling in juvenile and adult mice. Reduction of JNK activity significantly reduced cystic burden in kidneys of juvenile Pkd2 mutant mice. This correlated with reduced tubule cell proliferation and reduced kidney fibrosis. The improvement in cystic phenotype was driven primarily by Jnk1 deletion rather than Jnk2. JNK signaling inhibition in adult Pkd2 mutants significantly reduced liver cysts when mice were aged six months. JNK inhibition reduces the severity of cystic disease caused by the loss of Pkd2 suggesting that the JNK pathway should be explored as a potential therapeutic target for ADPKD.

No description available.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Atherosclerosis preferentially develops in vascular regions of low or disturbed flow and high spatial gradients. Endothelial cells that line the vessel walls actively participate in translating mechanical stimuli, shear stress due to fluid flow, into intracellular signals to regulate cellular activities. Atherosclerosis is a chronic disease. During its development, a cascade of inflammatory signals alters the arterial endothelial homeostatic functions. Death-associated protein (DAP) kinase and its correlated pathway have been associated with cell apoptosis, turnover, and cytoskeleton remodeling in cellular networks, ultimately leading to changes in cell motility and vascular wall permeability. DAP-kinase is also highly regulated by inflammatory triggers such as TNF-α. This thesis investigates DAP-kinase modulation due to shear stress, and the role of DAP-kinase activity in endothelial responses toward applied shear stress. Using bovine aortic endothelial cells (BAEC), DAP-kinase expression is demonstrated in both sheared (10 dynes/cm2) and static conditions. Overall DAPK expression increased with extended shearing, while the presence of phosphorylated DAPK decreased with applied shear stress, as demonstrated in Western blot analysis. In correlation, DAPK RNA expression profiles were explored to understand pre-translational behavior and to understand just how shear stress influences DAPK expression over time. There is a temporal increase in DAPK mRNA, occurring at earlier time points when compared to DAPK protein expression, displaying the precedence of mRNA expression leading to increased translation into protein. From our apoptosis assay results, shear stress reduces apoptotic and late stage/necrotic cell fractions. The exposure of shear stress potentially plays a role in inhibiting apoptosis activation and TNF-α induced death cascade. Overall, the apoptosis activity influenced by shear further exhibits a possible connection between shear stress and apoptosis inhibition. The shear stress ultimately decreases overall apoptosis, while DAPK expression is increased. Therefore, DAPK may have a function in other possible mechano-transduction cascades, when endothelial cells are exposed to constant shear. Our data suggests shear stress modulation of DAP-kinase expression and activity, and the potential crosstalk of mechano-transduction and DAPK/apoptosis pathway, may lead to further understanding the responsibility of DAPK in endothelial cell function.

Phosphorylation-dependent relief of Rad inhibition of cardiac Caᵥ1.2 channels underlies β-adrenergic increase in heart contraction essential for the fight-or-flight response. Prevailing evidence outline 4 steps involved in PKA-dependent relief of Caᵥ1.2 inhibition by Rad: basally, Rad inhibits Caᵥ1.2 by binding Caᵥβ and the plasma membrane using the G-domain and C-terminus, respectively (step 0), PKA-dependent phosphorylation of Ser residues in Rad C-terminus disengages Rad from the plasma membrane (step 1) and decreases affinity for Caᵥβ (step 2), potentially leading to Rad loss from Caᵥ1.2 nanodomain (step 3). It is unclear which steps and Rad structural determinants are necessary and sufficient for PKA regulation of CaV channels and the mechanism linking steps 1 and 2 is not entirely understood. Moreover, there is an apparent Rad-concentration-dependence to Caᵥ1.2 regulation wherein PKA activation is unable to overcome over-expressed Rad inhibition of the channel. The basis of this effect is unknown and constitutes a significant gap in our complete understanding of convergent regulation of Caᵥ channels, by Rad and PKA. We developed a systematic protein engineering-based approach to dissect the distinct steps and determinants involved in PKA modulation of Rad-inhibited Caᵥ channels. Fusing Rad C-terminus to Caᵥβ₃ generated β3-CT which was tethered to the plasma membrane when expressed alone in HEK293 cells and yielded constitutively inhibited channels when co-expressed with CaV2.2. Unexpectedly, PKA activation with forskolin further deepened inhibition of Caᵥ2.2 currents despite being sufficient to release β₃-CT from the plasma membrane. Phosphomimetic mutations in β₃-CT 6SD yielded deeply inhibited Caᵥ2.2 currents that were not further affected by forskolin. Two CaVβ-binding nanobodies fused to Rad C-terminus, F3-CT and B11-CT, were membrane-targeted yet yielded Caᵥ2.2 currents that were not basally inhibited and decreased by forskolin. Over-expressing wildtype Rad C-terminus (WTCT) by itself with Caᵥ1.2 produced basally inhibited channels that were further reduced by forskolin and co-expression of Caᵥ1.2 with a phosphomimetic Rad C-terminus (CTD) also produced constitutively inhibited channels. Truncated Rad lacking the C-terminus (Rad[1-276]) displayed low affinity to Caᵥβ, discounting a direct role for phosphorylated Rad C-terminus in linking steps 1 and 2. Fusing the protein kinase C C1 domain to Rad[1-276] yielded Rad₂₇₆-C1 which was cytosolic and displayed low affinity to Caᵥβ. Exposure to PdBu recruited Rad₂₇₆-C1 to the plasma membrane, increased affinity for Caᵥβ, and concomitantly inhibited Caᵥ1.2 currents. These results reveal that all 4 steps are necessary for PKA regulation of Caᵥ channels, membrane association regulates Rad affinity for CaVβ, and the Rad G-domain and C-terminus are replaceable with modular units that mimic their function. Our findings deepen understanding of PKA modulation of Caᵥ channels and provide new insights for developing chemo-genetic Caᵥ channel regulators.

The availability of completely sequenced genomes for a number of organisms provides an opportunity to understand the molecular basis of physiology, metabolism, regulation and evolution of these organisms. Significant understanding of the complexity of organisms can be obtained from the functional characterization of repertoire of proteins encoded in their genomes. Computational approaches for recognition of function of proteins of unknown function encoded in genomes often rely on ability to detect well characterized homologues. Homology searches based on pair-wise sequence comparisons can reliably detect homologues with sequence identity more than 30%. However, detecting homologues characterized by sequence identity below 30% is difficult using these methods. Distant homology relationship can be established using profiles or position specific scoring matrices, which encapsulate information about structurally and functionally conserved residues. These conserved residues imply high constraints at a particular amino acid residue site due to their involvement in structural stability, enzymatic activity, ligand binding, protein folding or protein–protein interactions. In addition, information on three dimensional structures of proteins also aid in detection of remote homologues, as tertiary structures of proteins are conserved better than the primary structures of proteins. The gross objective of the work reported in this thesis is to employ various sensitive remote homology detection methods to recognize relevant functional information of proteins encoded mainly in pathogenic organisms. Since proteins do not work in isolation in a cell, it has become essential to understand the in vivo context of functions of proteins. For this purpose, it is essential to have an understanding of all molecules that interact with a particular protein. Thus, another major area of bioinformatics has been to integrate protein-protein interaction information to enable better understanding of context of functional events. Protein-protein interaction analysis for host-pathogen can lead to useful insight into mode of pathogenesis and subsequent consequences in host cell. Chapters 2-6 of the thesis discuss the sequence and structural characteristics along with remote evolutionary relationships and functional implications of uncharacterized proteins encoded in genomes of following pathogens: Helicobacter pylori, Plasmodium falciparum and Leishmania donovani. The Chapters 6-8 discuss mainly various sequence, structural and functional aspects of protein kinases encoded in genomes of various prokaryotes and viruses. Chapter 1 discusses background information and literature survey in the areas of homology detection and prediction of protein-protein interactions. The growth of genomic data and need for processing genomic data to infer context of various functional events have been highlighted. Different approaches to recognize functions of proteins (experimental as well as computational) have been discussed. Various experimental and computational approaches to detect/predict protein-protein interactions have been mentioned. Chapter 2 discusses recognition of non-trivial remote homology relationships involving proteins of Helicobacter pylori and their implications for function recognition. H. pylori is microaerophilic, Gram negative bacterial pathogen. It colonizes human gastric mucosa and is a causative agent of gastroduodenal disease. The pathogen infects about 50% of the human population. It can lead to development of Mucosa-associated lymphoid tissue lymphoma. About 10% of the infected population develop gastric or duodenal ulcer and approximately 1% develop gastric cancer. H. pylori has been classified as class I carcinogen by WHO. Pathogen is characterized by type IV secretion system. The complete genomic sequences of three widely studied strains including 26695, J99 and HPAG1 of Helicobacter pylori are available. According to the genome analysis, the number of predicted open reading frames in strain 26695, J99 and HPAG1 are 1590, 1495 and 1536 respectively. Out of predicted H. pylori proteins from 26695, J99 and HPAG1 strains, numbers of proteins with no functional domain assignments in Pfam database (Protein family database) are 453, 357 and 400 respectively. There are proteins in different strains of H. pylori genomes where one part of the protein is associated with at least one protein domain of known function and hence preliminary indication of their functions is available whereas rest of the region is not associated with any function. There are 772, 803 and 790 such segments in proteins from strains 26695, J99 and HPAG1 respectively with at least 45 residues with no functional assignment currently available. Sensitive remote homology detection methods have been employed to establish relationships for 294 amino acid sequences and results have been grouped into 4 categories. Results of homology detection have been further confirmed by studying conservation of amino acid residues which are important for functioning of the proteins concerned. (i) Remote relationship has been established involving protein domain families for which no bonafide member is currently known in H. pylori. For example: DNA binding protein domain (Kor_B) has been assigned to a H. pylori protein at sequence identity of 20%. Study involving secondary structure prediction and conservation of amino acid residues confirms the results of homology detection methods. (ii) Remote relationship has been established involving H. pylori hypothetical proteins and protein domain families, for which paralogous members are present in Helicobacter pylori. For example, Cytochrome_C, an electron transfer protein domain could be associated with a Helicobacter pylori protein sequence which shows a sequence identity of 14% with sequences of bonafide cytochrome C. (iii) “Missing” metabolic proteins of H. pylori have also been recognized. For example, Aspartoacylase (EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartic acid to produce acetate and L-aspartate. This enzyme in aspartate metabolism pathway has not been reported so far from H. pylori. A remote evolutionary relationship between a H. pylori protein and Aspartoacylase domain has been established at sequence identity of 17% thus filling the gap in this metabolic pathway in the pathogen. (iv) New functional assignments for domains in H. pylori sequences with prior assignment of domains for the rest of the sequences have been made. For example, DNA methylase domain has been assigned to C-terminal region of H. pylori protein which already had Helicase domain assigned to the N-terminal region of the protein. All these information should open avenues for further probing by carrying out experiments which will impact the design of inhibitor against this pathogen and will result in better understanding of pathogenesis of this organism in human. Chapter 3 describes prediction of protein–protein interactions between Helicobacter pylori and the human host. A lack of information on protein-protein interactions at the host-pathogen interface is impeding the understanding of the pathogenesis process. A recently developed, homology search-based method to predict protein-protein interactions is applied to the gastric pathogen, Helicobacter pylori to predict the interactions between proteins of H. pylori and human proteins in vitro. Many of the predicted interactions could potentially occur between the pathogen and its human host during pathogenesis as we focused mainly on the H. pylori proteins that have a transmembrane region or are encoded in the pathogenic island and those which are known to be secreted into the human host. By applying the homology search approach to protein-protein interaction databases DIP and iPfam, in vitro interactions for a total of 623 H. pylori proteins with 6559 human proteins could be predicted. The predicted interactions include 549 hypothetical proteins of as yet unknown function encoded in the H. pylori genome and 13 experimentally verified secreted proteins. A total of 833 interactions involving the extracellular domains of transmembrane proteins of H. pylori could be predicted. Structural analysis of some of the examples reveals that the predicted interactions are consistent with the structural compatibility of binding partners. Various probable interactions with discernible biological relevance are discussed in this chapter. For example, interaction between CFTR protein (NP_000483) and multidrug resistance protein (HP1206) has been predicted. The structure of the CFTR intracellular domain is known in the homomeric form and consists of five AAA transport domains in tandem (PDB code 1XMI). Out of the five identical subunits, two subunits (the B chain and the E chain in the PDB structure) have been selected. The structure of multidrug resistance protein of the pathogen based on the B chain (sequence identity 32%) of the template has been modeled. This exercise suggests that interface residues in the model are congenial for interaction. This makes the structural complex feasible in in vitro conditions and suggests that the pathogen protein may compete for occupancy with the host protein. Chapter 4 describes recognition of Plasmodium-specific protein domain families and their roles in Plasmodium falciparum life cycle. Malaria in humans is caused by the parasites of intracellular, eukaryotic protozoan of apicomplexan nature belonging to the genus Plasmodium. Out of five species of Plasmodium, namely, P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi which infects human, P. falciparum causes lethal infection. P. falciparum proteins have diverged extensively during the course of evolution. Pathogen genome is rich in A+T composition which larger than the homologous proteins from other organisms due to presence of low complexity regions. Organism specific families are important as they play roles in peculiar life style of an organism. If the organism is a pathogen, then these family members may play roles in pathogenesis. Inhibiting these specific proteins is unlikely to interfere with host system as no homolog may be present in host. In the present work we identify Plasmodium specific protein families and their role in different stages of life cycle of the pathogen. A total of 5086 amino acid sequences (full length sequences/fragments of proteins) show homology only with amino acid sequences from Plasmodium organisms and hence are Plasmodium-specific. These Plasmodium-specific amino acid sequences cluster into 106 Plasmodium-specific families (≥2 members per family). 14 Plasmodium-specific protein domain families with known physico-chemical properties are observed. These Plasmodium-specific protein domain families are involved in various important functions such as rosetting and sequestering of infected erythrocytes, binding to surface of host cell and invasion process in life cycle of pathogen. Also, 89 new Plasmodium-specific protein domain families have been recognized. Analysis of various aspects of members of Plasmodium-specific proteins domain families such as their potential to target apicoplast, protein-protein interaction, expression profile and domain organization has been performed to derive relevant information about function. New Plasmodium specific domain families for which no function can be associated could provide some insight into much diverged Plasmodium species. These proteins may play role in parasite-specific life style. Experimental work on these Plasmodium-specific proteins might fill the gaps of less understood physiology of this parasite. Chapter 5 presents genome-wide compilation of low complexity regions (LCR) in proteins. An indepth analysis of the nature, structure, and functional role of the proteins containing low complexity regions in Plasmodium falciparum, was undertaken given the high prevalence of LCRs in the proteome of this organism. Low complexity regions and repeat patterns have been recognized in proteins encoded in 986 genomes (68 archaea, 896 prokaryotes and 22 eukaryotes). Low complexity regions have been classified into following three categories: a) Composition of LCRs: (i) LCRs can be stretches of homo amino acid residues (ii) LCRs can be stretches of more than one amino acid residue type b) Periodicity of amino acids in LCRs: Certain amino acid residues can be observed at certain specific periodicity in proteins. c) Repeat patterns: Certain motif of amino acid residues are repeated in protein. 850 Plasmodium falciparum proteins are observed to have at least one repeat pattern where the repeating unit is at least 5 amino acid residues long. Statistical analysis on single amino acid residue repeats indicate that occurrence of stretches of homo amino acid residues is not a random event. Studies on recognition of functions, protein protein interactions and organization of tethered domain(s) in proteins containing LCR suggest that these proteins are part of variety of functional events such as signal transduction, enzymatic processes, cell differentiation, pyrimidine biosynthesis, fatty acid biosynthesis and chromosomal replication. Representations of low complexity regions of Plasmodium falciparum in protein data bank suggest that LCRs can take conformation of regular secondary structure (apart from disordered regions) in 3-D structures of proteins. Chapter 6 describes sequence analysis, structural modeling and evolutionary studies of Leishmania donovani hypusine pathway enzymes. Leishmania is an eukaryotic kinetoplastid protozoan parasite which causes leishmaniasis in humans. Hypusine is a non standard polyaminederived amino acid Nε-(4-amino-2-hydroxybutyl) lysine and is named after its two structural components, hydroxyputrescine and lysine. The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein containing hypusine. Synthesis of hypusine is critical for the function of elF5A and is essential for eukaryotic cell proliferation and survival. Formation of hypusine is the result of a two step post-translational modification process involving enzymes (i) deoxyhypusine synthase (DHS) (ii) deoxyhypusine hydroxylase (DOHH). DHS, the first enzyme involved in hypusine pathway catalyzes the NAD-dependent transfer of the butylamino moiety of spermidine (substrate) to the ε-amino group of a specific lysine residue of eIF5A precursor and generates deoxyhypusine containing intermediate. DOHH, the second enzyme in same pathway catalyzes the hydroxylation of deoxyhypusine-containing intermediate, generating hypusine-containing mature eIF5A. Two putative deoxyhypusine synthase (DHS) sequences DHS34 and DHS20 have been identified in Leishmania donovani, by Professor Madhubala and coworkers (Jawaharlal Nehru University, New Delhi) with whom the work embodied in this chapter was done in collaboration. Detailed comparison of DHS34 sequence from Leishmania with human DHS protein indicated conservation of functionally important residues. 3D structural modeling studies of protein suggested that residues around the active site were absolutely conserved. NAD binding regions are located spatially closer, however, one NAD binding region was observed in a large (225 amino acid residues long) insertion. Based on these observations, DHS34 was predicted to have enzymatic activity. Experimental studies done by our collaborators confirmed preliminary results of computational analysis. Based on sequence and structural analysis of DHS20 and DOHH proteins, DHS20 and DOHH were proposed to be catalytically inactive and active respectively. Experimental studies on these proteins supported results of computational analysis. Deoxyhypusine synthase (DHS) and Deoxyhypusine hydroxylase (DOHH) are key proteins conserved in the hypusine synthesis pathways of eukaryotes. Because they are highly conserved, they could be coevolving. Comparison of the genetic distance matrices of DHS and DOHH proteins reveals that their evolutionary rates are better correlated when compared to the rate of an unrelated protein such as Cytochrome C. This indicates that they are coevolving, further serving as an indicator that, even non-interacting proteins that are functionally coupled, experience correlated evolution. However, this correlation does not extend to their tree topologies. Chapter 7 provides a classification scheme for protein kinases encoded in genomes of prokaryotic organisms. Overwhelming majority of the Ser/Thr protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Ser/Thr protein kinases prokaryotic Ser/Thr protein kinases. Traditional sequence alignment and phylogenetic approaches have been used to identify and classify prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence databases, it is recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly, subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, it was also possible to identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a degree of complexity in protein-protein interactions and the signaling pathways in these microbes. Chapter 8 focuses on recognition, classification of protein kinases encoded in genomes of viruses and their implications in various functions and diseases. Protein kinases encoded by viral genomes play a major role in infection, replication and survival of viruses. Using traditional sequence homology detection tools, sequence alignment methods and phylogenetic approaches, protein kinases were recognized. 646123 protein sequences from 35799 viral genomes (including strains) have been used in this analysis. Protein kinases are identified using a combination of profile-based search methods such as PSI-BLAST, RPS-BLAST and HMMER approaches. Based upon sequence similarity over the length of catalytic kinase domains, 479 protein kinase domains recognized in 244 viral genomes have been clustered into 46 subfamilies with minimum sequence identity of 35% within a subfamily. Viral protein kinases are encoded in genomes of retro-transcribing viruses or viruses which possess double stranded DNA as genetic material. Based on the available functional information present for one or more members of a subfamily, a putative function has been assigned to other members of the subfamily. Information regarding interaction of viral protein kinases with viral/host protein has also been considered for enhancing understanding of function of kinases in a subfamily. Out of 46 subfamilies, 14 subfamilies are characterized by various functions. Kinases belonging to UL97, US69, UL13 and BGLF subfamilies are virus specific. For 7 subfamilies, nearest neighbors are from well characterized eukaryotic protein kinase groups such as AGC, CAMK and CDK. Out of 25 new uncharacterized subfamilies observed in this analysis, 13 subfamilies are virus specific. Different subfamilies have been characterized by various functions which are crucial for viral infection such as synthesis of structural unit, replication of genetic material, modification of cellular components, alteration in host immune system, competing with cellular protein for efficient usage of host machinery. Also, many viral kinases share very high sequence identity (~97%) with their eukaryotic counterpart and represent disease state. For example, a protein kinase encoded in Avian erythroblastosis virus shares 97% sequence identity with catalytic domain of human epidermal growth factor receptor tyrosine kinase. Leucine at position 861 in human protein is substituted by Gln in cancer conditions; the viral protein kinase sequence possesses Gln at corresponding position and thus represents disease state. Chapter 9 provides study of dependency on the ability of 3-D structural features of comparative models and crystal structures of inactive forms of enzymes to predict enzymes by considering protein kinases as case study. With the advent of structural genomics initiatives, there is a surge in the number of proteins with 3-D structural information even before functional features are understood on many of these proteins. One of the useful annotations of a protein is the demarcation of a protein into an enzyme or non-enzyme solely from the knowledge of 3-D structure. This is facilitated by the identification of active sites and ligand binding sites in a protein. In this work, which was carried out in collaboration with Dr Jim Warwicker of Manchester University, UK, an approach developed by Warwicker and coworkers has been used. In the 3D structure of proteins, the largest clefts are generally considered to be ligand binding sites. This feature along with other sequence alignment independent properties such as residue preferences, fraction of surface residues and secondary structure elements have been considered to differentiate enzymes from non-enzymes. Electrostatic potential at the active site is one of the key properties utilized in this respect. Active sites in enzymes are generally associated with ionizable groups which can take part in catalysis. In addition to the feature of large clefts in enzymes, active site residues are in buried environments and show larger deviation in pKa values than surface residues. The method proposed by Warwicker and co-workers distinguish proteins in to enzymes and non-enzymes considering the electrostatic features at clefts along with the sequence profile of the protein concerned. Conformation of the inactive state of an enzyme is not congenial to the catalytic function. In an ideal situation, a method should be capable of predicting an enzyme irrespective of whether determined structure corresponds to active or inactive state. Peak potential values have been calculated by using Warwicker program for a set of 15 protein kinases for which 3-D structures are present in active as well in inactive conformations. Comparison of peak potential values calculated for active and inactive conformations suggests that algorithm can differentiate between active and inactive conformations as value for active conformations are generally higher than corresponding values for inactive conformations. However, the peak potential values are high enough for even the inactive conformations to be predicted as enzyme. Peak potential values calculated for generated homology models of protein kinases (for which crystal structures are already available) at different sequence identities with template sequences predict protein kinases as enzymes and their peak potential values are comparable to corresponding values for X-ray structures. This suggests that proteins for which there are no crystal or NMR structures yet available and no good template with high sequence identity are present, peak potential values for models generated at low sequence identity can still give insight into probable function of protein as an enzyme. The enzyme/non-enzyme prediction algorithm was also found to be useful in confirming enzyme functionality using 3-D models of putative viral kinases. Initially, putative function of kinase has been assigned to these viral proteins based solely upon their sequence characteristics such as presence of residues/motifs which are important for activity of the protein. The enzyme recognition method which is not directly sensitive to these motifs confirmed that all the analyzed putative viral kinases are enzymes. Chapter 10 presents conclusions of work embodied in the entire thesis. Very briefly, various computational approaches have been used to analyze and understand structural and functional properties of repertoire of proteins of pathogenic organisms. Analysis of uncharacterized protein domain families has helped to understand the functional implications of constituent proteins. Experimental validation of these results can further facilitate unraveling of functional aspects of proteins encoded in various pathogenic organisms. Apart from studies embodied in the thesis, author has been involved in two other studies, which are provided as appendices. Appendix 1 describes comparison of substitution pattern of amino acid residues of protein encoded in P. falciparum genome with substitution pattern of corresponding homologous proteins from non-Plasmodium organisms. Salient differences have been highlighted. Appendix 2 discusses study of bacterial tyrosine kinases with an objective of recognition of all putative protein tyrosine kinases in E. coli. Computational study suggests that protein SopA can be a potential tyrosine kinase and this conclusion is being tested experimentally in collaborator’s laboratory.

The c-Jun NH2-terminal kinase (JNK) group and the p38 group of mitogen-activated protein kinases (MAPK) are stress-activated protein kinases that regulate cell proliferation, differentiation, development, and apoptosis. These protein kinases are involved in a signal transduction cascade that includes a MAP kinase (MAPK), a MAP kinase kinase (MAP2K), and a MAP kinase kinase kinase (MAP3K). MAPK are phosphorylated and activated by the MAP2K, which are phosphorylated and activated by various MAP3K. The work presented in this dissertation focuses on understanding the regulation and function of the JNK and p38 MAPK pathways. Two different strategies were utilized. First, I used molecular and biochemical techniques to examine how MAP2K and MAP3K mediate signaling specificity and to define their role in the MAPK pathway. Second, I used gene targeted disruption studies to determine the in vivo role ofMAP2K and MAP3K in MAPK activation. I specifically used these approaches to examine: (1) docking interactions between p38 MAPK and MAP2K [MKK3 and MKK6 (Chapter II)]; (2) the differential activation of p38 MAPK by MAP2K [MKK3, MKK4, and MKK6 (Chapter III)]; and (3) the selective involvement of the mixed lineage kinase (MLK) group of MAP3K in JNK and p38 MAPK activation (Chapter IV and Appendix). In addition, I analyzed the role of the MKK3 and MKK6 MAP2K in cell proliferation and the role of the MLK MAP3K in adipocyte differentiation (Chapter III and Chapter IV). Together, these data provide insight into the regulation and function of the stress-activated MAPK signal transduction pathways.

Plants are constantly exposed to pathogens and various environmental stresses, such as cold, salinity and drought. Plants normally respond rapidly to these biotic and abiotic stresses. Efficient perception of biotic and abiotic stresses and cell programmed signaling mechanisms for appropriate responses are important for growth and survival of plants. Calcium is an important second messenger in signaling pathways that respond to environmental stresses, pathogen attack as well as hormonal stimuli (For review, see DeFalco et al., 2010; Reddy and Reddy, 2004; Sanders et al., 2002). The transient increase of cytosolic free calcium concentration has been shown in a variety of external signals (Reddy, 2001), which in turn triggers many signal transduction pathways leading to a variety of cellular responses (Bush, 1995). Any calcium mediated signal transduction process involves generation of signal-specific calcium signature in the cytosol (Scrase-Field and Knight, 2003). These changes in cytosolic calcium level or ‘calcium signatures’ are sensed by the specific group of proteins called the ‘calcium sensors’. Different calcium sensors recognize specific calcium signatures and transduce them into downstream effects, including altered protein phosphorylation and gene expression patterns. In plants the protein kinases are a large and differentiated group of calcium sensors. After analyzing 1264 protein kinase sequences, a superfamily of protein kinase called CDPK/SnRK family of protein kinase were defined (Hrabak et al., 2003). CDPK/SnRK family of protein kinases encompasses five subfamilies viz., calcium-dependant protein kinases, (CPKs), calcium/calmodulin dependant protein kinases (CCaMKs), calmodulin-dependant protein kinases (CaMKs), CPKrelated kinases (CRKs), and SnF1 related kinase 3 (SnRK3) and are regulated by calcium directly or indirectly. Among these, in plants, calcium-dependant protein kinases (CPKs) are predominant calcium sensors, which are shown to be involved in myriads of physiological responses. They are Ser/Thr family of protein kinases typically made up of five domains with an Nterminal variable domain followed by catalytic protein kinase domain, an autoinhibitory/ junction domain, a regulatory calmodulin-like domain (CaMLD) and a Cterminal domain of variable length. The CPKs are unique due to the presence of CaMLD which couples the calcium sensor directly to its responder (kinase domain). Although CPKs are highly conserved, there are several features that distinguish different members of the plant CDPK family. In an attempt to investigate the role of a CPK isoform, in the present work we bring out the results and inferences on isolation and characterization of a novel cDNA encoding a calcium-dependant protein kinase isoform ScCPK1 from Swainsonacanescens, a pharmaceutically important Australian herb known to produce an anticancer drug, swainsonine. Initially, we have cloned an 800 bp partial CPK cDNA from S. canescens by reverse transcription polymerase chain reaction (RTPCR) using degenerate oligonucleotide primers designed based on conserved regions of the other known CPKs. A 2.1 kb full length CPK was obtained using 5` and 3` RACE which was designated as ScCPK1. An open reading frame (ORF) of 1659 bp was detected that encodes a protein of 552 amino acids with a calculated molecular mass of 61.8 kDa. Comparison of the deduced amino acid sequence of ScCPK1 with sequences of other CPKs revealed the highest identity (>90%) to Glycine max and Vigna radiate CPKs. As described for other CPKs, ScCPK1 has a long variable domain (88 aa), an auto-inhibitory domain (31 aa) and a C-terminal calmodulin domain (145 aa) containing four EF-hand calcium binding motifs, which is found in many CPKs. Phylogenetic tree analysis showed that ScCPK1 was closely related to StCPK4 , CmCPK1 and CmCPK2. The entire coding region of ScCPK1 was cloned into pRSETA expression vector and expressed as fusion protein in E.coli. The recombinant ScCPK1 protein was purified to homogeneity by NiNTA affinity chromatography. The recombinant purified ScCPK1 was catalytically active in a calcium-dependent manner. The recombinant ScCPK1 phosphorylated itself and histone IIIS as substrate only in the presence of Ca2+. Phosphoaminoacid analyses showed that ScCPK1 phosphorylates serine and threonine residues of histone IIIS and its autophosphorylation also occurs on serine and threonine residues. ScCPK1 has a pH and temperature optima of 7.5 and 37 °C, respectively. It showed high affinity to histone III-S with a Km of 4.8 µM and had a Vmax of 4.700 pmoles of γ32P incorporation/min/mg at saturating substrate concentrations. The ScCPK1 is ~100fold active and showed 10fold higher affinities to histone III-S than CaCPK1 and CaCPK2, CPKs which were characterized from Cicer arietinum previously in our laboratory (Prakash and Jayabaskaran, 2006). From literature it is known that many CPKs are activated or inhibited by metal ions. (PutnamEvans etal., 1990; Anil and Rao, 2001). The influence of Na+ and Mg2+on the in vitro substrate phosphorylation activity of the recombinant ScCPK1 was tested in this work. Addition of NaCl strongly inhibited ScCPK1 activity. The inhibition of substrate phosphorylation activity by salt implies ionic interactions between the positively charged substrate and the enzyme’s active site. The optimum concentration of Mg2+ for ScCPK1 substrate phosphorylation activity was found to be 810 mM, similar to CaCPK1 and CaCPK2 (Prakash and Jayabaskaran, 2006). However, the activity was inhibited above 10 mM Mg2+suggesting the disruption of ionic interactions between the enzyme and the substrate. The kinase and autophosphorylation activities of the recombinant ScCPK1 were calmodulin independent and sensitive to CaM antagonists’ calmidazolium and W7 (N(6aminohexyl)5chloronaphthalene sulphonate). This indicates that the activation was supported by calmodulin-like domain, which is typical of CPK family. Farmer and Choi (1999), showed that DcCPK1 activity was inhibited by polyamines vizspermine and spermidine, and polylysine. We found that substrate phosphorylation activity of ScCPK1 was inhibited by polyLLysine with an IC50 of 8 M but not the polyamines, spermine and spermidine. An interesting aspect that makes CPKs attractive for research is their functional similarity to mammalian PKCs. There are no structural PKC analogues found in plant genomic data. Similar to PKCs, CPKs are regulated by intracellular Ca2+ signals. There is also experimental evidence that some of the CPKs are additionally activated by phospholipids (Farmer and Choi, 1999; Szczegielniak etal., 2000). We investigated the effects of lipid molecules on the activity of ScCPK1. Phosphorylation of histone IIIS by ScCPK1 was stimulated by phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol in the presence of Ca2+, where as lysophosphatidylcholine, phosphatidylcholine and phosphatidic acid did not increase the enzyme activity. Our data that shows interaction of ScCPK1 with phospholipids supports the idea that this protein kinase could be associated with the membrane. The work from Farmer and Choi (1999), with DcCPK1 suggested that some of the PKClike activities observed in plants may be attributed to CPKs. They also demonstrated that DcCPK1 phosphorylated PKC pseudosubstrate peptide and also was sensitive to staurosporine inhibition. However, the protein kinase inhibitor, staurosporine inhibited the substrate phosphorylation activity of ScCPK1 completely with an IC50 value of 700 nM invitro. But PKC inhibitor PMA was less effective, inhibiting the substrate phosphorylation activity of ScCPK1 to a maximum of 50%, but at a very high concentration (200 nM). Our data suggests that ScCPK1 may not have any features attributable to PKC. We investigated subcellular localization of the ScCPK1. To gain a better understanding of the subcellular localization of the ScCPK1, we generated GFP fusion protein of ScCPK1 and transiently expressed it in Agrobacterium-mediated transformed tobacco BY2 cells. Analysis of the GFP expression patterns in transformed tobacco BY2 cells revealed ScCPK1 localization in the plasma membrane of the transformed tobacco BY2 cells despite lacking consensus myristoylation and palmitoylation motifs (as per in silico analyses). Taking together, our data have demonstrated that ScCPK1 is a Ser/Thr protein kinase and its sub-cellular localization studies revealed that it is localized to plasma membrane. We propose that ScCPK1 is a key component of one or more signaling pathways and plays vital roles in plant development, responses to environmental stimuli and/ or in secondary metabolite biosynthetic gene expression. The involvement of the ScCPK1 as a component of signaling pathways warrants further studies.