Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization

Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core.     

Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death

Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to such injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well.     

The role of metal ions in substrate recognition and stability of concanavalin A: a molecular dynamics study

The binding of carbohydrate substrates to concanavalin A (Canavalia ensiformis agglutinin (ConA)) is essential for its interaction with various glycoproteins. Even though metal ions are known to control the sugar binding ability of legume lectins, the interplay between sugar and metal ion binding to ConA has not been elucidated in a detailed manner at the atomic level. We have carried out long, explicit solvent molecular dynamics simulations for tetrameric, dimeric, and monomeric forms of ConA in both the presence and absence of trimannoside and metal ions. Detailed analyses of these trajectories for various oligomeric forms under different environmental conditions have revealed dynamic conformational changes associated with the demetalization of ConA. We found that demetalization of ConA leads to large conformational changes in the ion binding loop, with some of the loop residues moving as far as 17 Å with respect to their positions in the native trimannoside and metal ion-bound crystal structure. However, the β-sheet core of the protein remains relatively unperturbed. In addition, the high mobility of the ion binding loop results in drifting of the substrates in the absence of bound metal ions. These simulations provide a theoretical rationale for previous experimental observations regarding the abolition of the sugar binding ability upon demetalization. We also found that the amino acid stretches of ConA, having high B-factor values in the crystal structure, show relatively greater mobility in the simulations. The overall agreement of the results of our simulations with various experimental studies suggests that the force field parameters and length of simulations used in our study are adequate to mimic the dynamic structural changes in the ConA protein.     

A p53-like protein from a freshwater mollusc Lamellidens corrianus

p53 is the most frequently mutated protein in human cancers and the accumulation of its high levels is a potential novel marker for malignancy. Recently, its homologues such as p63 and p73 have been reported in human, mice and fish. Environmentally induced alterations in p53 protein have been reported to contribute to pathogenesis of leukemia in soft-shell clam Mya arenaria inhabiting polluted water, suggesting that p53 proteins can also be used as pollution markers. In the present study, the presence of p53 protein or its homologues was investigated in tissues of bivalve molluscs Lamellidens corrianus that are predominant in the freshwater riverine environment and are well suited to act as test organisms for evaluation of habitat degradation. The molluscs were collected live from the river Ganga at three sampling sites viz., Kanpur, Allahabad and Varanasi and different tissues (foot, gill and mantle) were collected. Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On immunoblot analysis, a 45 kDa protein (p45) was recognized by the monoclonal anti-p53 antibody in the molluscan tissues. The p45 showed immunoreactivity in all the three tissues of molluscs collected at Kanpur, in foot and gill tissues in those collected at Allahabad, and in foot tissue only, in those collected at Varanasi. Since monoclonal anti-p53 recognizes a denaturation-resistant epitope on the p53 (53 kDa) nuclear protein and does not react with other cellular proteins, the molluscan p45 is a p53-homologue or p53-like protein. Further, the differential expression of p45 in the different organs might serve as a useful biomarker that would help in establishing pollution gradient for environmental monitoring in the large aquatic ecosystems.     

Differential Sensitivity of Macrocarpa and Microcarpa Types of Chickpea （Cicer arietinum L.） to Water Stress： Association of Contrasting Stress Response with Oxidative Injury

Chickpea (Cicer arietinum L.) is particularly sensitive to water stress at its reproductive phase and, under conditions of water stress, will abort flowers and pods, thus reducing yield potential. There are two types of chickpea: (i) Macrocarpa (“Kabuli”), which has large, rams head‐shaped, light brown seeds; and (ii) Microcarpa (“Desi”), which has small, angular and dark‐brown seeds. Relatively speaking, “Kabuli” has been reported to be more sensitive to water stress than “Desi”. The underlying mechanisms associated with contrasting sensitivity to water stress at the metabolic level are not well understood. We hypothesized that one of the reasons for contrasting water stress sensitivity in the two types of chickpea may be a variation in oxidative injury. In the present study, plants of both types were water stressed at the reproductive stage for 14 d. As a result of the stress, the “Kabuli” type exhibited an 80% reduction in seed yield over control compared with a 64% reduction observed for the “Desi” type. The decrease in leaf water potential (Ψ_w) was faster in the “Kabuli” compared with the “Desi” type. At the end of the water stress period, Ψ_w was reduced to ?2.9 and ?3.1 MPa in the “Desi” and “Kabuli” types, respectively, without any significant difference between them. On the last day of stress, “Kabuli” experienced 20% more membrane injury than “Desi”. The chlorophyll content and photosynthetic rate were significantly greater in “Desi” compared with “Kabuli”. The malondialdehyde and H₂O₂ content were markedly higher at the end of the water stress in “Kabuli” compared with “Desi”, indicating greater oxidative stress in the former. Levels of anti‐oxidants, such as ascorbic acid and glutathione, were significantly higher in “Desi” than “Kabuli”. Superoxide dismutase and catalase activity did not differ significantly between the two types of chickpea, whereas on the 10th day, the activities of ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase were higher in “Desi”. These findings indicate that the greater stress tolerance in the “Desi” type may be ascribed to its superior ability to maintain better water status, which results in less oxidative damage. In addition, laboratory studies conducted by subjecting both types of chickpea to similar levels of polyethylene glycol‐induced water stress and to 10 μ.mol/L abscisic acid indicated a greater capacity of the “Desi” type to deal with oxidative stress than the “Kabuli” type. (Managing editor: Ping He)     

Down-regulation of lignin biosynthesis in transgenic Leucaena leucocephala harboring O-methyltransferase gene

In the present study, a 0.47 kb OMT gene construct from aspen, encoding for an enzyme O-methyltransferase (OMT, EC 2.1.1.6), in antisense orientation was used to down-regulate lignin biosynthesis in Leucaena leucocephala. The plants were transformed with Agrobacterium tumefaciens strain harboring the antisense gene, and the transformation was confirmed by PCR amplification of the npt II gene. The integration of a heterologous antisense OMT gene construct in transformed plants led to a maximum of 60% reduction in OMT activity relative to control. The evaluation of total lignin content by the Klason method revealed a maximum of 28% reduction. Histochemical analyses of stem sections depicted a reduction in lignin content and normal xylem development. The results also suggested a probable increase in aldehyde levels and a decrease in syringyl units. Lignin down-regulation was accompanied by an increase in methanol soluble phenolics to an extent that had no impact on wood discoloration, and the plants displayed a normal phenotype. Concomitantly, an increase of up to 9% in cellulose content was also observed. Upon alkali extraction, modified lignin was more extractable as evident from reduced Klason lignin in saponified residue and increased alkali soluble phenolics. The results together suggested that the extent of down-regulation of OMT activity achieved may lead to quality amelioration of Leucaena with respect to its applicability in pulp and paper manufacture as well as nutritive and easily digestible forage production.     

A ClpP protein model as tuberculosis target for screening marine compounds

ATP-dependent Clp protease (ClpP) is a core unit of a major bacterial protease complex employing as a new attractive drug target for that isolates, which are resistant to antibiotics. Mycobacterium tuberculosis, a gram-positive bacterium, is one of the major causes of hospital acquired infections. ClpP in Mycobacterium tuberculosis is usually tightly regulated and strictly requires a member of the family of Clp-ATPase and often further accessory proteins for proteolytic activation. Inhibition of ClpP eliminates these safeguards and start proteolytic degradation. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death. In order to inhibit Clp protease, at first three dimensional structure model of ClpP in Mycobacterium tuberculosis was determined by comparative homology modeling program MODELLER based on crystal structure of the proteolytic component of the caseinolytic Clp protease (ClpP) from E. coli as a template protein and has 55%sequence identity with ClpP protein. The computed model's energy was minimized and validated using PROCHECK to obtain a stable model structure and is submitted in Protein Model Database (PMDB-ID: PM0075741). Stable model was further used for virtual screening against marine derived bioactive compound database through molecular docking studies using AutoDock 3.05. The docked complexes were validated and enumerated based on the AutoDock Scoring function to pick out the best marine inhibitors based on docked Energy. Thus from the entire 186 Marine compounds which were Docked, we got best 5 of them with optimal docked Energy (Ara-A: -14.31 kcal/mol, Dysinosin C: - 14.90kcal/mol, Nagelamide A: -20.49 kcal/mol, Strobilin: -8.02 kcal/mol, Manoalide: -8.81 kcal/mol). Further the five best-docked complexes were analyzed through Python Molecular Viewer software for their interaction studies. Thus from the Complex scoring and binding ability its deciphered that these Marine compounds could be promising inhibitors for ClpP as Drug target yet pharmacological studies have to confirm it.     

Mining and characterization of EST derived microsatellites in Curcuma longa L

Turmeric (Curcuma longa L.) (Family: Zingiberaceae) is a perennial rhizomatous herbaceous plant often used as a spice since time immemorial. Turmeric plants are also widely known for its medicinal applications. Recently EST-derived SSRs (Simple sequence repeats) are a free by-product of the currently expanding EST (Expressed Sequence Tag) databases. SSRs have been widely applied as molecular markers in genetic studies. Development of high throughput method for detection of SSRs has given a new dimension in their use as molecular markers. A software tool SciRoKo was used to mine class I SSR in Curcuma EST database comprising 12953 sequences. A total of 568 non-redundant SSR loci were detected with an average of one SSR per 14.73 Kb of EST. Furthermore, trinucleotide was found to be the most abundant repeat type among 1-6-nucleotide repeat types. It accounted for 41.19% of the total, followed by the mononucleotide (20.07%) and hexanucleotide repeats (15.14%). Among all the repeat motifs, (A/T)n accounted for the highest proportion followed by (AGG)n. These detected SSRs can be greatly used for designing primers that can be used as markers for constructing saturated genetic maps and conducting comparative genomic studies in different Curcuma species.