Biomedical subjects

Deepak Kumar Saini

Publications and source records attributed to Deepak Kumar Saini.

4 recordsLinked to original sources

PubMed2005

High-throughput microplate phosphorylation assays based on DevR-DevS/Rv2027c 2-component signal transduction pathway to screen for novel antitubercular compounds.

DevR-DevS (Rv3133c-Rv3132c) and DevR-Rv2027c have been established through their autophosphorylation and phospho-transfer properties to constitute bonafide regulatory 2-component systems of Mycobacterium tuberculosis. DevR has also been shown by others to play a key regulatory role in the expression of M. tuberculosis genes comprising the dormancy regulon. The authors describe high-throughput phosphorylation assays in a microplate format using DevS and Rv2027c histidine kinases and DevR response regulator proteins from M. tuberculosis. The assays were designed to measure [gamma-(32)P]ATP-dependent autophosphorylation of DevS/Rv2027c and also the phosphotransfer reaction to DevR. First, the optimal reaction conditions were established using the conventional method of radiolabeling the 2-component proteins by [gamma-(32)P]ATP and followed by gel electrophoresis-based analysis. Next, the assays were converted to a high-throughput format in which the radiolabeled protein retained on a filter using mixed cellulose ester-based 96-well filter plates was analyzed for radioactivity retention by scintillation counting. The utility of these assays to screen for inhibitors is illustrated using 2-mercaptobenzimidazole, ethidium bromide, and EDTA. The high quality and flexibility of these assays will enable their use in high-throughput screening for new antitubercular compounds directed against 2-component systems that comprise a novel target in dormant mycobacteria.

Antitubercular Agents↗

PubMed2004

Cross talk between DevS sensor kinase homologue, Rv2027c, and DevR response regulator of Mycobacterium tuberculosis.

Rv2027c is a putative orphan histidine sensor kinase that bears strong homology to DevS of the hypoxia-responsive DevR-DevS two-component system in M. tuberculosis. The cytosolic C-terminal domain of Rv2027c protein (Rv2027c(194)) was overexpressed in E. coli and biochemically characterized. Rv2027c(194) underwent autophosphorylation at a conserved His(392) residue and engaged in phosphotransfer with DevR response regulator. The rates of autophosphorylation and the stabilities of the phosphorylated species were broadly similar in Rv2027c and DevS. However, unlike DevS, Rv2027c utilized Ca(2+) as an alternative divalent ion during autophosphorylation. In contrast to DevS which completed phosphotransfer to DevR in 5-10 min, phosphotransfer from Rv2027c approximately P was only partial at 30 min. Unlike devS transcription that was hypoxia-responsive, Rv2027c transcript levels were not upregulated from basal levels during hypoxia. The differential regulation of devS and Rv2027c genes, the ability of Rv2027c to utilize Ca(2+) as a divalent cation in autophosphorylation at physiological concentrations and to engage in phosphotransfer with DevR suggests that the DevR regulon could be modulated by more than one environmental cue relayed through DevS and Rv2027c.

Bacterial Proteins↗

PubMed2004

DevR-DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR.

Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS(201)) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of Mycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His(6)-tagged proteins from Escherichia coli. DevS(201) underwent autophosphorylation and participated in rapid phosphotransfer to DevR in a Mg(2+)-dependent manner. Chemical stability analysis and site-directed mutagenesis implicated the highly conserved residues His(395) and Asp(54) as the sites of phosphorylation in DevS and DevR, respectively. Mutations in Asp(8) and Asp(9) residues, postulated to form the acidic Mg(2+)-binding pocket, and the invariant Lys(104) of DevR, abrogated phosphoryl transfer from DevS(201) to DevR. DevR-DevS was thus established as a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Expression of the Rv3134c-devR-devS operon was induced at the RNA level in hypoxic cultures of M. tuberculosis H37Rv and was associated with an increase in the level of DevR protein. However, in a devR mutant strain expressing the N-terminal domain of DevR, induction was observed at the level of RNA expression but not at that of protein. DevS was translated independently of DevR and induction of devS transcripts was not associated with an increase in protein level in either wild-type or mutant strains, reflecting differential regulation of this locus during hypoxia.

Bacterial Proteins↗

PubMed2002

Cloning, overexpression, purification, and matrix-assisted refolding of DevS (Rv 3132c) histidine protein kinase of Mycobacterium tuberculosis.

The devR-devS (Rv 3133c-Rv 3132c) two-component system of Mycobacterium tuberculosis was identified in our laboratory by RNA subtractive hybridization. This genetic system was predicted to encode a response regulator and histidine protein kinase, respectively. The putative histidine kinase protein DevS was overexpressed to high levels in Escherichia coli as a fusion protein with a hexahistidine tag, His(6)-DevS201, in the form of inclusion bodies. Here we report a "redox-based" method of matrix-bound renaturation of DevS protein. The refolded protein was biochemically active in an autophosphorylation reaction characteristic of histidine kinases and was suitable for the generation of polyclonal antibodies and as an antigen in ELISA.

Animals↗