Proteome and phosphoproteome analysis of the serine / threonine protein kinase E mutant of Mycobacterium tuberculosis.

Kumar, D.; Sharma, P.; Bisht, D.; Narayanan, S.

Life Sci ences; 2014; 109; 116-126.

Abstract: Aims: Serine/threonine protein kinases (STPKs) have prominent roles in the survival mechanisms of Mycobacterium tuberculosis ( M. tuberculosis ). Previous studies from our laboratory underscored the role of PknE, an STPK in virulence, adaptation and the suppression of host cell apoptosis. In this study, two-dimensional gel electrophoresis was used to study the proteome and phosphoproteome profiles of wild type M. tuberculosis and its isogenic pkn E deletion mutant (∇pknE) during growth in Middlebrook 7H9 and nitric oxide stress.

Main methods: Wild-type M. tuberculosis and its isogenic pknE deletion mutant strain were grown in Middlebrook 7H9 as well as subjected to nitric oxide stress using sodium nitroprusside. Whole cell lysates were prepared and analyzed by 2D-gel electrophoresis. Phosphoproteomes were analyzed using phospho serine and phospho threonine antibodies after subjecting the 2D-gels to western blotting. Proteins of interest were identified using mass spectrometry.

Key findings: Our analysis provides insights into the targets that impose pro-apoptotic as well as altered cellular phenotypes on ?pknE, revealing novel substrates and functions for PknE.

Significance: For the first time, our proteome and phosphoproteome data decipher the function of PknE in cell division, virulence, dormancy, suppression of sigma factor B and its regulated genes, suppression of two component systems and in the metabolic activity of M. tuberculosis .

Keywords: PknE; Mycobacterium tuberculosis ; Proteome; Phosphoproteome; Middlebrook 7H9; Nitrate stress; 2D gel electrophoresis; Western blotting; Sigma B

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