1. Identification of Novel Physiological Substrates of Mycobacterium bovis BCG Protein Kinase G (PknG) by Label-free Quantitative Phosphoproteomics.
- Author
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Nakedi KC, Calder B, Banerjee M, Giddey A, Nel AJM, Garnett S, Blackburn JM, and Soares NC
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Mycobacterium bovis growth & development, Phosphopeptides chemistry, Phosphopeptides metabolism, Phosphorylation, Reproducibility of Results, Staining and Labeling, Substrate Specificity, Bacterial Proteins metabolism, Cyclic GMP-Dependent Protein Kinases metabolism, Mycobacterium bovis metabolism, Phosphoproteins metabolism, Proteomics methods
- Abstract
Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. Linking kinases to the substrates they phosphorylate in vivo , thereby elucidating their exact functions, is still a challenge. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BCG versus the respective PknG knock-out mutant (ΔPknG-BCG) strains. We employed TiO
2 phosphopeptide enrichment techniques combined with label-free quantitative phosphoproteomics workflow on LC-MS/MS. The comprehensive analysis of label-free data identified 603 phosphopeptides on 307 phosphoproteins with high confidence. Fifty-five phosphopeptides were differentially phosphorylated, of these, 23 phosphopeptides were phosphorylated in M. bovis BCG wild-type only and not in the mutant. These were further validated through targeted mass spectrometry assays (PRMs). Kinase-peptide docking studies based on a published crystal structure of PknG in complex with GarA revealed that the majority of identified phosphosites presented docking scores close to that seen in previously described PknG substrates, GarA, and ribosomal protein L13. Six out of the 22 phosphoproteins had higher docking scores than GarA, consistent with the proteins identified here being true PknG substrates. Based on protein functional analysis of the PknG substrates identified, this study confirms that PknG plays an important regulatory role in mycobacterial metabolism, through phosphorylation of ATP binding proteins and enzymes in the TCA cycle. This work also reinforces PknG's regulation of protein translation and folding machinery., (© 2018 Nakedi et al.)- Published
- 2018
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