Dehydroalanine Containing MAPK Peptide Modification Inhibits Activity Of OspF/SpvC, And The Mechanism Of Nuclear Translocation

OspF from Shigella and SpvC from Salmonella, define a family of conserved gram-negative pathogenic effectors with a unique phosphothreonine lyase activity, irreversibly inactivates the dual-phosphorylated host MAPKs, through P-elimination reaction to remove the phosphate moiety from the phosphothreonine.No eukaryotic phosphothreonine lyase has been identified, making this enzyme an attractive narrow spectrum antibiotic target. The inhibition of the pathogenic effectors differs from the many existing antibiotic paradigms and may elicit less resistance.OspF localizes to the nucleus of shigella-infected cells, however, the mechanism remains obscure. Based on above questions, we start our following research work:A deprotonated state is a prerequisite for lysine to act as a base. The deprotonated lysine can react with α,β-unsaturated compounds through 1,4-nucleophilic addition reaction. The nucleophilic addition reaction with dehydroalanine is more efficient than with β-methyldehydroalanine. SpvC can react with the unsaturated MAPK peptides, and results of tandem mass spectrometry analysis indicate that the addition reaction occurred between unsaturated Cα=Cβ bond and K136. The deprotonated state of K136 and peptide binding ability affect the adduction efficiency. Y158F mutantion elevates pKa of K136 and reduces the efficiency of reaction. Addition product with dehydroalanine containing Erk5 peptide was relatively higher than with dehydroalanine containing Erk2 peptide or P38 peptide. Modification of K136 inhibits phosphothreonine lyase activity, and addition with dehydroalanine containing Erk5 peptide almost completely inactivates SpvC.OspF accumulated in the nucleus in transfected Hela cells, and the Erk binding-deficient mutants still localized to the nucleus, indicats that nuclear localization of OspF was independent of binding with Erk. C-terminal 30 amino acid deletion construct is mainly localized in cytoplasm and perinucleus, insertion of the C-terminal 30 amino acid resulted in almost complete nuclear relocalization of PAK4 protein. GST pull-down assays exhibited the NLS interaction with Kapal.Taken together, these results indicated that: 1) Dehydroalanine containing MAPK peptides react with K136 of SpvC and inhibits its catalytic activity; 2) The dehydroalanine containing Erk5 peptide may be a leader compound to development inhibitors of phosphothreonine lyase; 3) OspF contains a nuclear localization signal and interacts with Kapal, suggesting that the Kapal mediates nuclear import of OspF.