Biochemical Characterization of the Yersinia Effector Protein, YopJ
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Yersinia species, the causal agent of plague and gastroenteritis, uses a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ disrupts the mitogen-activated protein kinase and the nuclear factor κ B signaling pathways used in innate immune response by preventing activation of the family of mitogen-activated protein kinase kinases. The catalytic domain of YopJ is similar to Clan CE of cysteine proteases, and mutating the putative catalytic cysteine disrupts YopJ's inhibitory activity. YopJ binds mitogenactivated protein kinase kinases, including MKK1 through MKK6, and the related kinase, IκB kinase beta, however, the mechanism by which this binding leads to inactivation of these kinases is unknown. An in vitro cell-free signaling system was developed to recapitulate the inhibition of eukaryotic signaling by YopJ. Mass spectrometric studies were undertaken to determine the biochemical nature of modification of the mitogen-activated protein kinase kinases in the presence of YopJ. Based on the observations, a simple, molecular mechanism utilized by YopJ to block the signaling pathways was discovered. YopJ acted as an acetyltransferase, using acetyl coenzyme A, to modify the critical serine and threonine residues in the activation loop of mitogen-activated protein kinase kinases and thereby blocking phosphorylation. The acetylation on the kinase directly competed with phosphorylation, preventing activation of the modified protein. An essential characteristic feature of bacterial effector proteins is that they usurp or mimic a eukaryotic activity and refine this activity to produce an extremely efficient mechanism to combat eukaryotic signaling. Therefore, modification of amino acids, other than lysine, by acetylation could be a commonly used eukaryotic mechanism that has been undetected previously. The acetylation of these amino acids may compete with various other types of posttranslational modifications, such as ubiquitination, SUMOylation and glycosylation. Several questions that still need to be addressed are: Is this modification reversible? What are the eukaryotic proteins that add and remove this type of posttranslational modification? How do bacterial effectors use this activity? The characterization of a bacterial effector as a serine or threonine acetyltransferase presents a previously unknown paradigm to be considered for other biological signaling pathways.