The microbial pathogen Yersinia pestis, was the causal agent for the devastating plagues killing millions of people in Europe, India, China and even the United States. The pathogen persists in flea/rodent ecosystems on almost every continent and can cause a short but extremely painful death in untreated victims. This pathogen is a potential weapon that may be used for Biowarfare and therefore it is important to understand how this toxin works. Yersinia's modus operandi includes evading phagocytosis, destroying the host defense system and inducing programmed cell death in the target host cell. Only six proteins called Yersinia outer proteins (Yops) encoded on a virulence plasmid efficiently carry out these activities; one of them, YopJ, both blocks cytokine production and promotes apoptosis in target host cells. YopJ, a 32kD protein, blocks all major signaling pathways involved in cytokine production including all the MAPK signaling pathways and the NFKappaB pathway. The family of YopJ proteins, composed of effectors from plant and animal pathogens, has structural homology to a clan of cysteine proteases that includes adenoviral proteases and the ubiquitin-like protein proteases. Consistent with the proposal that YopJ is a hydrolase, an intact catalytic site is required for YopJ to block the activation of the evolutionarily conserved super family of MAPK kinases (MKK). Based on these observations, we propose that YopJ uses a novel mechanism to disrupt a component of the signaling machinery that is required for the activity of all of the aforementioned kinase-driven signaling cascades. We propose three Specific Aims to investigate our hypothesis: (i) To characterize the target(s) of the Yersinia effector YopJ in the mammalian MAPK signaling pathway. (ii) To characterize the target(s) of the Yersinia effector YopJ in the NFkappaB signaling pathway. (iii) To characterize the hydrolytic activity of YopJ in vitro. Results from these studies will identify the targets of YopJ and characterize the inhibitory activity of YopJ that will lead to the understanding of an evolutionarily conserved mechanism of regulation that is necessary for intracellular transmission of signals. Discovery of this mechanism is essential for understanding how signaling pathways are regulated in both plants and animals.
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