This application is for the renewal of the R01 AI056404-01 entitled "Biochemical Characterization of Yersinia Effector YopJ" that was funded from 9/30/03 1/31/08 and currently has less than a year of funding remaining. The bacterial effector YopJ inhibits MAPK and NFkB signaling pathways by preventing activation of MKKs and IKKb. As described in our Progress Report, we discovered that the bacterial effector YopJ is an acetyltransferase that modifies serine and threonine residues on the activation loop of MKKs, thereby, directly competing with and preventing activation of these kinases by phosphorylation. The overall goal of this grant is to elucidate the biochemical mechanism for the pathogenic effector expressed by Yersinia, YopJ that uses the newly discovered posttranslational modification of serine/threonine acetylation to modify proteins and to explore the possibility that this is a regulatory mechanism used by eukaryotes. We predict this activity is an evolutionarily conserved regulatory mechanism. Support for this claim comes from the discovery of the enzymatic properties of four other Yersinia effectors: YopH, a tyrosine phosphatase;YopE, a GAP;YpkA, a serine kinase;YopT, papain-like protease. Bacterial pathogens, as with viral pathogen's, usurp critical activities from their hosts and modified them to suit their own needs. In the case of Yersinia pestis, these virulence factors promote the pathogens proliferation at the deadly expense of the host. Clearly these effectors are important to study because all of them have had a profound impact on our way of thinking about eukaryotic signaling. Herein, we describe the Specific Aims that we will use to study this newly discovered posttranslational modification on serine and threonine residues. In these Aims we will use a yeast system that we established over the last funding period to analyze how YopJ interacts with MKKs and its mechanism of inhibition. These studies will provide insight into, not only the inhibitory activity of YopJ, but also the mechanisms that regulate MKK activation. We will also investigate the enzymatic mechanism used by the family of YopJ proteins to acetylate their substrates using kinetic studies. Finally, we will develop tools to study this newly discovered posttranslational modification and its role in eukaryotic signaling, including the discovery of eukaryotic enzymes that maybe involved in acteylation of serine and threonine residues and the possible reversal of this posttranslational modification.Yersinia pestis is the infectious agent that caused the Black Death in the Middle Ages, and Y. pseudotuberculosis and Y. enterocolitica (two closely related food borne pathogens) are causal agents of gastrointestinal disorders. One of the bacterial effectors produced by Yersinia, YopJ, is a 32kD protein that possesses the remarkable capacity to block multiple signaling pathways including all of the MAPK signaling pathways and the NFkB pathways, resulting in inhibition of the innate immune response and promotion of cell death in host cells. This application is for the Renewal of the R01 AI056404-01 grant where we discovered that YopJ utilizes a new type of posttranslational modification to inhibit signaling. YopJ is a serine and threonine acetyltransferase that modifies serine and threonine residues on the activation loop of MKKs, thereby, directly competing with and preventing phosphorylation by upstream kinases. The three Specific Aims proposed for the renewal of this grant are designed to use genetics and biochemistry to study this novel inhibitory mechanism and to investigate its possible use in eukaryotic signaling pathways.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI056404-10
Application #
8204785
Study Section
Special Emphasis Panel (ZRG1-CB-G (01))
Program Officer
Mukhopadhyay, Suman
Project Start
2003-09-30
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2013-12-31
Support Year
10
Fiscal Year
2012
Total Cost
$307,751
Indirect Cost
$111,731
Name
University of Texas Sw Medical Center Dallas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Lewallen, Daniel M; Sreelatha, Anju; Dharmarajan, Venkatasubramanian et al. (2014) Inhibiting AMPylation: a novel screen to identify the first small molecule inhibitors of protein AMPylation. ACS Chem Biol 9:433-42
Yu, Xiaobo; Woolery, Andrew R; Luong, Phi et al. (2014) Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host AMPylation on self-assembled human protein microarrays. Mol Cell Proteomics 13:3164-76
Salomon, Dor; Kinch, Lisa N; Trudgian, David C et al. (2014) Marker for type VI secretion system effectors. Proc Natl Acad Sci U S A 111:9271-6
Calder, Thomas; Kinch, Lisa N; Fernandez, Jessie et al. (2014) Vibrio type III effector VPA1380 is related to the cysteine protease domain of large bacterial toxins. PLoS One 9:e104387
de Souza Santos, Marcela; Orth, Kim (2014) Intracellular Vibrio parahaemolyticus escapes the vacuole and establishes a replicative niche in the cytosol of epithelial cells. MBio 5:e01506-14
Salomon, Dor; Klimko, John A; Orth, Kim (2014) H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1. Microbiology 160:1867-73
Zhang, Lingling; Orth, Kim (2013) Virulence determinants for Vibrio parahaemolyticus infection. Curr Opin Microbiol 16:70-7
Sreelatha, Anju; Bennett, Terry L; Zheng, Hui et al. (2013) Vibrio effector protein, VopQ, forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux. Proc Natl Acad Sci U S A 110:11559-64
Krachler, Anne Marie; Orth, Kim (2013) Targeting the bacteria-host interface: strategies in anti-adhesion therapy. Virulence 4:284-94
Salomon, Dor; Gonzalez, Herman; Updegraff, Barrett L et al. (2013) Vibrio parahaemolyticus type VI secretion system 1 is activated in marine conditions to target bacteria, and is differentially regulated from system 2. PLoS One 8:e61086

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