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Mol Cell Proteomics. 2014 Nov;13(11):3164-76. doi: 10.1074/mcp.M114.041103. Epub 2014 Jul 29.

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host AMPylation on self-assembled human protein microarrays.

Author information

1
From the ‡The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA;
2
§Department of Molecular Biology, UT Southwestern Medical Center, Dallas, Texas 75390-9148, USA;
3
¶The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York 10065, USA.
4
From the ‡The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA; jlabaer@asu.edu.

Abstract

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.

PMID:
25073739
PMCID:
PMC4223499
DOI:
10.1074/mcp.M114.041103
[Indexed for MEDLINE]
Free PMC Article

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