Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

Xiaobo Yu, Andrew R. Woolery, Phi Luong, Yi Heng Hao, Markus Grammel, Nathan Westcott, Jin Park, Jie Wang, Xiaofang Bian, Gokhan Demirkan, Howard C. Hang, Kim Orth, Joshua LaBaer

Research output: Contribution to journalArticle

25 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)3164-3176
Number of pages13
JournalMolecular and Cellular Proteomics
Volume13
Issue number11
DOIs
StatePublished - Nov 1 2014

Fingerprint

Click Chemistry
Protein Array Analysis
Alkynes
Azides
Cycloaddition
Cycloaddition Reaction
Pathogens
Microarrays
Vibrio parahaemolyticus
Copper
Pasteurellaceae
Substrates
Host-Pathogen Interactions
Proteins
src-Family Kinases
Threonine
Post Translational Protein Processing
Cell signaling
Cell Count
Phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays. / Yu, Xiaobo; Woolery, Andrew R.; Luong, Phi; Hao, Yi Heng; Grammel, Markus; Westcott, Nathan; Park, Jin; Wang, Jie; Bian, Xiaofang; Demirkan, Gokhan; Hang, Howard C.; Orth, Kim; LaBaer, Joshua.

In: Molecular and Cellular Proteomics, Vol. 13, No. 11, 01.11.2014, p. 3164-3176.

Research output: Contribution to journalArticle

Yu, Xiaobo ; Woolery, Andrew R. ; Luong, Phi ; Hao, Yi Heng ; Grammel, Markus ; Westcott, Nathan ; Park, Jin ; Wang, Jie ; Bian, Xiaofang ; Demirkan, Gokhan ; Hang, Howard C. ; Orth, Kim ; LaBaer, Joshua. / Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays. In: Molecular and Cellular Proteomics. 2014 ; Vol. 13, No. 11. pp. 3164-3176.
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