title: Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host AMPylation on self-assembled human protein microarrays.
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.
authors: From the ‡The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA;
authors: Xiaobo
authors: X
authors: Yu
authors: §Department of Molecular Biology, UT Southwestern Medical Center, Dallas, Texas 75390-9148, USA;
authors: Andrew R
authors: AR
authors: Woolery
authors: §Department of Molecular Biology, UT Southwestern Medical Center, Dallas, Texas 75390-9148, USA;
authors: Phi
authors: P
authors: Luong
authors: §Department of Molecular Biology, UT Southwestern Medical Center, Dallas, Texas 75390-9148, USA;
authors: Yi Heng
authors: YH
authors: Hao
authors: ¶The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York 10065, USA.
authors: Markus
authors: M
authors: Grammel
authors: ¶The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York 10065, USA.
authors: Nathan
authors: N
authors: Westcott
authors: From the ‡The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA;
authors: Jin
authors: J
authors: Park
authors: From the ‡The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA;
authors: Jie
authors: J
authors: Wang
authors: From the ‡The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA;
authors: Xiaofang
authors: X
authors: Bian
authors: From the ‡The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA;
authors: Gokhan
authors: G
authors: Demirkan
authors: ¶The Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York 10065, USA.
authors: Howard C
authors: HC
authors: Hang
authors: §Department of Molecular Biology, UT Southwestern Medical Center, Dallas, Texas 75390-9148, USA;
authors: Kim
authors: K
authors: Orth
authors: From the ‡The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA; jlabaer@asu.edu.
authors: Joshua
authors: J
authors: LaBaer
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D057930:Click Chemistry / Q000379:methods*; D003300:Copper / Q000737:chemistry; D061565:Cycloaddition Reaction; D054884:Host-Pathogen Interactions; D006801:Humans; D015661:Pasteurellaceae / Q000378:metabolism; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D014735:Vibrio Infections / Q000473:pathology; D014736:Vibrio parahaemolyticus / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta / Q000378:metabolism; D000097766:RAC2 GTP-Binding Protein
chemical_list: D000249:Adenosine Monophosphate; D001426:Bacterial Proteins; D003300:Copper; D020744:rac GTP-Binding Proteins; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D000097766:RAC2 GTP-Binding Protein; C569658:ARHGDIB protein, human; C411563:RAC3 protein, human