title: Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress.
abstract: ADP-ribosylation is a post-translational modification that is known to be involved in cellular homeostasis and stress but has been challenging to analyze biochemically. To facilitate the detection of ADP-ribosylated proteins, we show that an alkyne-adenosine analog, N6-propargyl adenosine (N6pA), is metabolically incorporated in mammalian cells and enables fluorescence detection and proteomic analysis of ADP-ribosylated proteins. Notably, our analysis of N6pA-labeled proteins that are upregulated by oxidative stress revealed differential ADP-ribosylation of small GTPases. We discovered that oxidative stress induced ADP-ribosylation of Hras on Cys181 and Cys184 in the C-terminal hypervariable region, which are normally S-fatty-acylated. Downstream Hras signaling is impaired by ADP-ribosylation during oxidative stress, but is rescued by ADP-ribosyltransferase inhibitors. Our study demonstrates that ADP-ribosylation of small GTPases not only is mediated by bacterial toxins but is endogenously regulated in mammalian cells. N6pA provides a useful tool to characterize ADP-ribosylated proteins and their regulatory mechanisms in cells.
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York, USA.
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Westcott
authors.affiliation: Proteomics Resource Center, The Rockefeller University, New York, New York, USA.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Fernandez
authors.affiliation: Proteomics Resource Center, The Rockefeller University, New York, New York, USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York, USA.
authors.forename: Howard C
authors.initials: HC
authors.lastname: Hang
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D015394:Molecular Structure; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D018384:Oxidative Stress; D040901:Proteomics
chemical_list: D000244:Adenosine Diphosphate; D020559:Monomeric GTP-Binding Proteins