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Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress

Nature Chemical Biology volume 13, pages 302–308 (2017)Cite this article

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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.

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Figure 1: N6pA labeling schematic.
Figure 2: N6pA labels ADP-ribosylated proteins in mammalian cells.
Figure 3: Summary and validation of N6pA-labeled proteins.
Figure 4: Oxidative stress induces ADP-ribosylation of Hras at S-palmitoylation sites.
Figure 5: Oxidative stress induces Hras removal of S-palmitoylation.
Figure 6: Hras ADP-ribosylation and signaling during oxidative stress.

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Acknowledgements

N.P.W. is a Leukemia and Lymphoma Society postdoctoral fellow. The authors thank R. Pagano (Mayo Clinic) for GFP–HA–Rab7, G. Wahl (Salk Institute) for the histone H2B–GFP, M. Phillips (New York University) for the HA–Kras4A and Flag–Kras4B constructs and V. Schreiber (University of Strasbourg) for the ARTD1−/− (PARP1−/−) MEFs. H.C.H. acknowledges support from NIH-NIGMS R01 GM087544 grant.

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Authors and Affiliations

  1. Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York, USA

    Nathan P Westcott & Howard C Hang

  2. Proteomics Resource Center, The Rockefeller University, New York, New York, USA

    Joseph P Fernandez & Henrik Molina

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Contributions

H.C.H. and N.P.W. designed the experiments, analyzed data and wrote the manuscript. H.M. helped with mass spectrometry analysis. J.P.F. performed and helped analyze the NAD metabolite mass spectrometry. N.P.W. performed all other experiments.

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Correspondence to Howard C Hang.

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Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Figures 1–22. (PDF 2533 kb)

Supplementary Dataset 1

List of proteins with a fudge factor greater than 1 and a false discovery rate of 0.05 for the unstressed samples. (XLSX 115 kb)

Supplementary Dataset 2

List of proteins with a fudge factor greater than 1 and a false discovery rate of 0.05 for the stressed samples. (XLSX 111 kb)

Supplementary Dataset 3

List of proteins with a difference of means greater than 2 with the probability of that difference P <0.05 for the stressed vs. the unstressed samples. (XLSX 54 kb)

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Westcott, N., Fernandez, J., Molina, H. et al. Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress. Nat Chem Biol 13, 302–308 (2017). https://doi.org/10.1038/nchembio.2280

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