Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the sites in the proteome that are most sensitive to electrophilic modification requires more quantitative methods. Here we describe a competitive activity–based profiling method for quantifying the reactivity of electrophilic compounds against >1,000 cysteines in parallel in the human proteome. Using this approach, we identified a select set of proteins that constitute 'hot spots' for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxy-2-nonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site–proximal cysteine and that the resulting enzyme inhibition creates a negative feedback mechanism that can suppress the activation of JNK pathways normally induced by oxidative stress.
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We thank K. Backus, D. Bachovchin, B. Lanning, K. Tsuboi and A. Adibekian from the Cravatt Lab for providing reagents, and the Marletta lab at The Scripps Research Institute for sharing instrumentation for data collection. This work was supported by the US National Institutes of Health (NIH) (CA087660), an NIH/NIEHS K99/R00 Pathways to Independence Postdoctoral Award (K99ES020851, C.W.), a Pfizer Postdoctoral Fellowship (E.W.), a US National Science Foundation predoctoral fellowship (M.B.) and the Skaggs Institute for Chemical Biology.
The authors declare no competing financial interests.
Supplementary Figures 1–8 (PDF 771 kb)
In vitro profiling with each of the three LDEs at 100 μM in MDA-MB-231 and Ramos proteomes (XLS 6769 kb)
List of cysteines competed by one or more LDE with competition ratios > 5 (XLSX 51 kb)
In vitro profiling with HNE at 5 different concentrations in MDA-MB-231 proteomes (XLS 711 kb)
In situ profiling with HNE at 50 and 100 μM in MDA-MB-231 cells (XLS 422 kb)
List of kinases with reactive cysteines quantified by in vitro profiling with 100 μM HNE in MDA-MB-231 proteomes (XLSX 53 kb)
SILAC-ABPP ratios for acylphosphate-ATP probe-labeled proteins in proteomes from HEK-293T cells transfected with ZAK and treated with DMSO or 100 μM HNE in vitro (XLSX 1817 kb)
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Wang, C., Weerapana, E., Blewett, M. et al. A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles. Nat Methods 11, 79–85 (2014). https://doi.org/10.1038/nmeth.2759
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