A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles

Nat Methods. 2014 Jan;11(1):79-85. doi: 10.1038/nmeth.2759. Epub 2013 Dec 1.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Breast Neoplasms / metabolism
  • Catalytic Domain
  • Cell Line, Tumor
  • Cysteine / chemistry
  • Dose-Response Relationship, Drug
  • Electrochemistry / methods
  • Female
  • Gene Expression Regulation, Neoplastic
  • HEK293 Cells
  • Humans
  • Inhibitory Concentration 50
  • Lipids / chemistry
  • Mass Spectrometry / methods
  • Molecular Sequence Data
  • Oxidative Stress
  • Prostaglandin D2 / analogs & derivatives
  • Prostaglandin D2 / chemistry
  • Protein Kinases / metabolism*
  • Protein Processing, Post-Translational
  • Proteome
  • Proteomics / methods*
  • Sequence Homology, Amino Acid

Substances

  • 15-deoxyprostaglandin J2
  • Lipids
  • Proteome
  • Protein Kinases
  • ZAK protein, human
  • Cysteine
  • Prostaglandin D2