Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human RKO and THP-1 cells treated with alkynyl analogs of the lipid electrophiles 4-hydroxynonenal (HNE) and 4-oxononenal (ONE). Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions.  We further used RNA-seq analysis to characterize transcriptome responses in RKO cells induced by HNE at subcytotoxic and cytotoxic doses. RNA-seq data analysis combining gene and coding DNA sequence (CDS) levels yielded an informative and comprehensive picture of gradually evolving response networks with increasing HNE doses, from cell protection against oxidative injury at low dose, initiation of cell apoptosis and DNA damage response at intermediate dose to significant deregulation of cellular functions at high dose.  These datasets reveal a hierarchical organization of both damage susceptibility and response networks in response to electrophile-mediated protein alkylation.  (Supported by NIH Grants ES013125, ES000267, ES0007028 and CA159988.)

Systems analysis of protein damage by lipid electrophiles

248th National Meeting (2014)

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