Alkylation Damage by Lipid Electrophiles Targets Functional Protein Systems

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 cells treated with lipid electrophile probes. 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. 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 cells treated with lipid electrophile probes. 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. Covalent protein alkylation by reactive electrophiles was identified as a key triggering event in chemical toxicity over 40 years ago (1Miller E.C. Miller J.A. Sapp R.W. Weber G.M. Studies on the protein-bound aminoazo dyes formed in vivo from 4-dimethylaminoazobenzene and its C-monomethyl derivatives.Cancer Res. 1949; 9: 336-343PubMed Google Scholar, 2Jollow D.J. Mitchell J.R. Potter W.Z. Davis D.C. Gillette J.R. Brodie B.B. Acetaminophen-induced hepatic necrosis. II. Role of covalent binding in vivo.J. Pharmacol. Exp. Ther. 1973; 187: 195-202PubMed Google Scholar). These reactions remain a major cause of drug toxicity and represent a longstanding problem in drug safety (3Evans D.C. Baillie T.A. Minimizing the potential for metabolic activation as an integral part of drug design.Curr. Opin. Drug Discov. Devel. 2005; 8: 44-50PubMed Google Scholar). Oxidative stress also generates reactive lipid electrophiles, which covalently modify proteins and trigger stress signaling and cell death associated with inflammation and degenerative diseases (4Rudolph T.K. Freeman B.A. Transduction of redox signaling by electrophile-protein reactions.Sci. Signal. 2009; 2: re7Crossref PubMed Scopus (176) Google Scholar, 5Schopfer F.J. Cipollina C. Freeman B.A. Formation and signaling actions of electrophilic lipids.Chem. Rev. 2011; 111: 5997-6021Crossref PubMed Scopus (241) Google Scholar, 6West J.D. Marnett L.J. Endogenous reactive intermediates as modulators of cell signaling and cell death.Chem. Res. Toxicol. 2006; 19: 173-194Crossref PubMed Scopus (253) Google Scholar). Despite the broad importance of this problem, the mechanisms by which protein alkylation drives toxicity remain largely unexplained. Interestingly, some drug molecules produce significant protein covalent binding without causing toxicity (7Streeter A.J. Bjorge S.M. Axworthy D.B. Nelson S.D. Baillie T.A. The microsomal metabolism and site of covalent binding to protein of 3′-hydroxyacetanilide, a nonhepatotoxic positional isomer of acetaminophen.Drug Metab. Dispos. 1984; 12: 565-576PubMed Google Scholar, 8Bauman J.N. Kelly J.M. Tripathy S. Zhao S.X. Lam W.W. Kalgutkar A.S. Obach R.S. Can in vitro metabolism-dependent covalent binding data distinguish hepatotoxic from nonhepatotoxic drugs? An analysis using human hepatocytes and liver S-9 fraction.Chem. Res. Toxicol. 2009; 22: 332-340Crossref PubMed Scopus (113) Google Scholar), which suggests that a only critical subset of protein alkylation events contribute to injury. Thus, the major challenge in this field is to distinguish toxic from nontoxic protein alkylation damage. We and others have explored this problem by combining electrophile probes and MS-based proteomics to perform systematic, global inventories of protein alkylation damage. These studies revealed electrophile-specific patterns of protein alkylation, yet also indicated that many proteins are targets of multiple electrophiles (9Weerapana E. Simon G.M. Cravatt B.F. Disparate proteome reactivity profiles of carbon electrophiles.Nat. Chem. Biol. 2008; 4: 405-407Crossref PubMed Scopus (195) Google Scholar) (10Dennehy M.K. Richards K.A. Wernke G.R. Shyr Y. Liebler D.C. Cytosolic and nuclear protein targets of thiol-reactive electrophiles.Chem. Res. Toxicol. 2006; 19: 20-29Crossref PubMed Scopus (133) Google Scholar, 11Shin N.Y. Liu Q. Stamer S.L. Liebler D.C. Protein targets of reactive electrophiles in human liver microsomes.Chem. Res. Toxicol. 2007; 20: 859-867Crossref PubMed Scopus (80) Google Scholar, 12Wong H.L. Liebler D.C. Mitochondrial protein targets of thiol-reactive electrophiles.Chem. Res. Toxicol. 2008; 21: 796-804Crossref PubMed Scopus (69) Google Scholar, 13Codreanu S.G. Zhang B. Sobecki S.M. Billheimer D.D. Liebler D.C. Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal.Mol. Cell. Proteomics. 2009; 8: 670-680Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Inference of possible toxicity mechanisms from such inventories is complicated by the diversity of biological processes represented by electrophile protein targets. However, we have recently reported that combined analysis of protein target inventories with transcriptome expression profiles enables inference of candidate networks associated with activation of stress responses (14Zhang B. Shi Z. Duncan D.T. Prodduturi N. Marnett L.J. Liebler D.C. Relating protein adduction to gene expression changes: a systems approach.Mol. Biosyst. 2011; 7: 2118-2127Crossref PubMed Scopus (23) Google Scholar). Thus, functional readouts of damage effects could guide interpretation of protein adduct profiles. An important limitation of most previous protein adduct profiling studies is that they were done in subcellular fractions or cell lysates, which lack intact detoxification mechanisms for electrophiles, particularly glutathione (GSH) 1The abbreviations used are:GSHglutathioneONE4-oxo-2-nonenalHNE4-hydroxy-2-nonenalFDRfalse discovery rate. 1The abbreviations used are:GSHglutathioneONE4-oxo-2-nonenalHNE4-hydroxy-2-nonenalFDRfalse discovery rate.-dependent conjugation. The onset of toxicity in both cell and animal models often coincides with GSH depletion and an increase in covalent protein binding (15DeLeve L.D. Kaplowitz N. Glutathione metabolism and its role in hepatotoxicity.Pharmacol. Ther. 1991; 52: 287-305Crossref PubMed Scopus (436) Google Scholar). Analysis of protein adduct profile changes accompanying GSH depletion thus may provide another means to interpret the effects of protein damage. glutathione 4-oxo-2-nonenal 4-hydroxy-2-nonenal false discovery rate. glutathione 4-oxo-2-nonenal 4-hydroxy-2-nonenal false discovery rate. To address these questions, we performed a series of quantitative adduct profiling experiments with alkynyl analogs of the prototypical lipid electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). The alkynyl analogs (aHNE and aONE, supplemental Fig. S1A and S1B) form adducts that can be biotinylated during by for for analysis K.A. Liebler D.C. Marnett L.J. of protein targets of using chemistry for vivo of and alkynyl Res. Toxicol. 2008; 21: PubMed Scopus Google Scholar). of a enables from thus with proteins K.A. Liebler D.C. An for in the of protein adducts of electrophiles by and Cell. Proteomics. 2009; 8: Full Text Full Text PDF PubMed Scopus (80) Google Scholar). were from were from was from was from was as K.A. Liebler D.C. Marnett L.J. of protein targets of using chemistry for vivo of and alkynyl Res. 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Zhang B. data over biological PubMed Scopus Google Scholar) to the impact of protein alkylation by and in both cell proteins in a interaction S. B. on the 2011; PubMed Scopus Google Scholar) in a on the of the and the the of the target proteome and protein adduct inventories over the interaction as and enables of and of the proteins in the and However, of the of protein adduction in the target proteome and cell damage across the protein interaction of the target proteome are in as indicated by in Fig. The of these are to and patterns for cell and electrophiles are with these of for protein adduct inventories as a of electrophile that electrophile concentrations impact protein for the An and of the by a in Fig. the electrophile for protein adduction in cells in which in The of proteins in the were in the cell of to in but to and in The impact of and on proteins a susceptibility of this in expression of these proteins in both electrophiles selectively alkylated proteins in protein interaction networks, we this of damage could selectively impact functional biological We used to biological of and protein targets for electrophile in both cell models of the are in supplemental protein targets in reactivity were for biological targets in cells were for metabolic processes and and targets in both and cells were for protein in both cell and electrophile targets were for processes involved in cytoskeletal and and targets were for processes involved in protein synthesis and biological processes involved in cytoskeletal mapped to both and processes processes associated with protein synthesis and turnover protein the and of protein targets are alkylated at the electrophile significant damage to cytoskeletal systems at electrophile damage to systems associated with protein synthesis and turnover at toxic electrophile the reported of protein damage in intact cells and is the to the accumulation of damage as from to toxic electrophile most important is that electrophiles target functional protein systems in a Proteins in the susceptibility were for biological processes associated with cytoskeletal proteins in the susceptibility were associated with protein synthesis and Damage to targets coincides with the onset of cell which suggests that damage to these protein systems injury. Damage to such as cytoskeletal protein at electrophile concentrations and may be The of proteins to electrophile alkylation suggests that reactivity electrophiles may functions. The data that damage susceptibility functional protein and a of critical protein for by (10Dennehy M.K. Richards K.A. Wernke G.R. Shyr Y. Liebler D.C. Cytosolic and nuclear protein targets of thiol-reactive electrophiles.Chem. Res. Toxicol. 2006; 19: 20-29Crossref PubMed Scopus (133) Google Scholar, 11Shin N.Y. Liu Q. Stamer S.L. Liebler D.C. Protein targets of reactive electrophiles in human liver microsomes.Chem. Res. Toxicol. 2007; 20: 859-867Crossref PubMed Scopus (80) Google Scholar, 12Wong H.L. Liebler D.C. Mitochondrial protein targets of thiol-reactive electrophiles.Chem. Res. Toxicol. 2008; 21: 796-804Crossref PubMed Scopus (69) Google Scholar, 13Codreanu S.G. Zhang B. Sobecki S.M. Billheimer D.D. Liebler D.C. Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal.Mol. Cell. Proteomics. 2009; 8: 670-680Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, Marnett L.J. Liebler D.C. to electrophile adduction of by Res. Toxicol. 2011; PubMed Scopus Google Scholar) and others (9Weerapana E. Simon G.M. Cravatt B.F. Disparate proteome reactivity profiles of carbon electrophiles.Nat. Chem. Biol. 2008; 4: 405-407Crossref PubMed Scopus (195) Google Scholar, E. C. Simon G.M. S. Cravatt B.F. reactivity profiling functional in PubMed Scopus Google Scholar, J.A. Z. of protein by in a of liver Pharmacol. Exp. Ther. 2005; PubMed Scopus Google Scholar, protein adducts of in human protein is by Res. Toxicol. PubMed Scopus Google Scholar, Cipollina C. T.K. Freeman B.A. F.J. generates from Chem. Biol. PubMed Scopus Google Scholar) identified as the targets of used quantitative reactivity profiling of human cell to a subset of protein that were for or functional E. C. Simon G.M. S. Cravatt B.F. reactivity profiling functional in PubMed Scopus Google Scholar). We identified electrophile protein targets that these and that they were protein targets in the by GSH However, in most of these proteins were or which that GSH and reactivity as of protein damage GSH profiles of covalent protein alkylation in both the and cell the GSH protein adduction and toxicity suggests that GSH the but the which have most protein targets are alkylated only at the electrophile in which have only most protein targets are alkylated at electrophile concentrations is with the of GSH as a critical (15DeLeve L.D. Kaplowitz N. Glutathione metabolism and its role in hepatotoxicity.Pharmacol. Ther. 1991; 52: 287-305Crossref PubMed Scopus (436) Google Scholar). electrophile concentrations were in the cells to produce a of protein adduction as in the These in susceptibility to covalent binding are that the for both and in both cell models are K.A. Marnett L.J. analysis of lipid electrophiles associated with and Res. Toxicol. 2011; PubMed Scopus Google Scholar). of a of protein damage thus for the onset of injury. cells at and aONE, and proteins were in cells at the electrophile only and proteins were and these only to and at Thus, the adduction in the toxic for cells was only of that in GSH at toxic electrophile concentrations was which is over in cells and toxic electrophile the of protein adduction in both cell is significant adduction of proteins involved in protein synthesis and Thus, of protein damage to critical as to protein alkylation to with cell of damage susceptibility may represent the of functional protein systems to from and cells to Mitochondrial systems are highly resistant to alkylation and are to Damage to protein and turnover systems only at toxic electrophile concentrations and the lower reactivity of these systems may these critical functions. protein and metabolic the most systems and the of cells to damage to these may be a key to cell death at the electrophile concentrations we we have activation of stress, and stress signaling responses at sublethal concentrations in cells J.D. Marnett L.J. in gene expression by the lipid Res. Toxicol. 2005; PubMed Scopus Google Scholar, Marnett L.J. critical role for protein and of Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, Marnett L.J. expression in cells of Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, damage to cytoskeletal systems may be by responses that contribute to cell on alkylation damage by analogs of lipid electrophiles associated with oxidative stress, an is a systems for susceptibility to oxidative damage redox that for alkylation damage. that protein may have to functional to oxidative stress E. C. Simon G.M. S. Cravatt B.F. reactivity profiling functional in PubMed Scopus Google Scholar). could be with studies of redox such as and of which are to analysis by probes and Click analysis and for Rev. PubMed Scopus Google Scholar, of Proteomics. PubMed Scopus Google Scholar). Studies to could a systems the accumulation of redox in models of oxidative damage. data to longstanding drug toxicity. covalent protein alkylation is a of many hepatotoxic for the of binding distinguish toxic from binding J.N. Kelly J.M. Tripathy S. Zhao S.X. Lam W.W. Kalgutkar A.S. Obach R.S. Can in vitro metabolism-dependent covalent binding data distinguish hepatotoxic from nonhepatotoxic drugs? An analysis using human hepatocytes and liver S-9 fraction.Chem. Res. Toxicol. 2009; 22: 332-340Crossref PubMed Scopus (113) Google Scholar, J.N. Obach R.S. Kalgutkar A.S. of the potential of the and with a and Metab. Dispos. 2008; PubMed Scopus Google Scholar). data that covalent binding may largely be damage to cytoskeletal and protein toxic covalent binding by protein synthesis and and studies with molecules for toxic and could these and provide a for protein alkylation in drug safety data with an to and protein and a are Click the the and the The are in the with