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Protein-selective capture to analyze electrophile adduction of hsp90 by 4-hydroxynonenal.

ABSTRACT: The analysis of protein modification by electrophiles is a challenging problem. Most reported protein-electrophile adducts have been characterized from in vitro reactions or through affinity capture of the adduct moiety, which enables global analyses but is poorly suited to targeted studies of specific proteins. We employed a targeted molecular probe approach to study modifications of the molecular chaperone heat shock protein 90 (Hsp90), which regulates diverse client proteins. Noncovalent affinity capture with a biotinyl-geldanamycin probe isolated both isoforms of the native protein (Hsp90? and Hsp90?) from human RKO colorectal cancer cells. Geldanamycin-biotin capture afforded higher purity Hsp90 than did immunoprecipitation and enabled detection of endogenously phosphorylated protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We applied this approach to map and quantify adducts formed on Hsp90 by 4-hydroxynonenal (HNE) in RKO cells. LC-MS/MS analyses of tryptic digests by identified His(450) and His(490) of Hsp90? as having a 158 Da modification, corresponding to NaBH(4)-reduced HNE adducts. Five histidine residues were also adducted on Hsp90?: His(171), His(442), His(458), His(625), and His(632). The rates of adduction at these sites were determined with Hsp90 protein in vitro and with Hsp90 in HNE-treated cells with a LC-MS/MS-based, label-free relative quantitation method. During in vitro and cell treatment with HNE, residues on Hsp90? and Hsp90? displayed adduction rates ranging from 3.0 × 10(-5) h(-1) to 1.08 ± 0.17 h(-1). Within the middle client-binding domain of Hsp90?, residue His(450) demonstrated the most rapid adduction with k(obs) of 1.08 ± 0.17 h(-1) in HNE-treated cells. The homologous residue on Hsp90?, His(442), was adducted more rapidly than the N-terminal residue, His(171), despite very similar predicted pK(a) values of both residues. The Hsp90 middle client-binding domain thus may play a signicant role in HNE-mediated disruption of Hsp90-client protein interactions. The results illustrate the utility of a protein-selective affinity capture approach for targeted analysis of electrophile adducts and their biological effects.

SUBMITTER: Connor RE

PROVIDER: S-EPMC3155980 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Protein-selective capture to analyze electrophile adduction of hsp90 by 4-hydroxynonenal.

Connor Rebecca E RE Marnett Lawrence J LJ Liebler Daniel C DC

Chemical research in toxicology 20110727 8

The analysis of protein modification by electrophiles is a challenging problem. Most reported protein-electrophile adducts have been characterized from in vitro reactions or through affinity capture of the adduct moiety, which enables global analyses but is poorly suited to targeted studies of specific proteins. We employed a targeted molecular probe approach to study modifications of the molecular chaperone heat shock protein 90 (Hsp90), which regulates diverse client proteins. Noncovalent affi ...[more]

PMID: 21749116

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Project description:Cholesterol cycles between free cholesterol (unesterified) found predominantly in membranes and cholesteryl esters (CEs) stored in cytoplasmic lipid droplets. Only free cholesterol is effluxed from macrophages via ATP-binding cassette (ABC) transporters to extracellular acceptors. Carboxylesterase 1 (CES1), proposed to hydrolyze CEs, is inactivated by oxon metabolites of organophosphorus pesticides and by the lipid electrophile 4-hydroxynonenal (HNE). We assessed the ability of these compounds to reduce cholesterol efflux from foam cells. Human THP-1 macrophages were loaded with [(3)H]-cholesterol/acetylated LDL and then allowed to equilibrate to enable [(3)H]-cholesterol to distribute into its various cellular pools. The cholesterol-engorged cells were then treated with toxicants in the absence of cholesterol acceptors for 24 h, followed by a 24 h efflux period in the presence of toxicant. A concentration-dependent reduction in [(3)H]-cholesterol efflux via ABCA1 (up to 50%) was found for paraoxon (0.1-10 ?M), whereas treatment with HNE had no effect. A modest reduction in [(3)H]-cholesterol efflux via ABCG1 (25%) was found after treatment with either paraoxon or chlorpyrifos oxon (10 ?M each) but not HNE. No difference in efflux rates was found after treatments with either paraoxon or HNE when the universal cholesterol acceptor 10% (v/v) fetal bovine serum was used. When the re-esterification arm of the CE cycle was disabled in foam cells, paraoxon treatment increased CE levels, suggesting the neutral CE hydrolysis arm of the cycle had been inhibited by the toxicant. However, paraoxon also partially inhibited lysosomal acid lipase, which generates cholesterol for efflux, and reduced the expression of ABCA1 protein. Paradoxically, silencing CES1 expression in macrophages did not affect the percent of [(3)H]-cholesterol efflux. However, CES1 mRNA knockdown markedly reduced cholesterol uptake by macrophages, with SR-A and CD36 mRNA reduced 3- and 4-fold, respectively. Immunoblots confirmed SR-A and CD36 protein downregulation. Together, these results suggest that toxicants, e.g., oxons, may interfere with macrophage cholesterol homeostasis/metabolism.

Dataset Information

Protein-selective capture to analyze electrophile adduction of hsp90 by 4-hydroxynonenal.

Publications

Protein-selective capture to analyze electrophile adduction of hsp90 by 4-hydroxynonenal.

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