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Protein damage and chemical toxicity

Since the initial discovery by James and Elizabeth Miller in the late 1940s that hepatocarcinogenic dyes became covalently bound to liver proteins, investigators have tried to understand how such protein damage leads to stress responses, toxicity and cell death.  Reactive electrophiles are produced by metabolism of drugs and other chemicals and appear to mediate many of their toxic effects.  This phenomenon also is thought to contribute to many disease processes that involve oxidative stress and inflammation (neurodegenerative diseases, atherosclerosis, cancer, metabolic diseases) because endogenous lipid electrophiles form protein adducts.

The starting point for understanding the effects of protein damage—identifying the protein targets of electrophiles—seemed for many years to be an intractable problem.  The introduction of mass spectrometry-based proteomics in the late 1990s provided a new opportunity to characterize protein damage on a global scale, to map specific adduct sites on proteins and to identify sensors that initiate adaptive and toxic responses.  For a perspective on how MS-based proteomics has transformed this field, see this recent review.

Because electrophile adduction affects a small fraction of the proteome, enrichment strategies are needed to characterize adducts.  We initially employed biotinyl electrophiles as model compounds and developed a two-pronged strategy to identify their protein targets and map the adducts to specific sequences.   Avidin capture of biotinylated (adducted) proteins followed by digestion and shotgun proteomics (A) confidently identifies the captured proteins, but does not efficiently enable mapping of adducts.  Avidin capture after digestion (B) selectively captures only the adducted peptides and enables efficient adduct site mapping.

Since 2005, we have worked with the laboratories of Ned Porter, Larry Marnett, Bing Zhang and Alex Brown on a program project awarded by the National Institute of Environmental Health Sciences.  The overall goal of the program is to characterize the toxic effects of reactive lipid electrophiles, especially the formation and consequences of protein adducts.  We have employed alkynyl lipid electrophiles (see affinity labeled electrophile probes) and shotgun proteomics to identify the protein targets of electrophiles.

An important direction for our work is to integrate protein adduct identifications, protein expression data and transcriptome profiling to identify electrophile response networks that mediate cellular responses to stress and toxicity.  This work is done in collaboration with Bing Zhang’s group.

Inference from network and pathway analysis leads to the identification of specific proteins that may act as sensors for protein damage and triggers for downstream signaling.  We have previously used MS/MS-based mapping to characterize adducts of the sensor protein Keap1 and are applying this approach to study other candidate protein damage sensors.

Representative references from our work and collaborations

Hong, F., Sekhar, K. R., Freeman, M. L., and Liebler, D. C. (2005) Specific patterns of electrophile adduction trigger Keap1 ubiquitination and Nrf2 activation. J. Biol. Chem., 280, 31768-31775.  PubMed

Dennehy, M. K., Richards, K. A., Wernke, G. R., Shyr, Y., and Liebler, D. C. (2006) Cytosolic and nuclear protein targets of thiol-reactive electrophiles. Chem. Res. Toxicol., 19, 20-29.  PubMed

Szapacs, M. E., Riggins, J. N., Zimmerman, L. J., and Liebler, D. C. (2006) Covalent adduction of human serum albumin by 4-hydroxy-2-nonenal: kinetic analysis of competing alkylation reactions. Biochemistry, 45, 10521-10528.  PubMed

Orton, C. R., and Liebler, D. C. (2007) Analysis of protein adduction kinetics by quantitative mass spectrometry: competing adduction reactions of glutathione-S-transferase P1-1 with electrophiles. Chem.-Biol. Interact., 168, 117-127.  PubMed

Shin, N. Y., Liu, Q., Stamer, S. L., and Liebler, D. C. (2007) Protein targets of reactive electrophiles in human liver microsomes. Chem. Res. Toxicol., 20, 859-867.  PubMed

Tallman, K. A., Kim, H. Y., Ji, J. X., Szapacs, M. E., Yin, H., McIntosh, T. J., Liebler, D. C., and Porter, N. A. (2007) Phospholipid-protein adducts of lipid peroxidation: synthesis and study of new biotinylated phosphatidylcholines. Chem. Res. Toxicol., 20, 227-234.  PubMed

Lin, D., Saleh, S., and Liebler, D. C. (2008) Reversibility of covalent electrophile-protein adducts and chemical toxicity. Chem. Res. Toxicol., 21, 2361-2369.  PubMed

Rachakonda, G., Xiong, Y., Sekhar, K. R., Stamer, S. L., Liebler, D. C., and Freeman, M. L. (2008) Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3. Chem. Res. Toxicol., 21, 705-710.  PubMed

Szapacs, M. E., Kim, H. Y., Porter, N. A., and Liebler, D. C. (2008) Identification of proteins adducted by lipid peroxidation products in plasma and modifications of apolipoprotein A1 with a novel biotinylated phospholipid probe. J. Proteome Res., 7, 4237-4246.  PubMed

Vila, A., Tallman, K. A., Jacobs, A. T., Liebler, D. C., Porter, N. A., and Marnett, L. J. (2008) Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives. Chem. Res. Toxicol., 21, 432-444.  PubMed

Wong, H. L., and Liebler, D. C. (2008) Mitochondrial protein targets of thiol-reactive electrophiles. Chem. Res. Toxicol., 21, 796-804.  PubMed

Codreanu, S. G., Zhang, B., Sobecki, S. M., Billheimer, D. D., and Liebler, D. C. (2009) Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal. Mol. Cell. Proteomics, 8, 670-680.  PubMed

Kim, H. Y., Tallman, K. A., Liebler, D. C., and Porter, N. A. (2009) An azido-biotin reagent for use in the isolation of protein adducts of lipid-derived electrophiles by streptavidin catch and photorelease. Mol. Cell. Proteomics, 8, 2080-2089.  PubMed

Dasari, S., Chambers, M. C., Codreanu, S. G., Liebler, D. C., Collins, B. C., Pennington, S. R., Gallagher, W. M., and Tabb, D. L. (2011) Sequence tagging reveals unexpected modifications in toxicoproteomics. Chem. Res. Toxicol., 24, 204-216.  PubMed

Michaelson-Richie, E. D., Ming, X., Codreanu, S. G., Loeber, R. L., Liebler, D. C., Campbell, C., and Tretyakova, N. Y. (2011) Mechlorethamine-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. J. Proteome Res., 10, 2785-2796.  PubMed

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