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Sites of alkylation of human Keap1 by natural chemoprevention agents

  • Yan Luo
  • Aimee L. Eggler
  • Dongting Liu
  • Guowen Liu
  • Andrew D. Mesecar
  • Richard B. van Breemen
Articles

Abstract

Under basal conditions, the interaction of the cytosolic protein Keap1 (Kelch-like ECH-associated protein 1) with the transcription factor nuclear factor-E2-related factor 2 (Nrf2) results in a low level of expression of cytoprotective genes whose promoter region contains the antioxidant response element (ARE). Alkylation of one or more of the 27 cysteine sulfhydryl groups of human Keap1 is proposed to lead to Nrf2 nuclear accumulation, to upregulation of cytoprotective gene expression by the ARE, and to prevention of degenerative diseases, such as cancer. Therefore, identification of the most reactive of these cysteine residues toward specific electrophiles should help clarify this mechanism of cancer prevention, also known as chemoprevention. To address this issue, preliminary analyses of tryptic digests of Keap1 alkylated by the model electrophile 1-biotinamido-4-(4′-[maleimidoethyl-cyclohexane]-carboxamido) butane were carried out using liquid chromatographic-tandem mass spectrometry (LC-MS/MS) with a cylindrical ion trap mass spectrometer and also using LC-MS/MS with a hybrid linear ion trap FT ICR mass spectrometer. Because the FT ICR instrument provided more complete peptide sequencing coverage and enabled the identification of more alkylated cysteine residues, only this instrument was used in subsequent studies of Keap1 alkylation by three electrophilic natural products that can upregulate the ARE, xanthohumol, isoliquiritigenin, and 10-shogaol. Among the various cysteine residues of Keap1, C151 was most reactive toward these three electrophiles. These in vitro results agree with evidence from in vivo experiments, and indicate that C151 is the most important site of alkylation on Keap1 by chemoprevention agents that function by activating the ARE through Nrf2.

Keywords

Sulforaphane Ylated Ebselen Antioxidant Response Element Xanthohumol 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Itoh, K.; Wakabayashi, N.; Katoh, Y.; Ishii, T.; Igarashi, K.; Engel, J. D.; Yamamoto, M. Keap1 Represses Nuclear Activation of Antioxidant Responsive Elements by Nrf2 through Binding to the Amino-Terminal Neh2 Domain. Genes Dev. 1999, 1, 76–86.CrossRefGoogle Scholar
  2. 2.
    Dinkova-Kostova, A. T.; Holtzclaw, W. D.; Cole, R. N.; Itoh, K.; Wakabayashi, N.; Katoh, Y.; Yamamoto, M.; Talalay, P. Direct Evidence That Sulfhydryl Groups of Keap1 Are the Sensors Regulating Induction of Phase 2 Enzymes that Protect Against Carcinogens and Oxidants. Proc. Natl. Acad. Sci. U.S.A. 2002, 18, 11908–11913.CrossRefGoogle Scholar
  3. 3.
    Zhang, D. D.; Hannink, M. Distinct Cysteine Residues in Keap1 are Required for Keap1-Dependent Ubiquitination of Nrf2 and for Stabilization of Nrf2 by Chemopreventive Agents and Oxidative Stress. Mol. Cell. Biol. 2003, 22, 8137–8151.CrossRefGoogle Scholar
  4. 4.
    Talalay, P.; Fahey, J. W.; Holtzclaw, W. D.; Prestera, T.; Zhang, Y. Chemoprotection Against Cancer by Phase 2 Enzyme Induction. Toxicol. Lett. 1995, 75, 173–179.CrossRefGoogle Scholar
  5. 5.
    Cullinan, S. B.; Gordan, J. D.; Jin, J.; Harper, J. W.; Diehl, J. A. The Keap1-BTB Protein Is an Adaptor that Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase. Mol. Cell. Biol. 2004, 19, 8477–8486.CrossRefGoogle Scholar
  6. 6.
    Furukawa, M.; Xiong, Y. BTB Protein Keap1 Targets Antioxidant Transcription Factor Nrf2 for Ubiquitination by the Cullin 3-Roc1 Ligase. Mol. Cell. Biol. 2005, 1, 162–171.CrossRefGoogle Scholar
  7. 7.
    Zhang, D. D.; Lo, S. C.; Cross, J. V.; Templeton, D. J.; Hannink, M. Keap1 is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex. Mol. Cell. Biol. 2004, 24, 10941–10953.CrossRefGoogle Scholar
  8. 8.
    Itoh, K.; Wakabayashi, N.; Katoh, Y.; Ishii, T.; O’Connor, T.; Yamamoto, M. Keap1 Regulates both Cytoplasmic-Nuclear Shuttling and Degradation of Nrf2 in Response to Electrophiles. Genes Cells 2003, 4, 379–391.CrossRefGoogle Scholar
  9. 9.
    Zipper, L. M.; Mulcahy, R. T. The Keap1 BTB/POZ Dimerization Function is Required to Sequester Nrf2 in Cytoplasm. J. Biol. Chem. 2002, 39, 36544–36552.CrossRefGoogle Scholar
  10. 10.
    Dinkova-Kostova, A. T.; Holtzclaw, W. D.; Kensler, T. W. The Role of Keap1 in Cellular Protective Responses. Chem. Res. Toxicol. 2005, 12, 1779–1791.CrossRefGoogle Scholar
  11. 11.
    Wakabayashi, N.; Dinkova-Kostova, A. T.; Holtzclaw, W. D.; Kang, M. I.; Kobayashi, A.; Yamamoto, M.; Kensler, T. W.; Talalay, P. Protection Against Electrophile and Oxidant Stress by Induction of the Phase 2 Response: Fate of Cysteines of the Keap1 Sensor Modified by Inducers. Proc. Natl. Acad. Sci. U.S.A. 2004, 7, 2040–2045.CrossRefGoogle Scholar
  12. 12.
    Hong, F.; Freeman, M. L.; Liebler, D. C. Identification of Sensor Cysteines in Human Keap1 Modified by the Cancer Chemopreventive Agent Sulforaphane. Chem. Res. Toxicol. 2005, 12, 1917–1926.CrossRefGoogle Scholar
  13. 13.
    Hong, F.; Sekhar, K. R.; Freeman, M. L.; Liebler, D. C. Specific Patterns of Electrophile Adduction Trigger Keap1 Ubiquitination and Nrf2 Activation. J. Biol. Chem. 2005, 36, 31768–31775.CrossRefGoogle Scholar
  14. 14.
    Gerhauser, C. Broad Spectrum Anti-Infective Potential of Xanthohumol from Hop (Humulus Lupulus L.) in Comparison with Activities of Other Hop Constituents and Xanthohumol Metabolites. Mol. Nutr. Food Res. 2005, 9, 827–831.CrossRefGoogle Scholar
  15. 15.
    Gerhauser, C. Beer Constituents as Potential Cancer Chemopreventive Agents. Eur. J. Cancer 2005, 13, 1941–1954.CrossRefGoogle Scholar
  16. 16.
    Stevens, J. F.; Page, J. E. Xanthohumol and Related Prenylflavonoids from Hops and Beer: To Your Good Health! Phytochemistry 2004, 10, 1317–1330.CrossRefGoogle Scholar
  17. 17.
    Albini, A.; Dell’Eva, R.; Vene, R.; Ferrari, N.; Buhler, D. R.; Noonan, D. M.; Fassina, G. Mechanisms of the Antiangiogenic Activity by the Hop Flavonoid Xanthohumol: NF-kappaB and Akt as Targets. FASEB J. 2006, 3, 527–529.Google Scholar
  18. 18.
    Kim, D. C.; Choi, S. Y.; Kim, S. H.; Yun, B. S.; Yoo, I. D.; Reddy, N. R.; Yoon, H. S.; Kim, K. T. Isoliquiritigenin Selectively Inhibits H(2) Histamine Receptor Signaling. Mol. Pharmacol. 2006, 2, 493–500.CrossRefGoogle Scholar
  19. 19.
    Jang, D. S.; Park, E. J.; Kang, Y. H.; Hawthorne, M. E.; Vigo, J. S.; Graham, J. G.; Cabieses, F.; Fong, H. H.; Mehta, R. G.; Pezzuto, J. M.; Kinghorn, A. D. Potential Cancer Chemopreventive Flavonoids from the Stems of Tephrosia Toxicaria. J. Nat. Prod. 2003, 9, 1166–1170.CrossRefGoogle Scholar
  20. 20.
    Baba, M.; Asano, R.; Takigami, I.; Takahashi, T.; Ohmura, M.; Okada, Y.; Sugimoto, H.; Arika, T.; Nishino, H.; Okuyama, T. Studies on Cancer Chemoprevention by Traditional Folk Medicines: XXV. Inhibitory Effect of Isoliquiritigenin on Azoxymethane-induced Murine Colon Aberrant Crypt Focus Formation and Carcinogenesis. Biol. Pharm. Bull. 2002, 2, 247–250.CrossRefGoogle Scholar
  21. 21.
    Yamazaki, S.; Morita, T.; Endo, H.; Hamamoto, T.; Baba, M.; Joichi, Y.; Kaneko, S.; Okada, Y.; Okuyama, T.; Nishino, H.; Tokue, A. Isoliquiritigenin Suppresses Pulmonary Metastasis of Mouse Renal Cell Carcinoma. Cancer Lett. 2002, 1, 23–30.CrossRefGoogle Scholar
  22. 22.
    Cuendet, M.; Oteham, C. P.; Moon, R. C.; Pezzuto, J. M. Quinone Reductase Induction as a Biomarker for Cancer Chemoprevention. J. Nat. Prod. 2006, 3, 460–463.CrossRefGoogle Scholar
  23. 23.
    Iwasaki, Y.; Morita, A.; Iwasawa, T.; Kobata, K.; Sekiwa, Y.; Morimitsu, Y.; Kubota, K.; Watanabe, T. A Nonpungent Component of Steamed Ginger—[10]-Shogaol—Increases Adrenaline Secretion via the Activation of TRPV1. Nutr. Neurosci. 2006, 3–4, 169–178.CrossRefGoogle Scholar
  24. 24.
    Burton, A. Chemoprevention: Eat Ginger, Rub on Pomegranate. Lancet Oncol. 2003, 12, 715.CrossRefGoogle Scholar
  25. 25.
    Ihlaseh, S. M.; de Oliveira, M. L.; Teran, E.; de Camargo, J. L.; Barbisan, L. F. Chemopreventive Property of Dietary Ginger in Rat Urinary Bladder Chemical Carcinogenesis. World J. Urol. 2006, 5, 591–596.CrossRefGoogle Scholar
  26. 26.
    Manju, V.; Nalini, N. Chemopreventive Efficacy of Ginger, a Naturally Occurring Anticarcinogen during the Initiation, Post-Initiation Stages of 1,2 Dimethylhydrazine-induced Colon Cancer. Clin. Chim. Acta 2005, 1–2, 60–67.CrossRefGoogle Scholar
  27. 27.
    Park, E. J.; Pezzuto, J. M. Botanicals in Cancer Chemoprevention. Cancer Metastasis Rev. 2002, 3–4, 231–255.CrossRefGoogle Scholar
  28. 28.
    Dietz, B. M.; Kang, Y. H.; Liu, G.; Eggler, A. L.; Yao, P.; Chadwick, L. R.; Pauli, G. F.; Farnsworth, N. R.; Mesecar, A. D.; van Breemen, R. B.; Bolton, J. L. Xanthohumol Isolated from Humulus Lupulus Inhibits Menadione-induced DNA Damage through Induction of Quinone Reductase. Chem. Res. Toxicol. 2005, 8, 1296–1305.CrossRefGoogle Scholar
  29. 29.
    Tao, Y. Characterization of Cyclooxygenase-2 Inhibitors as Anti-Inflammatory Agents from Ginger Dietary Supplements and In Vitro Metabolism Studies of Gingerol-Related Compounds, Ph.D. dissertation; University of Illinois at Chicago, 2007; p. 127.Google Scholar
  30. 30.
    Eggler, A. L.; Liu, G.; Pezzuto, J. M.; van Breemen, R. B.; Mesecar, A. D. Modifying Specific Cysteines of the Electrophile-Sensing Human Keap1 Protein Is Insufficient to Disrupt Binding to the Nrf2 Domain Neh2. Proc. Natl. Acad. Sci. U.S.A. 2005, 29, 10070–10075.CrossRefGoogle Scholar
  31. 31.
    Rybak, J. N.; Scheurer, S. B.; Neri, D.; Elia, G. Purification of Biotinylated Proteins on Streptavidin Resin: A Protocol for Quantitative Elution. Proteomics 2004, 8, 2296–2299.CrossRefGoogle Scholar
  32. 32.
    Talalay, P.; De Long, M. J.; Prochaska, H. J. Identification of a Common Chemical Signal Regulating the Induction of Enzymes That Protect Against Chemical Carcinogenesis. Proc. Natl. Acad. Sci. U.S.A. 1988, 21, 8261–8265.CrossRefGoogle Scholar
  33. 33.
    Xue, F.; Cooley, L. Kelch Encodes a Component of Intercellular Bridges in Drosophila Egg Chambers. Cell 1993, 5, 681–693.CrossRefGoogle Scholar
  34. 34.
    Santos, R. L.; Hassan, M. J.; Sikandar, S.; Lee, K.; Ali, G.; Martin, P. E., Jr.; Wambangco, M. A.; Ahmad, W.; Leal, S. M. DFNB68, a Novel Autosomal Recessive Non-Syndromic Hearing Impairment Locus at Chromosomal Region 19p13. 2. Hum. Genet. 2006, 1, 85–92.CrossRefGoogle Scholar
  35. 35.
    Sakurai, T.; Kanayama, M.; Shibata, T.; Itoh, K.; Kobayashi, A.; Yamamoto, M.; Uchida, K.; Ebselen, A. Seleno-Organic Antioxidant, as an Electrophile. Chem. Res. Toxicol. 2006, 9, 1196–1204.CrossRefGoogle Scholar
  36. 36.
    Eggler, A. L.; Luo, Y.; van Breemen, R. B.; Mesecar, A. D. Identification of the Highly Reactive Cysteine 151 in the Chemopreventive Agent-Sensor Keap1 Protein is Method-Dependent. Chem. Res. Toxicol. 2007, 10 (E-pub ahead of print).Google Scholar

Copyright information

© American Society for Mass Spectrometry 2007

Authors and Affiliations

  • Yan Luo
    • 1
  • Aimee L. Eggler
    • 1
  • Dongting Liu
    • 1
  • Guowen Liu
    • 1
  • Andrew D. Mesecar
    • 1
  • Richard B. van Breemen
    • 1
  1. 1.Department of Medicinal Chemistry and PharmacognosyUniversity of Illinois College of PharmacyChicagoUSA

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