Keap1 cysteine 288 as a potential target for diallyl trisulfide-induced Nrf2 activation.
Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) and
quinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.
Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea.
Sanghyun
S
Kim
Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea.
Hee-Geum
HG
Lee
Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea.
Sin-Aye
SA
Park
College of Pharmacy, Keimyung University, Daegu, South Korea.
Joydeb Kumar
JK
Kundu
College of Pharmacy, Dongguk University, Ilsan, South Korea.
Young-Sam
YS
Keum
College of Medicine, Inha University, Incheon, South Korea.
Young-Nam
YN
Cha
Department of Food and Nutrition, Sungshin Women's University, Seoul, South Korea.
Hye-Kyung
HK
Na
Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea ; Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul, South Korea ; Cancer Research Institute, Seoul National University, Seoul, South Korea.
Young-Joon
YJ
Surh
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000498:Allyl Compounds / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D000072019:Kelch-Like ECH-Associated Protein 1; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D013440:Sulfides / Q000494:pharmacology*
D048868:Adaptor Proteins, Signal Transducing; D000498:Allyl Compounds; D003598:Cytoskeletal Proteins; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; D013440:Sulfides; C042577:diallyl trisulfide; D003545:Cysteine