Keap1 cysteine 288 as a potential target for diallyl trisulfide-induced Nrf2 activation

Sanghyun Kim; Hee-Geum Lee; Sin-Aye Park; Joydeb Kumar Kundu; Young-Sam Keum; Young-Nam Cha; Hye-Kyung Na; Young-Joon Surh

doi:10.1371/journal.pone.0085984

Keap1 cysteine 288 as a potential target for diallyl trisulfide-induced Nrf2 activation

PLoS One. 2014 Jan 28;9(1):e85984. doi: 10.1371/journal.pone.0085984. eCollection 2014.

Authors

Sanghyun Kim¹, Hee-Geum Lee¹, Sin-Aye Park¹, Joydeb Kumar Kundu², Young-Sam Keum³, Young-Nam Cha⁴, Hye-Kyung Na⁵, Young-Joon Surh⁶

Affiliations

¹ Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea.
² College of Pharmacy, Keimyung University, Daegu, South Korea.
³ College of Pharmacy, Dongguk University, Ilsan, South Korea.
⁴ College of Medicine, Inha University, Incheon, South Korea.
⁵ Department of Food and Nutrition, Sungshin Women's University, Seoul, South Korea.
⁶ 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.

Abstract

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

Nad(p)h: 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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adaptor Proteins, Signal Transducing / genetics
Adaptor Proteins, Signal Transducing / metabolism*
Allyl Compounds / pharmacology*
Animals
Blotting, Western
Cell Line
Cells, Cultured
Cysteine / chemistry*
Cytoskeletal Proteins / genetics
Cytoskeletal Proteins / metabolism*
Female
Humans
Immunohistochemistry
Kelch-Like ECH-Associated Protein 1
Mice
NF-E2-Related Factor 2 / genetics
NF-E2-Related Factor 2 / metabolism*
Sulfides / pharmacology*

Substances

Adaptor Proteins, Signal Transducing
Allyl Compounds
Cytoskeletal Proteins
Keap1 protein, mouse
Kelch-Like ECH-Associated Protein 1
NF-E2-Related Factor 2
Sulfides
diallyl trisulfide
Cysteine

Grants and funding

This work was supported by the Global Core Research Center grant from the National Research Foundation, Republic of Korea. The funders have no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.