Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1''s ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.     

Oxidative and electrophilic stresses activate Nrf2 through inhibition of ubiquitination activity of Keap1

The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.     

Glucosinolates and isothiocyanates in health and disease

Glucosinolates and isothiocyanates have both been objects of research for more than half a century. Interest in these unique phytochemicals escalated following the discovery that sulforaphane, an isothiocyanate from broccoli, potently induces mammalian cytoprotective proteins through the Keap1-Nrf2-ARE pathway. In parallel with the advances in understanding the molecular regulation of this pathway and its critical role in protection against electrophiles and oxidants, there have been increased efforts toward translating this knowledge to improve human health and combat disease. This review focuses on the animal studies demonstrating the beneficial effects of glucosinolates and isothiocyanates in models of carcinogenesis, and cardiovascular and neurological diseases, as well as on the intervention studies of their safety, pharmacokinetics, and efficacy in humans.     

Effects of different exercise durations on Keap1-Nrf2-ARE pathway activation in mouse skeletal muscle

Abstract

The purpose of this study was to investigate the effects of acute exercise stress on the nuclear factor-erythroid2 p45-related factor 2 (Nrf2)/antioxidant response element (ARE) transactivation, Kelch-like ECH-associated protein 1 (Keap1) cytosolic protein and Nrf2 nucleoprotein expressions, Nrf2 target genes mRNA expressions, and glutathione redox (GSH/GSSG) ratio level; with a particular focus on the changes in Keap1-Nrf2-ARE pathway activation following different durations of exercise. Wild-type mice (C57BL/6J, two months old) were separated into one-hour and six-hour treadmill running groups, as well as a non-exercise control group (n = 10 in each group). Measurements of Nrf2/ARE transactivation, Nrf2 nucleoprotein expressions, Keap1 cytosolic protein expression, Nrf2 target genes’ mRNA expressions (superoxide dismutase-1 [SOD1], superoxide dismutase-2 [SOD2], γ-glutamyl cysteine ligase-modulatory [GCLm], γ-glutamyl cysteine ligase-catalytic [GCLc], glutathione reductase [GR], glutathione peroxidase-1 [Gpx1], catalase [CAT], and hemoxygenase-1 [Ho-1]), and GSH/GSSG ratio were carried out immediately after exercise. The results showed significant increases in Keap1-Nrf2-ARE pathway activation and the mRNA expressions of six measured enzymes in skeletal muscle after six hours of exercise; while in the one-hour exercise group, there was no change in Keap1-Nrf2-ARE pathway activation and only two enzymes’ mRNA expressions were increased. It is suggested that the changes in Keap1-Nrf2-ARE pathway activation and its target genes’ mRNA expressions were dependent on the exercise duration, with longer duration associated with higher responses.     

Nearest neighbor analysis of the SecYEG complex. 1. Identification of a SecY-SecG interface

Integral membrane components SecY, SecE, and SecG of protein translocase form a complex in the Escherichia coli plasma membrane. To characterize subunit interactions of the SecYEG complex, a series of SecY variants having a single cysteine in its cytoplasmic (C1-C6) or periplasmic (P1-P5) domain were subjected to site-specific cross-linking experiments using bifunctional agents with thiol-amine reactivity. Experiments using inverted membrane vesicles revealed specific cross-linkings between a cysteine residue placed in the C2 or C3 domain of SecY and the cytosolic lysine (Lys26) near the first transmembrane segment of SecG. These SecY Cys residues also formed a disulfide bond with an engineered cytosolic cysteine at position 28 of SecG. Thus, the C2-C3 region of SecY is in the proximity of the N-terminal half of the SecG cytoplasmic loop. Experiments using spheroplasts revealed the physical proximity of P2 (SecY) and the C-terminal periplasmic region of SecG. In addition, mutations in secG were isolated as suppressors against a cold-sensitive mutation (secY104) affecting the TM4-C3 boundary of SecY. These results collectively suggest that a C2-TM3-P2-TM4-C3 region of SecY serves as an interface with SecG.     

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 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.     

Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein

Induction of the phase 2 response, a major cellular reaction to oxidative/electrophile stress depends on a protein triad: actin-tethered Keap1 that binds to Nrf2. Inducers react with Keap1 releasing Nrf2 for nuclear translocation and activation of the antioxidant response element (ARE), which regulates phase 2 genes. The primary sensors for inducers are certain uniquely reactive cysteine thiols of Keap1. Recombinant murine Keap1 contains 0.9 zinc atoms per monomer as determined by inductively coupled plasma-optical emission spectrometry: its zinc content depends on the metal composition of the overexpression medium. Simultaneous direct measurement of bound zinc using a pyridazoresorcinol chelator and protein thiol groups using 4,4'-dipyridyl disulfide has established that (i) zinc is bound to reactive cysteine thiols of Keap1 and is displaced stoichiometrically by inducers, (ii) with these cysteines mutated to alanine, the affinity for zinc is reduced by nearly 2 orders of magnitude, and (iii) the association constant of Keap1 for zinc is 1.02 (+/-0.19) x 10(11) M(-)(1), consistent with a Zn(2+) metalloprotein. Co(2+) substitution for Zn(2+) yields an optical spectrum consistent with tetrahedral metal coordination. Coincident binding of inducers and release of zinc alters the conformation of Keap1, as shown by a profound decline of its tryptophan fluorescence and depression of fluorescence of a hydrophobicity probe. Thus, regulation of the phase 2 response involves chemical modification of critical cysteine residues of Keap1, whose reactivity is modulated by zinc binding. Keap1 is a zinc-thiol protein endowed with a delicate switch controlled by both metal-binding and thiol reactivity.