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.     

Discovery of benzo[g]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor

The Keap1-Nrf2 system is an attractive target for drug discovery regarding various unmet medical needs. Only covalent inhibitors for protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 have been approved or are under clinical trials, but such electrophilic compounds lack selectivity. Therefore, specific non-covalent Keap1-Nrf2 PPI inhibitors are expected to be safer Nrf2 activators. We found a novel class of non-covalent Keap1-Nrf2 PPI inhibitor that has a benzo[g]indole skeleton and an indole-3-hydroxamic acid moiety and that exhibits significant PPI inhibitory activity. Additionally, the benzo[g]indole-3-carbohydrazide derivatives were newly prepared. The benzo[g]indole derivatives showed a stronger Keap1-Nrf2 PPI inhibitory activity than Cpd16, a previously reported non-covalent PPI inhibitor. Moreover, most of the PPI inhibitors showed a high metabolic stability in a human microsome system with a low cytotoxicity against HepG2 cell lines, which suggests that novel benzo[g]indole-type Keap1-Nrf2 PPI inhibitors are expected to be biological tools or lead compounds for Nrf2 activators.     

The Nrf2 activator MIND4-17 protects retinal ganglion cells from high glucose-induced oxidative injury

Diabetic retinopathy (DR) is a leading cause of acquired blindness among adults. High glucose (HG) induces oxidative injury and apoptosis in retinal ganglion cells (RGCs), serving as a primary pathological mechanism of DR. MIND4-17 activates nuclear-factor-E2-related factor 2 (Nrf2) signaling via modifying one cysteine (C151) residue of Kelch-like ECH-associated protein 1 (Keap1). The current study tested its effect in HG-treated primary murine RGCs. We show that MIND4-17 disrupted Keap1–Nrf2 association, leading to Nrf2 protein stabilization and nuclear translocation, causing subsequent expression of key Nrf2 target genes, including heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1. Functional studies showed that MIND4-17 pretreatment significantly inhibited HG-induced cytotoxicity and apoptosis in primary murine RGCs. Reactive oxygen species production and oxidative injury in HG-treated murine RGCs were attenuated by MIND4-17. Nrf2 silencing (by targeted small interfering RNA) or knockout (by CRISPR/Cas9 method) abolished MIND4-17-induced RGC cytoprotection against HG. Additionally, Keap1 knockout or silencing mimicked and abolished MIND4-17-induced activity in RGCs. In vivo, MIND4-17 intravitreal injection activated Nrf2 signaling and attenuated retinal dysfunction by light damage in mice. We conclude that MIND4-17 activates Nrf2 signaling to protect murine RGCs from HG-induced oxidative injury.     

Nrf2 Enhances Cholangiocyte Expansion in Pten-Deficient Livers

Keap1-Nrf2 system plays a central role in the stress response. While Keap1 ubiquitinates Nrf2 for degradation under unstressed conditions, this Keap1 activity is abrogated in response to oxidative or electrophilic stresses, leading to Nrf2 stabilization and coordinated activation of cytoprotective genes. We recently found that nuclear accumulation of Nrf2 is significantly increased by simultaneous deletion of Pten and Keap1, resulting in the stronger activation of Nrf2 target genes. To clarify the impact of the cross talk between the Keap1-Nrf2 and Pten–phosphatidylinositide 3-kinase–Akt pathways on the liver pathophysiology, in this study we have conducted closer analysis of liver-specific Pten::Keap1 double-mutant mice (Pten::Keap1-Alb mice). The Pten::Keap1-Alb mice were lethal by 1 month after birth and displayed severe hepatomegaly with abnormal expansion of ductal structures comprising cholangiocytes in a Nrf2-dependent manner. Long-term observation of Pten::Keap1-Alb::Nrf2^+/− mice revealed that the Nrf2-heterozygous mice survived beyond 1 month but developed polycystic liver fibrosis by 6 months. Gsk3 directing the Keap1-independent degradation of Nrf2 was heavily phosphorylated and consequently inactivated by the double deletion of Pten and Keap1 genes. Thus, liver-specific disruption of Keap1 and Pten augments Nrf2 activity through inactivation of Keap1-dependent and -independent degradation of Nrf2 and establishes the Nrf2-dependent molecular network promoting the hepatomegaly and cholangiocyte expansion.     

p62/SQSTM1 protects against cisplatin-induced oxidative stress in kidneys by mediating the cross talk between autophagy and the Keap1-Nrf2 signalling pathway

Acute kidney injury (AKI) is a major kidney disease associated with poor clinical outcomes. Oxidative stress is predominantly involved in the pathogenesis of AKI. Autophagy and the Keap1-Nrf2 signalling pathway are both involved in the oxidative-stress response. However, the cross talk between these two pathways in AKI remains unknown. Here, we found that autophagy is upregulated during cisplatin-induced AKI. In contrast with previous studies, we observed a marked increase in p62. We also found that p62 knockdown reduces autophagosome formation and the expression of LC3II. To explore the cross talk between p62 and the Keap1-Nrf2 signalling pathway, HK-2 cells were transfected with siRNA targeting Nrf2, and we found that Nrf2 knockdown significantly reduced cisplatin-induced p62 expression. Moreover, p62 knockdown significantly decreased the protein expression of Nrf2, as well as Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase l (NQO1), whereas the expression of kelch-like ECH-associated protein 1 (Keap1) was upregulated. These results indicate that p62 creates a positive feedback loop in the Keap1-Nrf2 signalling pathway. Finally, we examined the role of p62 in cell protection during cisplatin-induced oxidative stress, and we found that p62 silencing in HK-2 cells increases apoptosis and reactive oxygen species (ROS) levels, which further indicates the protective role of p62 under oxidative stress and suggests that the cytoprotection 62 mediated is in part by regulating autophagic activity or the Keap1-Nrf2 signalling pathway. Taken together, our results have demonstrated a reciprocal regulation of p62, autophagy and the Keap1-Nrf2 signalling pathway under oxidative stress, which may be a potential therapeutic target against AKI.     

Physical and Functional Interaction of Sequestosome 1 with Keap1 Regulates the Keap1-Nrf2 Cell Defense Pathway

Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.     

Extract of Ginkgo biloba induces phase 2 genes through Keap1-Nrf2-ARE signaling pathway

The standard extract of Ginkgo biloba (EGb) has been demonstrated to possess remarkable antioxidant activity in both cell lines and animals. However, the molecular mechanism underlying this effect is not fully understood. Phase 2 enzymes play important roles in the antioxidant system by reducing electrophiles and reactive oxygen species (ROS). We demonstrated that EGb induced typical phase 2 genes: glutamate cysteine ligase catalytic subunit (GCLC) and glutathione-S-transferase subunit-P1 (GST-P1), by real-time PCR. To investigate the molecular mechanism of this induction, we used quinone oxidoreductase 1 (NQO1) -- Antioxidant response element (ARE) reporter assay and found that EGb activated the activity of the wild type but not the one with ARE mutated. It indicated that EGb induced these genes through ARE, a cis-acting motif located in the promoter region of nearly all phase 2 genes. Since nuclear factor erythroid 2-related factor 2 (Nrf2) binds ARE to enhance the expression of phase 2 genes, we detected the Nrf2 content in nucleus and found an accumulation of Nrf2 stimulated by EGb. In a further test of Kelch-like ECH-associated protein 1 (Keap1), the repression protein of Nrf2 in the cytosol under resting condition, we found that Keap1 content was inhibited by EGb and then more Nrf2 would be released to translocate into nucleus. Thus, EGb was testified for the first time to induce the phase 2 genes through the Keap1-Nrf2-ARE signaling pathway, which is (or part of) the antioxidant mechanism of EGb.     

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.     

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.     

Structural and mechanistic insights into the Keap1-Nrf2 system as a route to drug discovery

The proteins Keap1 and Nrf2 together act as a cytoprotective mechanism that enables cells to overcome electrophilic and oxidative stress. Research has shown that manipulating this system by modulating the Keap1-Nrf2 interaction either through inhibition at the binding interface or via the covalent modification of Keap1 could provide a powerful therapeutic strategy for a range of diseases. However, despite intensive investigation of the system and significant progress in the development of inhibitory small molecules, there is still much to learn about the pathways associated with the Keap1-Nrf2 system and the structural details underpinning its mechanism of action. In this review, we discuss how a deeper understanding could prove revolutionary in the development of new inhibitors and activators as well as guiding how to best harness Keap1 for targeted protein degradation.