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.     

p62/SQSTM1 is required for the protection against endoplasmic reticulum stress-induced apoptotic cell death

Endoplasmic reticulum (ER) stress is triggered by various cellular stresses that disturb protein folding or calcium homeostasis in the ER. To cope with these stresses, ER stress activates the unfolded protein response (UPR) pathway, but unresolved ER stress induces reactive oxygen species (ROS) accumulation leading to apoptotic cell death. However, the mechanisms that underlie protection from ER stress-induced cell death are not clearly defined. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway plays a crucial role in the protection of cells against ROS-mediated oxidative damage. Keap1 acts as a negative regulator of Nrf2 activation. In this study, we investigated the role of the Nrf2-Keap1 pathway in protection from ER stress-induced cell death using tunicamycin (TM) as an ER stress inducer. We found that Nrf2 is an essential protein for the prevention from TM-induced apoptotic cell death and its activation is driven by autophagic Keap1 degradation. Furthermore, ablation of p62, an adapter protein in the autophagy process, attenuates the Keap1 degradation and Nrf2 activation that was induced by TM treatment, and thereby increases susceptibility to apoptotic cell death. Conversely, reinforcement of p62 alleviated TM-induced cell death in p62-deficient cells. Taken together, these results demonstrate that p62 plays an important role in protecting cells from TM-induced cell death through Nrf2 activation.     

氧化和化学应激的防御性转导通路——Nrf2/ARE

Nrf2/ARE是近年新发现的机体抵抗内外界氧化和化学等刺激的防御性转导通路．生理条件下,NF-E2相关因子2(Nrf2,NF-E2-related factor 2)在细胞质中与Keap1结合处于非活性、易降解的状态.在内外界自由基和化学物质刺激时,Keap1的构象改变或者Nrf2直接被磷酸化,导致Nrf2与Keap1解离而活化．活化的Nrf2进入细胞核,与抗氧化反应元件(ARE)结合,启动ARE下游的Ⅱ相解毒酶、抗氧化蛋白、蛋白酶体/分子伴侣等基因转录和表达以抵抗内外界的有害刺激．MAPK、PI3K/AKT、PKC等信号通路分子广泛参与了Nrf2的活化和核转位过程,但是具体何种通路被激动、何种通路发挥主导作用,取决于刺激物种类、刺激方式和细胞类型．本文就Nrf2分子结构、Nrf2活化机制、Nrf2/ARE调控的下游基因、与Nrf2相关的信号通路分子以及其在肿瘤、炎症、衰老等应用领域的最新进展进行综述．     

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.     

核转录因子相关因子2的活化在肿瘤代谢中的作用

核转录因子(NF-E2)相关因子2（nuclear factor erythroid 2 related factor 2, Nrf2）是细胞应对外界应激的主要调控因子,通过调控多种靶基因的表达,在生理条件下减轻氧化应激,维持细胞稳态。其上游受多种因素调控,包括氧化与亲电应激、外界营养状态、细胞内代谢中间产物和能量状态等。在肿瘤细胞中,异常活跃的Nrf2使其抗氧化能力增强,并且通过介导代谢重编程（metabolic reprogramming）,促进肿瘤细胞增殖和生长。Keap1 (Kelch-like ECH-associated protein 1)是氧化和亲电应激感受器,通过募集泛素降解系统,对Nrf2的活性起主要调控作用。本文介绍Keap1依赖与非依赖条件下Nrf2的活化途径,着重介绍在肿瘤中Nrf2的异常活化,以及如何调控代谢重编程进而调节肿瘤细胞的合成代谢,最终促进肿瘤的进展。     

Molecular basis of the Keap1–Nrf2 system

Nrf2 (NF-E2-related factor 2) is a master regulator of cellular responses against environmental stresses. Nrf2 induces the expression of detoxification and antioxidant enzymes, and Keap1 (Kelch-like ECH-associated protein 1), an adaptor subunit of Cullin 3-based E3 ubiquitin ligase, regulates Nrf2 activity. Keap1 also acts as a sensor for oxidative and electrophilic stresses. Keap1 retains multiple sensor cysteine residues that detect various stress stimuli. Increasing attention has been paid to the roles that Nrf2 plays in the protection of our bodies against drug toxicity and stress-induced diseases. On the other hand, Nrf2 is found to promote both oncogenesis and cancer cell resistance against chemotherapeutic drugs. Thus, although Nrf2 acts to protect our body from deleterious stresses, cancer cells hijack the Nrf2 activity to support their malignant growth. Nrf2 has emerged as a new therapeutic target, and both inducers and inhibitors of Nrf2 are awaited. Studies challenging the molecular basis of the Keap1–Nrf2 system functions are now critically important to improve translational studies of the system. Indeed, recent studies identified cross talk between Nrf2 and other signaling pathways, which provides new insights into the mechanisms by which the Keap1–Nrf2 system serves as a potent regulator of our health and disease.     

Nrf2及天然产物导向的Nrf2激活剂研究进展

氧化应激能够破坏细胞内氧化还原平衡,造成系统和组织损伤,最终引起一系列疾病的产生.转录因子E2相关因子2(Nrf2),受Kelch样环氧氯丙烷相关蛋白1(Keap1)蛋白的调控,是细胞氧化应激反应中的关键因子,在氧化应激条件下,Nrf2从Keap1中分离,然后进入细胞核与抗氧化反应元件(ARE)结合,增加了Ⅱ相解毒酶的...     

Caffeic acid phenethyl ester activation of Nrf2 pathway is enhanced under oxidative state: Structural analysis and potential as a pathologically targeted therapeutic agent in treatment of colonic inflammation

Caffeic acid phenethyl ester (CAPE) is a polyphenolic natural product that possesses numerous biological activities including anti-inflammatory effects. CAPE-mediated nuclear factor-erythroid 2 p45 (NF-E2)-related factor 2 (Nrf2) activation is likely responsible for some of its biological effects. CAPE was chemically modified to yield CAPE analogues that were subjected to experiments examining cellular Nrf2 activity. CAPE and the CAPE analogue with a catechol moiety, but not the other analogues, activated the Nrf2 pathway. In addition, only biotin-labeled CAPE analogues with the catechol moiety precipitated Kelch-like ECH associated protein 1 (Keap1) when incubated with cell lysates and streptavidin agarose beads. Sodium hypochlorite (NaOCl) oxidation of the catechol moiety in CAPE produced an oxidized, electrophilic form of CAPE (Oxi-CAPE) and greatly enhanced the ability of CAPE to activate Nrf2 and to bind to Keap1. Rectal administration of CAPE ameliorated 2,4,6-trinitrobenzene sulfonic acid-induced rat colitis and activated the Nrf2 pathway in the inflamed colon, and incubation of CAPE in the lumen of the inflamed distal colon generated Oxi-CAPE. However, these biological effects and chemical change of CAPE were not observed in the normal colon. Our data suggest that CAPE requires the catechol moiety for the oxidation-enhanced activation of the Nrf2 pathway and has potential as a pathologically targeted Nrf2-activating agent that is exclusively activated in pathological states with oxidative stress such as colonic inflammation.