http://www.jbc.org/content/284/20/13291.full.pdf

The Nrf2-Antioxidant Response Element Signaling Pathway and Its Activation by Oxidative Stress

Impaired selective turnover of p62 by autophagy causes severe liver injury accompanied by the formation of p62-positive inclusions and upregulation of detoxifying enzymes. These phenotypes correspond closely to the pathological conditions seen in human liver diseases, including alcoholic hepatitis and hepatocellular carcinoma. However, the molecular mechanisms and pathophysiological processes in these events are still unknown. Here we report the identification of a novel regulatory mechanism by p62 of the transcription factor Nrf2, whose target genes include antioxidant proteins and detoxification enzymes. p62 interacts with the Nrf2-binding site on Keap1, a component of Cullin-3-type ubiquitin ligase for Nrf2. Thus, an overproduction of p62 or a deficiency in autophagy competes with the interaction between Nrf2 and Keap1, resulting in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Our findings indicate that the pathological process associated with p62 accumulation results in hyperactivation of Nrf2 and delineates unexpected roles of selective autophagy in controlling the transcription of cellular defence enzyme genes.

The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1

The multifunctional regulator nuclear factor erythroid 2-related factor (Nrf2) is considered not only as a cytoprotective factor regulating the expression of genes coding for anti-oxidant, anti-inflammatory and detoxifying proteins, but it is also a powerful modulator of species longevity. The vertebrate Nrf2 belongs to Cap ‘n’ Collar (Cnc) bZIP family of transcription factors and shares a high homology with SKN-1 from Caenorhabditis elegans or CncC found in Drosophila melanogaster. The major characteristics of Nrf2 are to some extent mimicked by Nrf2-dependent genes and their proteins including heme oxygenase-1 (HO-1), which besides removing toxic heme, produces biliverdin, iron ions and carbon monoxide. HO-1 and their products exert beneficial effects through the protection against oxidative injury, regulation of apoptosis, modulation of inflammation as well as contribution to angiogenesis. On the other hand, the disturbances in the proper HO-1 level are associated with the pathogenesis of some age-dependent disorders, including neurodegeneration, cancer or macular degeneration. This review summarizes our knowledge about Nrf2 and HO-1 across different phyla suggesting their conservative role as stress-protective and anti-aging factors.

https://link.springer.com/content/pdf/10.1007%2Fs00018-016-2223-0.pdf

Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism

Hydrogen Peroxide Sensing and Signaling

The Keap1–Nrf2 regulatory pathway plays a central role in the protection of cells against oxidative and xenobiotic damage. Under unstressed conditions, Nrf2 is constantly ubiquitinated by the Cul3–Keap1 ubiquitin E3 ligase complex and rapidly degraded in proteasomes. Upon exposure to electrophilic and oxidative stresses, reactive cysteine residues of Keap1 become modified, leading to a decline in the E3 ligase activity, stabilization of Nrf2 and robust induction of a battery of cytoprotective genes. Biochemical and structural analyses have revealed that the intact Keap1 homodimer forms a cherry-bob structure in which one molecule of Nrf2 associates with two molecules of Keap1 by using two binding sites within the Neh2 domain of Nrf2. This two-site binding appears critical for Nrf2 ubiquitination. In many human cancers, missense mutations in KEAP1 and NRF2 genes have been identified. These mutations disrupt the Keap1–Nrf2 complex activity involved in ubiquitination and degradation of Nrf2 and result in constitutive activation of Nrf2. Elevated expression of Nrf2 target genes confers advantages in terms of stress resistance and cell proliferation in normal and cancer cells. Discovery and development of selective Nrf2 inhibitors should make a critical contribution to improved cancer therapy.

https://www.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1365-2443.2010.01473.x

Molecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution.

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.

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

Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous Keap1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, Keap1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of Keap1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole-treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that Keap1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous Keap1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2443.2007.01118.x

Yoriko Watai

Papers

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

Subcellular localization and cytoplasmic complex status of endogenous Keap1.