Keap1, the cysteine-based mammalian intracellular sensor for electrophiles and oxidants

The Kelch-like ECH associated protein 1 (Keap1) is a component of a Cullin3-based Cullin-RING E3 ubiquitin ligase (CRL) multisubunit protein complex. Within the CRL, homodimeric Keap1 functions as the Cullin3 adaptor, and importantly, it is also the critical component of the E3 ligase that performs the substrate recognition. The best-characterized substrate of Keap1 is transcription factor NF-E2 p45-related factor 2 (Nrf2), which orchestrates an elaborate transcriptional program in response to environmental challenges caused by oxidants, electrophiles and pro-inflammatory agents, allowing adaptation and survival under stress conditions. Keap1 is equipped with reactive cysteine residues that act as sensors for endogenously produced and exogenously encountered small molecules (termed inducers), which have a characteristic chemical signature, reactivity with sulfhydryl groups. Inducers modify the cysteine sensors of Keap1 and impair its ability to target Nrf2 for ubiquitination and degradation. Consequently, Nrf2 accumulates, enters the nucleus and drives the transcription of its target genes, which encode a large network of cytoprotective proteins. Here we summarize the early studies leading to the prediction of the existence of Keap1, followed by the discovery of Keap1 as the main negative regulator of Nrf2. We then describe the available structural information on Keap1, its assembly with Cullin3, and its interaction with Nrf2. We also discuss the multiple cysteine sensors of Keap1 that allow for detection of a wide range of endogenous and environmental inducers, and provide fine-tuning and tight control of the Keap1/Nrf2 stress-sensing response

The multifaceted role of Nrf2 in mitochondrial function

Oxidative ToxicologyThe transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) is the master regulator of the cellular redox homeostasis. Nrf2 target genes comprise of a large network of antioxidant enzymes, proteins involved in xenobiotic detoxification, repair and removal of damaged proteins, inhibition of inflammation, as well as other transcription factors. In recent years it has emerged that as part of its role as a regulator of cytoprotective gene expression, Nrf2 impacts mitochondrial function. Increased Nrf2 activity defends against mitochondrial toxins. Reduced glutathione, the principal small molecule antioxidant in the mammalian cell and a product of several of the downstream target genes of Nrf2, counterbalances mitochondrial ROS production. The function of Nrf2 is suppressed in mitochondria-related disorders, such as Parkinson's disease and Friedrich's ataxia. Studies using isolated mitochondria and cultured cells have demonstrated that Nrf2 deficiency leads to impaired mitochondrial fatty acid oxidation, respiration and ATP production. Small molecule activators of Nrf2 support mitochondrial integrity by promoting mitophagy and conferring resistance to oxidative stress-mediated permeability transition. Excitingly, recent studies have shown that Nrf2 also affects mitochondrial function in stem cells with implications for stem cell self-renewal, cardiomyocyte regeneration, and neural stem/progenitor cell survival.Peer reviewe

KEAP1 and done? Targeting the NRF2 pathway with sulforaphane:Targeting the NRF2 Pathway with Sulforaphane

Background: Since the re-discovery of sulforaphane in 1992 and the recognition of the bioactivity of this phytochemical, many studies have examined its mode of action in cells, animals and humans. Broccoli, especially as young sprouts, is a rich source of sulforaphane and broccoli-based preparations are now used in clinical studies probing efficacy in health preservation and disease mitigation. Many putative cellular targets are affected by sulforaphane although only one, KEAP1-NRF2 signaling, can be considered a validated target at this time. The transcription factor NRF2 is a master regulator of cell survival responses to endogenous and exogenous stressors.Scope and Approach: This review summarizes the chemical biology of sulforaphane as an inducer of NRF2 signaling and efficacy as an inhibitor of carcinogenesis. It also provides a summary of the current findings from clinical trials using a suite of broccoli sprout preparations on a series of short-term endpoints reflecting a diversity of molecular actions.Key Findings and Conclusions: Sulforaphane, as a pure chemical, protects against chemical-induced skin, oral, stomach, colon, lung and bladder carcinogenesis and in genetic models of colon and prostate carcinogenesis. In many of these settings the antitumorigenic efficacy of sulforaphane is dampened in Nrf2-disrupted animals. Broccoli preparations rich in glucoraphanin or sulforaphane exert demonstrable pharmacodynamic action in over a score of clinical trials. Measures of NRF2 pathway response and function are serving as guideposts for the optimization of dose, schedule and formulation as clinical trials with broccoli-based preparations become more commonplace and more rigorous in design and implementation.</p

Pharmacokinetics and pharmacodynamics of orally administered acetylenic tricyclic <i>bis</i>(cyanoenone), a highly potent Nrf2 activator with a reversible covalent mode of action

AbstractThe acetylenic tricyclic bis(cyanoenone) TBE-31 is a highly potent cysteine targeting compound with a reversible covalent mode of action; its best-characterized target being Kelch-like ECH-associated protein-1 (Keap1), the cellular sensor for oxidants and electrophiles. TBE-31 reacts with cysteines of Keap1, impairing its ability to target nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) for degradation. Consequently, Nrf2 accumulates and orchestrates cytoprotective gene expression. In this study we investigated the pharmacokinetic and pharmacodynamic properties of TBE-31 in C57BL/6 mice. After a single oral dose of 10 μmol/kg (∼200 nmol/animal), the concentration of TBE-31 in blood exhibited two peaks, at 22.3 nM and at 15.5 nM, 40 min and 4 h after dosing, respectively, as determined by a quantitative stable isotope dilution LC-MS/MS method. The AUC0–24h was 195.5 h/nmol/l, the terminal elimination half-life was 10.2 h, and the kel was 0.068 h−1. To assess the pharmacodynamics of Nrf2 activation by TBE-31, we determined the enzyme activity of its prototypic target, NAD(P)H:quinone oxidoreductase 1 (NQO1) and found it elevated by 2.4- and 1.5-fold in liver and heart, respectively. Continuous feeding for 18 days with diet delivering the same daily doses of TBE-31 under conditions of concurrent treatment with the immunosuppressive agent azathioprine had a similar effect on Nrf2 activation without any indications of toxicity. Together with previous reports showing the cytoprotective effects of TBE-31 in animal models of carcinogenesis, our results demonstrate the high potency, efficacy and suitability for chronic administration of cysteine targeting reversible covalent drugs

Abstracts Of The Proceedings And The Posters From The Third Scientific Session Of The Medical College Of Varna

October 2-3, 201