Monitoring Keap1–Nrf2 interactions in single live cells

Baird, Liam; Swift, Sam; Llères, David; Dinkova‐Kostova, Albena T.

doi:10.1016/j.biotechadv.2014.03.004

Cited by 128 publications

(118 citation statements)

References 118 publications

(105 reference statements)

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“…We speculate that at the concentrations used in the cell studies, RA839 affected mainly the weaker interaction between Keap1 and the DLG motif of Nrf2 and did not lead to a full dissociation. According to the hinge and latch model, as well as to the conformation cycling model, an interference of the interaction between Keap1 and the DLG motif of Nrf2 is already sufficient to prevent the degradation of Nrf2 (6,8). …”

Section: Discussionmentioning

confidence: 99%

“…According to the hinge and latch model, this weakens the interaction between Keap1 and the DLG motif of Nrf2 but does not lead to a release of Nrf2 (6,7). The conformation cycling model postulates a stabilization of the Keap1/Nrf2 interaction by the Keap1 modification, which prevents the release of Nrf2 (8). With that, both models propose that newly synthesized Nrf2 is not degraded but translocates to the nucleus.…”

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confidence: 99%

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Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling

Winkel

Engel

Margerie

et al. 2015

Journal of Biological Chemistry

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The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a K d of ϳ6 M, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 M RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.The transcription factor NF-E2-related factor 2 (Nrf2) 2 is a promising target for the treatment of oxidative and inflammatory stress-related disorders, such as neurodegenerative and microvascular diseases (1-5). Nrf2 regulates the expression of several cytoprotective anti-oxidative and anti-inflammatory proteins by binding to the cis-acting antioxidant response element (ARE) within gene promoters. Nrf2 itself is regulated by the kelch-like ECH-associated protein 1 (Keap1), which is a substrate recognition subunit for a cullin3-based ubiquitin E3 ligase and functions as a sensor for oxidative and electrophilic stress. Structural elements of the Keap1 protein include the C-terminal Nrf2-binding kelch domain, the intervening region, and the broad complex-Tramtrack-Bric-a-Brac domain. Keap1 binds as a dimer via its two Kelch domains to one molecule of Nrf2, specifically to the high affinity ETGE and the low affinity DLG motives at the N terminus of Nrf2. Without stressors, this leads to the ubiquitinylation and subsequent proteolytic degradation of the transcription factor. In the presence of oxidative or electrophilic stress, cysteine residues within the intervening region and broad complex-Tramtrack-Bric-a-Brac domain of Keap1 become modified. According to the hinge and latch model, this weakens the interaction between Keap1 and the DLG motif of Nrf2 but does not lead to a release of Nrf2 (6, 7). The conformation cycling model postulates a stabilization of the Keap1/Nrf2 interaction by the Keap1 modifica...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling

Winkel

Engel

Margerie

et al. 2015

Journal of Biological Chemistry

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“…Keap1 dimerizes and binds Cul3 through its BTB domain (Furukawa and Xiong, 2005), while its Kelch domain is able to interact with the Neh2 domain of Nrf2 (Itoh et al, 1999;McMahon et al, 2006). By a cyclical mechanism, Nrf2 is then ubiquitinated and transferred to the proteasome, where it is degraded, while Keap1 is regenerated Baird et al, 2014) (Figure 1A). In the Two main ubiquitin ligase systems are responsible for targeting Nrf2 for degradation in the proteasome.…”

Section: Nrf2 Structure and Regulationmentioning

confidence: 99%

Nrf2 activation in the treatment of neurodegenerative diseases: a focus on its role in mitochondrial bioenergetics and function

Esteras¹,

Dinkova‐Kostova²,

Abramov³

2016

Biological Chemistry

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132

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Abstract:The nuclear factor erythroid-derived 2 (NF-E2)-related factor 2 (Nrf2) is a transcription factor wellknown for its function in controlling the basal and inducible expression of a variety of antioxidant and detoxifying enzymes. As part of its cytoprotective activity, increasing evidence supports its role in metabolism and mitochondrial bioenergetics and function. Neurodegenerative diseases are excellent candidates for Nrf2-targeted treatments. Most neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia and Friedreich's ataxia are characterized by oxidative stress, misfolded protein aggregates, and chronic inflammation, the common targets of Nrf2 therapeutic strategies. Together with them, mitochondrial dysfunction is implicated in the pathogenesis of most neurodegenerative disorders. The recently recognized ability of Nrf2 to regulate intermediary metabolism and mitochondrial function makes Nrf2 activation an attractive and comprehensive strategy for the treatment of neurodegenerative disorders. This review aims to focus on the potential therapeutic role of Nrf2 activation in neurodegeneration, with special emphasis on mitochondrial bioenergetics and function, metabolism and the role of transporters, all of which collectively contribute to the cytoprotective activity of this transcription factor.

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“…31 Accordingly, we measured the mRNA levels of 3 well-characterized targets of NRF2, the Aldoketo reductase family 1 member C1 (AKR1C1), the Heme Oxygenase 1 (HMOX1), and the NAD(P)H dehydrogenase quinone 1 (NQO1) genes. A 74, 10 and 3 fold change in the mRNA levels of these genes was observed in cells plated under LD_hyper conditions, respectively (Fig.…”

Section: Redox Imbalance Is a Component Of The Dormant Statementioning

confidence: 99%

SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy

et al. 2015

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Abbreviations: BSO, buthionine sulfoximine; CE, cloning efficiency; DMEM-FCS, Dulbecco Modified Essential Medium supplemented with 10% Fetal Calf Serum and penicillin-streptomycin; LD_hyper, low cell density condition in DMEM-FCS medium; LD_hypo, low cell density condition in hypotonic medium made by addition of 25% water to DMEM-FCS; MD_hypo, medium cell density condition in hypotonic medium made by addition of 25% water to DMEM-FCS; MD_hyper, medium cell density condition in DMEM-FCS medium (slightly hypertonic); NAM, Normalized Averaging Method; ARM, Averaging Ratio Method.Metastasis involves the dissemination of single or small clumps of cancer cells through blood or lymphatic vessels and their extravasation into distant organs. Despite the strong regulation of metastases development by a cell dormancy phenomenon, the dormant state of cancer cells remains poorly characterized due to the difficulty of in vivo studies. We have recently shown in vitro that clonogenicity of prostate cancer cells is regulated by a dormancy phenomenon that is strongly induced when cells are cultured both at low cell density and in a slightly hypertonic medium. Here, we characterized by RT-qPCR a genetic expression signature of this dormant state which combines the presence of both stemness and differentiation markers. We showed that both TFGb/BMP signaling and redox imbalance are required for the full induction of this dormancy signature and cell quiescence. Moreover, reconstruction experiments showed that TFGb/BMP signaling and redox imbalance are sufficient to generate a pattern of genetic expression displaying all characteristic features of the dormancy signature. Finally, we observed that low cell density was sufficient to activate TGFb/BMP signaling and to generate a slight redox imbalance thus priming cells for dormancy that can be attained with a co-stimulus like hypertonicity, most likely through an increased redox imbalance. The identification of a dual regulation of dormancy provides a framework for the interpretation of previous reports showing a restricted ability of BMP signaling to regulate cancer cell dormancy in vivo and draws attention on the role of oxidative stress in the metastatic process.

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Monitoring Keap1–Nrf2 interactions in single live cells

Cited by 128 publications

References 118 publications

Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling

Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling

Nrf2 activation in the treatment of neurodegenerative diseases: a focus on its role in mitochondrial bioenergetics and function

SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy

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