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S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling

ABSTRACT: KEAP1, a cytoplasmic repressor of the oxidative stress responsive transcription factor NRF2, senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remains undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2 dependent transcription. This work identifies a novel posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress sensing machinery of the cell.

ORGANISM(S): Homo sapiens

PROVIDER: GSE230577 | GEO | 2023/05/01

REPOSITORIES: GEO

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Sensor Systems of KEAP1 Uniquely Detecting Oxidative and Electrophilic Stresses Separately In Vivo

Project description:In the KEAP1-NRF2 stress response system, KEAP1 acts as a sensor for oxidative and electrophilic stresses through formation of S-S bond and C-S bond, respectively. Of the many questions left related to the sensor activity, following three appear important; whether these KEAP1 sensor systems are operating in vivo, whether oxidative and electrophilic stresses are sensed by the similar or distinct systems, and how KEAP1 equips highly sensitive mechanisms detecting oxidative and electrophilic stresses in vivo. To address these questions, we conducted a series of analyses utilizing KEAP1-cysteine substitution mutant mice, conditional selenocysteine-tRNA (Trsp) knockout mice, and human cohort whole genome sequence (WGS) data. The Trsp-knockout provokes severe deficiency of selenoproteins and compensatory activation of NRF2. However, mice lacking homozygously a pair of critical oxidative stress sensor cysteine residues of KEAP1 fail to activate NRF2 in the Trsp-knockout livers. Secondly, this study provides evidence for the differential utilization of KEAP1 sensors for oxidative and electrophilic stresses in vivo. Thirdly, theoretical calculations show that the KEAP1 dimer equips quite sensitive sensor machinery in which modification of a single molecule of KEAP1 within the dimer is sufficient to affect the activity. WGS examinations of rare variants identified seven non-synonymous variants in the oxidative stress sensors in human KEAP1, while no variant was found in electrophilic sensor cysteine residues, supporting the fail-safe nature of the KEAP1 oxidative stress sensor activity. These results provide valuable information for our understanding how mammals respond to oxidative and electrophilic stresses efficiently.

2024-10-09 | GSE276818 | GEO

Chemical Modulation of Glycolysis Regulates the KEAP1-NRF2 Pathway Through a Metabolite-Induced Posttranslational Modification

Project description:Glycolysis is a master regulator of cellular energy, synthesis and redox regulation, however the mechanisms that underlie the responses to and regulation of changing glucose metabolism are incompletely understood. The NRF2-antioxidant response element (ARE) pathway serves as a central regulator of cellular stress by sensing and eliminating 2 chemically reactive species in the cell. A phenotypic high-throughput chemical screen for novel activators of NRF2 signaling identified an inhibitor of the glycolytic enzyme PGK1. Metabolomic and proteomic profiling experiments revealed that inhibition of PGK1 results in the accumulation of central glycolytic intermediates, as well as the reactive dicarbonyl metabolite methylglyoxal (MGx). MGx selectively modifies reactive residues on KEAP1, resulting in the formation of a covalent KEAP1 homodimer, accumulation of NRF2 and activation of the NRF2 transcriptional program. Proteomic and NMR experiments verified that KEAP1 homodimerization is mediated by a novel post-translational modification, termed MICA, formed by the reaction of methylglyoxal with proximal cysteine and arginine residues. The identified PGK1 inhibitor series was found to be efficacious in a NRF2-dependent mouse model of UV-damage, demonstrating that activation of KEAP1- NRF2 signaling through this mechanism is physiologically relevant. Together, these results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional program, which is mediated by a reactive metaboliteinduced post-translational modification (rmPTM). In addition, they provide new insight into metabolic regulation of cell stress response, and a potential therapeutic axis for controlling the cytoprotective antioxidant response in numerous human diseases.

2018-10-22 | GSE116642 | GEO

S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling

Project description:S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling

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Patient-derived KEAP1 superbinder mutants stabilize KEAP1 structure and sequester NRF2 in p62-dependent clusters

Project description:Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a pro-survival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a previous study of KEAP1 mutations observed in lung cancer, we classified 40% of the mutations as ‘superbinders’ (superbinders). These mutants bind and ubiquitylate NRF2 but do not promote NRF2 degradation. Here, we further investigated the molecular mechanism(s) driving the superbinder phenotype. BioID-based quantitative proteomic analysis of the R320Q and R470C superbinder mutations revealed increased co-complexed NRF2 without significant alteration to other KEAP1-associated proteins, including CUL3, VCP, and several ubiquitin receptors within the proteasome lid. Dynamic simulation modeling and limited proteolysis analyses suggest that superbinder mutations stabilize residues in KEAP1 that contact NRF2. In cells, KEAP1 R320Q and R470C mutants co-localize with NRF2, p62/SQSTM1 and polyubiquitin in spherical clusters that rapidly fuse and dissolve; KEAP1-NRF2 localization to these clusters requires p62. Expression of R320Q and R470C in lung cancer cells provided resistance to the reactive oxygen species-inducing drug bleomycin. We present a model wherein superbinder mutations alter the conformational dynamics of the KEAP1-NRF2 complex to alter the cycling of KEAP1 between open and closed conformations, thus inhibiting NRF2 degradation.

2018-11-07 | PXD008411 | Pride

Characterization of RA839, a non-covalent small-molecule binder to Keap1 and selective activator of Nrf2 signalling

Project description: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 kelch-like ECH-associated protein 1 (Keap1). Here, we describe RA839, a small molecule that binds non-covalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of approximately 6 µM, as demonstrated by X-ray co-crystallization and isothermal titration calorimetry. Whole-genome DNA arrays showed that at 10 µM RA839 significantly regulated 105 genes in bone marrow-derived macrophages. Canonical pathway mapping of these genes revealed an activation of pathways linked with Nrf2 signalling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knockout macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 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. Gene expression profile of bone marrow derived murine macrophages (BMDM) from Nrf2+/+ or Nrf2-/- mice treated with RA838, siKeap1-1 or siKeap1-2 were compared to untreated DMSO control or siControl. Four biological replicates were used for each sample group.

2015-10-31 | E-GEOD-71695 | biostudies-arrayexpress

Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription

Project description:Nrf2 (NF-E2-related factor-2) transcription factor regulates oxidative/xenobiotic stress response and also represses inflammation. However, the mechanisms how Nrf2 alleviates inflammation are still unclear. Here, we demonstrate that Nrf2 interferes with lipopolysaccharide-induced transcriptional upregulation of proinflammatory cytokines, including IL-6 and IL-1Î². ChIP-seq and ChIP-qPCR analyses revealed that Nrf2 binds to the proximity of these genes in macrophages and inhibits RNA Pol II recruitment. Further, we found that Nrf2-mediated inhibition is independent of the Nrf2 binding motif and reactive oxygen species level. Murine inflammatory models further demonstrated that Nrf2 interferes with IL6 induction and inflammatory phenotypes in vivo. Thus, contrary to the widely accepted view that Nrf2 suppresses inflammation through redox control, we demonstrate here that Nrf2 opposes transcriptional upregulation of proinflammatory cytokine genes. This study identifies Nrf2 as the upstream regulator of cytokine production and establishes a molecular basis for an Nrf2-mediated anti-inflammation approach. Gene expression in BMDMs obtained from wild-type and Keap1-CKO mice. In Keap1-CKO (Keap1 flox/flox::LysM-Cre) BMDMs, Nrf2 transcription factor is activated due to Keap1-deficiency. BMDMs were obtained by a culture of bone marrow cells in the presence of M-CSF for7 days. M1-activated BMDMs were obtained by stimulation with LPS and IFNg for 6 hours, while M2-activated BMDMs were obtained by a stimulation with IL-4 for 6 hours. Two independent BMDM cultures were performed, and each experiment contains samples obtained from one wild-type and one Keap1-CKO mice, respectively.

2016-05-12 | E-GEOD-71263 | biostudies-arrayexpress

Expression data from Keap1 overexpression and Nrf2 knockdown lung cancer cell

Project description:Keap1 overexpressed and Nrf2 depleted CL1-5 cells were used to identify genes regulated by Keap1/Nrf2 axis-dependent gene regulations We used microarrays to detail the global programme of gene expression underlying metastasis and identified distinct classes of Keap1/Nrf2-regulated genes during this process. CL1-5 cells stably expressed Keap1 expressing construct and Nrf2-specific shRNA were analyzed compared to control cells

2015-03-04 | E-GEOD-66473 | biostudies-arrayexpress

Characterization of RA839, a non-covalent small-molecule binder to Keap1 and selective activator of Nrf2 signalling

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Transcription profiling of mouse liver from conditional Keap1 knockout mice vs. genetic control mice

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2008-06-11 | E-GEOD-11287 | biostudies-arrayexpress

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