NFE2L2 Human
Description
Molecular Structure and Production
NFE2L2 Human Recombinant Protein ( ):
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Expression System: Escherichia coli (E. coli)
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Amino Acid Sequence: 625 residues (1-605 native sequence + 20-residue N-terminal His-tag)
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Molecular Mass: 69.9 kDa
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Purity: >90% via proprietary chromatography
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Formulation: 0.25 mg/ml in phosphate-buffered saline (pH 7.4) with 10% glycerol
Functional Roles in Cellular Physiology
NFE2L2 regulates:
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Antioxidant Response: Activates >200 genes with Antioxidant Response Elements (ARE), including G6PD, SOD, and HO-1
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Detoxification: Upregulates phase II enzymes (GST, UGT) and efflux transporters (MRPs)
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Autophagy: Directly induces autophagy-related genes (ULK1, SQSTM1/p62, Atg7)
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Proteostasis: Enhances proteasomal degradation of misfolded proteins
Regulatory Mechanism:
Genetic Mutations (4):
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De Novo Mutations (e.g., R34Q, R34P):
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Cause multisystem disorders with immunodeficiency, leukoencephalopathy, and hypohomocysteinemia
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Disrupt KEAP1 binding → constitutive NFE2L2 activation → redox imbalance
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Disease Associations:
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Cancer: Somatic mutations in NFE2L2 promote chemoresistance in lung, liver, and bladder cancers
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Diabetes: Elevated NFE2L2 expression correlates with diabetic retinopathy progression (3-fold increase in DR patients)
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Neurodegeneration: Impaired NFE2L2 activity linked to Parkinson’s and Alzheimer’s pathologies
Key Studies:
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NFE2L2-Associated Molecular Signature (NAMS) predicts recurrence-free survival (HR = 1.84, P < 0.001)
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Enriched pathways: TGF-β signaling, focal adhesion, ECM-receptor interaction
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Oxidative Stress in Diabetes ( ):
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Serum SOD and HO-1 inversely correlate with NFE2L2 levels in diabetic retinopathy (r = -0.68, P = 0.002)
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Therapeutics:
Transcript Variants and Isoforms (5)
| Transcript ID | Protein Length | Biotype | Flags |
|---|---|---|---|
| ENST00000397062.8 | 605 aa | Protein coding | MANE Select, Canonical |
| ENST00000699296.1 | 238 aa | Protein coding | Primary |
| ENST00000699405.1 | 275 aa | Protein coding | Basic |
Expression and Tissue Distribution
Product Specs
Q&A
What experimental strategies best elucidate NFE2L2's primary role in human oxidative stress response?
NFE2L2 orchestrates phase II detoxification through ARE (Antioxidant Response Element)-driven transcription. To map its activity:
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ARE-Luciferase Reporter Assays: Quantify transcriptional activation under H<sub>2</sub>O<sub>2</sub> or tBHQ stimulation . Include KEAP1 knockdown controls to confirm pathway specificity.
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Chromatin Immunoprecipitation Sequencing (ChIP-seq): Identify genome-wide NFE2L2 binding sites using validated antibodies (e.g., EP1808Y, Abcam). Cross-reference with ROS-scavenging genes like HMOX1 and NQO1 .
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Redox Metabolomics: Pair LC-MS measurements of glutathione/oxidized glutathione (GSH/GSSG) ratios with NFE2L2 activity readouts to establish quantitative stress-response thresholds .
Table 1: Core NFE2L2 Target Genes and Functional Assays
| Gene Target | Assay Platform | Stress Inducer | Validation Requirement |
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| HMOX1 | qRT-PCR | Cadmium | siRNA knockdown |
| SLC7A11 | Western Blot | Erastin | Ferroptosis inhibition |
| GCLM | EMSA | DEM | ARE motif mutation |
How should researchers resolve contradictions in NFE2L2 mutation data across cancer types?
Discordant findings (e.g., tumor-suppressive vs. oncogenic effects) arise from mutation heterogeneity and co-occurring β-catenin alterations :
What methodologies confirm NFE2L2-KEAP1 interaction dynamics in live human cells?
The "hinge-and-latch" binding mechanism requires real-time resolution:
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Bimolecular Fluorescence Complementation (BiFC): Fuse KEAP1 Kelch domains (aa 321-609) and NFE2L2 ETGE/DLG motifs to split Venus fragments. Track complex formation under confocal microscopy during paraquat exposure .
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Ubiquitination Pulse-Chase: Overexpress HA-tagged ubiquitin in HEK293T cells co-transfected with NFE2L2/KEAP1. Immunoprecipitate NFE2L2 and quantify polyubiquitination via anti-HA Western blot at 0/30/60-minute intervals .
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Cysteine Reactivity Profiling: Perform alkylation-based MS on KEAP1 C151A/C288A mutants to identify redox-sensitive residues critical for NFE2L2 release .
How do NFE2L2 gain-of-function mutations influence therapeutic resistance paradigms?
Recurrent mutations (e.g., L30P, R34P) confer chemoresistance through divergent mechanisms:
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Transcriptional Reprogramming: RNAseq of mutant-expressing hepatoblasts reveals SERPINE1 upregulation (6-fold vs. WT), driving ECM remodeling and cisplatin resistance .
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Ferroptosis Suppression: R34P mutants elevate SLC7A11 expression (3.2×), limiting lipid peroxidation. Validate using BODIPY 581/591 C11 staining and iron chelator deferoxamine .
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In Vivo Validation: Co-inject SB vectors encoding Δ90-β-catenin + NFE2L2 mutants into murine liver. Median survival drops from 13 weeks (WT) to 8 weeks (L30P), requiring necropsy-based tumor burden scoring .
Table 2: Murine Survival Outcomes by NFE2L2 Variant
What statistical approaches address heterogeneity in NFE2L2 activation biomarkers across human cohorts?
Inter-patient variability in NFE2L2 activity complicates clinical correlations:
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Multiplicity Adjustment: Apply Benjamini-Hochberg correction when testing ≥20 ARE genes (e.g., in TCGA pancancer analyses) .
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Latent Class Analysis: Cluster patients by NFE2L2/KEAP1/β-catenin mutation profiles using VarSelLCM in R. Associate clusters with recurrence-free survival .
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Digital Droplet PCR: Quantify low-frequency NFE2L2 CNVs in FFPE samples using RPP30-normalized assays. Thresholds: <3 copies (normal), 3-5 (gain), >5 (amplification) .
Which in vitro models best recapitulate NFE2L2-driven EMT in human epithelial cancers?
NFE2L2/KEAP1 imbalance promotes metastasis via TGF-β1 synergy:
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3D Spheroid Invasion: Seed MCF10A-keap1<sup>-/-</sup> cells in Matrigel. NFE2L2 stabilization increases invadopodia (2.3×) by upregulating MMP9 (q=1e-5) .
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Microfluidic Circulating Tumor Cell (CTC) Models: Subject NFE2L2-mutant HCC cells to shear stress (4 dyn/cm<sup>2</sup>). Monitor TWIST1 induction via GFP reporters during extravasation .
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Crispr Interference (dCas9-KRAB): Repress NFE2L2 in PDAC organoids. Single-cell RNAseq reveals MET reversal (E-cadherin<sup>+</sup> cells increase 44%) .
How can researchers mitigate off-target effects when pharmacologically activating NFE2L2?
Small-molecule inducers (e.g., sulforaphane) risk activating pro-tumorigenic pathways:
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Kinase Profiling: Screen inhibitors against 468 kinases (DiscoverX) to identify CDK4/6 co-activation (IC<sub>50</sub> shifts >50%).
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NQO1<sup>-/-</sup> Rescue Models: Treat NQO1-null mice with CDDO-Im. Absence of chemoprevention (tumor incidence 81% vs. 22% in WT) confirms on-target action .
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ATAC-seq Guided Dosing: Titrate bardoxolone methyl to maintain chromatin accessibility at canonical AREs (e.g., -2kb HMOX1) without inducing AP-1 sites .
Product Science Overview
NRF2 is a basic leucine zipper (bZIP) protein that binds to antioxidant response elements (AREs) in the promoter regions of genes encoding cytoprotective proteins . This binding induces the expression of various genes involved in the cellular response to oxidative stress, including phase II detoxifying enzymes . NRF2 is known to inhibit the NLRP3 inflammasome, which is involved in the inflammatory response .
The structure of NRF2 includes seven highly conserved domains known as NRF2-ECH homology (Neh) domains . These domains are responsible for various functions:
- Neh1: Allows heterodimerization with small Maf proteins.
- Neh2: Binds to the cytosolic repressor Keap1.
- Neh3: Plays a role in protein stability and acts as a transactivation domain.
- Neh4 and Neh5: Act as transactivation domains, binding to cAMP Response Element Binding Protein (CREB).
- Neh6: Contains a degron involved in the degradation of NRF2, even under stressed conditions .
NRF2 is involved in a complex regulatory network that affects metabolism, inflammation, autophagy, proteostasis, mitochondrial physiology, and immune responses . Due to its role in protecting cells from oxidative stress, NRF2 is a promising therapeutic target for diseases caused by oxidative damage, such as neurodegenerative diseases, cardiovascular diseases, and cancer .
Several drugs that stimulate the NFE2L2 pathway are currently being studied for their potential to treat these conditions . The ability of NRF2 to regulate the expression of cytoprotective genes makes it a valuable target for developing new therapeutic strategies.
In vitro studies have shown that NRF2 binds to AREs in the promoter regions of genes encoding cytoprotective proteins, leading to the upregulation of these genes . This has significant implications for developing treatments that can enhance the body’s natural defense mechanisms against oxidative stress.
Human recombinant NRF2 is used in research to study its effects on gene expression and its potential therapeutic applications. By understanding how NRF2 functions and regulates gene expression, researchers can develop new strategies to combat diseases associated with oxidative stress.
