Model organisms for investigating the functional involvement of NRF2 in non-communicable diseases

Abstract

Non-communicable chronic diseases (NCDs) are most commonly characterized by age-related loss of homeostasis and/or by cumulative exposures to environmental factors, which lead to low-grade sustained generation of reactive oxygen species (ROS), chronic inflammation and metabolic imbalance. Nuclear factor erythroid 2-like 2 (NRF2) is a basic leucine-zipper transcription factor that regulates the cellular redox homeostasis. NRF2 controls the expression of more than 250 human genes that share in their regulatory regions a cis-acting enhancer termed the antioxidant response element (ARE). The products of these genes participate in numerous functions including biotransformation and redox homeostasis, lipid and iron metabolism, inflammation, proteostasis, as well as mitochondrial dynamics and energetics. Thus, it is possible that a single pharmacological NRF2 modulator might mitigate the effect of the main hallmarks of NCDs, including oxidative, proteostatic, inflammatory and/or metabolic stress. Research on model organisms has provided tremendous knowledge of the molecular mechanisms by which NRF2 affects NCDs pathogenesis. This review is a comprehensive summary of the most commonly used model organisms of NCDs in which NRF2 has been genetically or pharmacologically modulated, paving the way for drug development to combat NCDs. We discuss the validity and use of these models and identify future challenges.

Keywords: Inflammation; Model organisms; NRF2; Non-communicable chronic diseases; Oxidative stress.

Fig. 1

Members of the small MAF and CNC transcription factor families in mammals. Small Maf proteins (sMafs), comprising MafG (sMaf), MafK, and MafF. The total number of amino acids is depicted to highlight that the molecular weight is different. The CNC family comprises NF-E2p45, NRF1-3, BACH1, and BACH2. Only mammalian family members are shown in this figure. CLS, cytoplasmic localization signal. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.

Fig. 2

The architecture of NRF2, KEAP1 and β-TrCP. A, NRF2 contains seven conserved NRF2-ECH homology NRF2-ECH homology (Neh) domains, Neh1-Neh7. Neh1 contains a basic leucine zipper (bZip) motif, where the basic region is responsible for DNA binding and the Zip dimerizes with other binding partners such as sMAFs. Neh2 contains ETGE and DLG motifs, which are required for the interaction with KEAP1 and subsequent KEAP1-mediated proteasomal degradation. Neh3, 4 and 5 domains are transactivation domains of NRF2. Neh6 contains two βTrCP degrons DSGIS and DSAPGS that are responsible for the β-TrCP mediated proteasomal degradation. B, KEAP1 contains five domains, amino terminal region (NTR), a broad complex, tramtrack, bric-a-brac (BTB) domain, an intervening region (IVR), six Kelch domains, and the C-terminal region (CTR). The Kelch domain and CTR mediate the interaction with NRF2, p62, DPP3, WTX, and PALB2 that contains ETGE motifs. The BTB domain homodimerizes with KEAP1 and contributes to the interaction of IVR with Cul3/RBX1 complex. Several functional important cysteine residues (C151, C226, C273 and C278) that sense reactive oxygen species (ROS) and electrophiles and modulate KEAP1-NRF2 interaction. C, βTrCP has three domains, dimerization domain (D) that forms homo- and heterodimers between βTrCP1 and βTrCP2, the F-box that recruits SKP1 for the binding of CUL1/RBX1 complex, and the WD40 repeat domain that binds βTrCP degrons DSGIS and DSAPGS in NRF2. βTrCP, β-transducing repeat-containing protein; CUL3, Cullin3; RBX1, RING-box protein; WD40, WD Repeat protein 40; RXRα, retinoic X receptor alpha; DPP3, dipeptidyl peptidase 3; WTX, Wilms tumor gene on X chromosome; PALB2, Partner and Localizer of BRCA2; GSK3, Glycogen synthase kinase-3; SKP1, S-phase kinase-associated protein-1. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.

Fig. 3

Maintenance of cellular homeostasis by NRF2-dependent modulation of redox balance and inflammation control. Redox imbalance and inflammation underlie development of non-communicable diseases (NCDs). NRF2 directly transactivates the expression of cytoprotective genes, including anti-oxidative and detoxifying enzymes, which restore proper tissue redox status. NRF2 also facilitates resolution of inflammation, by regulation of the expression of inflammatory genes, its interplay with NF-κB and ROS catabolism. BR, bilirubin; BV, biliverdin; CAT, catalase; G6PD, glucose-6-phosphate dehydrogenase; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modulatory subunit; GSH, glutathione; GSS, glutathione synthetase; GSR, glutathione disulfide reductase; Gpx, glutathione peroxidase; GR, glutathione reductase; HMGB1, high mobility group box 1; HMOX1, heme oxygenase-1; IDH1, isocitrate dehydrogenase 1; IL1β, interleukin 1 beta; IL6, interleukin 6; IL17D, interleukin 17D; MARCO, macrophage receptor with collagenous structure; ME1, malic enzyme 1; MMP9, matrix metallopeptidase 9; NCD, non-communicable disease; NETs, neutrophil extracellular traps; NQO1, NAD(P)H quinone dehydrogenase 1; NFKB, nuclear factor kappa B; NLRP3, NLR family pyrin domain containing 3; NOS2, nitric oxide synthase 2; PGD, phosphogluconate dehydrogenase; ROS, reactive oxygen species; SOD, superoxide dismutase; TXN, thioredoxin, TXRD1, thioredoxin reductase 1; VCAM1, vascular cell adhesion molecule-1. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.

Fig. 4

Summary of the evolutionary conservation of KEAP1-NRF2 pathway. KEAP1-NRF2 exhibits significant homology across species and specifically between D. melanogaster, zebrafish (D. rerio), and M. musculus. Additionally, in C. elegans, SKN-1 functions in a manner like NRF2, but an interesting divergence exists in the regulatory mechanism, as WDR-23 governs SKN-1 activity instead of KEAP1. In yeast, the transcription factor YAP1 is considered as the functional equivalent of NRF2. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.

Fig. 5

C. elegans as a model organism to study the role of NRF2 in NCDs. A, scheme showing the three isoforms transcribed from the worm Skn-1 gene to endorse an orchestrated protective response. B, summary of the main regulators of SKN1. C, toolbox of the main C. elegans strains employed to analyze the role of Skn-1 in NCDs. D, main advantages of C. elegans as a model organism. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.

Fig. 6

D. melanogaster as a model organism to study the role of NRF2 in NCDs. A, scheme showing the three isoforms transcribed from the fly Cnc gene. B, summary of the main regulators of CncC. C, toolbox of the key stocks of D. melanogaster employed to analyze the role of CncC in NCDs. D, main advantages of D. melanogaster as a model organism. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.

Fig. 7

D. rerio as a model organism to study the role of NRF2 in NCDs. A, scheme showing the genomic organization of KEAP1-NRF2 pathway in fish. B, summary of the main regulators of nrf2a. C, toolbox of the key stocks of D. rerio employed to analyze the role of nrf2a in NCDs. D, main advantages of D. rerio as a model organism. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.

Fig. 8

Domain structure of the different NRF2 knockout mouse models. At the top, the exonic structure of the Nfe2l2 mature mRNA is presented, indicating different exon-exon junctions. The shaded part corresponds to the coding sequence of the protein. The different domains in the structures below are aligned with the respective exons they are encoded in, considering the specific deletions in the Nfe2l2 gene each mouse model. Note that both Nfe2l2^−/− and Nfe2l2^fl/fl are transcriptional knockouts but retain the ability to bind KEAP1 through the Neh2 domain. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.

Fig. 9

Domain structure of the different KEAP1 knockout mouse models. At the top, the exonic structure of the Keap1 mature mRNA is presented, indicating different exon-exon junctions. The shaded part corresponds to the coding sequence of the protein. The different domains in the structures below are aligned with the respective exons they are encoded in, considering the specific deletions in the Keap1 gene each mouse model. Cysteine redox sensors are labelled in their approximate sites. Note that while in Keap1^−/− and Keap1^fl/fl B mice the ability to bind CUL3/RBX1 is lost, it is conserved in Keap1^fl/flA mice. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.

Fig. 10

M. musculus as a model organism to study the role of NRF2 in NCDs. A, scheme showing the genomic organization of KEAP1-NRF2 pathway in mammals. B, summary of the main regulators of NRF2. C, toolbox of transgenesis strategies to generate the myriads of mouse models. D, main advantages of M. musculus as a model organism. Created in BioRender. Rojo, A. (2024) BioRender.com/b33i790.