Nuclear factor erythroid 2-related factor 2 (NRF2), also known asnuclear factor erythroid-derived 2-like 2, is atranscription factor that in humans is encoded by theNFE2L2gene.[5] NRF2 is abasic leucine zipper (bZIP) protein that may regulate the expression ofantioxidant proteins that protect againstoxidative damage triggered by injury and inflammation, according to preliminary research.[6]In vitro, NRF2 binds to antioxidant response elements (AREs) in the promoter regions of genes encodingcytoprotective proteins.[7] NRF2 induces the expression ofheme oxygenase 1in vitro leading to an increase inphase II enzymes.[8] NRF2 also inhibits theNLRP3inflammasome.[9]
NRF2 appears to participate in a complex regulatory network and performs a pleiotropic role in the regulation of metabolism, inflammation,autophagy,proteostasis, mitochondrial physiology, andimmune responses.[10] Several drugs that stimulate the NFE2L2 pathway are being studied for treatment of diseases that are caused by oxidative stress.[6][11]
NFE2L2 and other genes, such asNFE2,NFE2L1 andNFE2L3, encode basicleucine zipper (bZIP)transcription factors. They share highly conserved regions that are distinct from other bZIP families, such asJUN andFOS, although remaining regions have diverged considerably from each other.[12][13]
NRF2 is a basic leucine zipper (bZip)transcription factor with a Cap "n" Collar (CNC) structure.[5] NRF2 possesses seven highly conserved domains called NRF2-ECH homology (Neh) domains. From theN-terminus to theC-terminus, they are:
Neh2 allows for binding of NRF2 to its cytosolic repressor Keap1,[14] through the conserved sites ETGE and DLG.[15]
Neh4 andNeh5 act as transactivation domains by binding to cAMP Response Element Binding Protein (CREB), which possesses intrinsichistone acetyltransferase activity.[14]
Neh7 is involved in the repression of Nrf2 transcriptional activity by the retinoid X receptor α through a physical association between the two proteins.[15]
Neh6 may contain adegron that is involved in a redox-insensitive process of degradation of NRF2. This occurs even in stressed cells, which normally extend the half-life of NRF2 protein relative to unstressed conditions by suppressing other degradation pathways.[16] Its two conserved motifs, DSGIS and DSAPGS, are recognized by β-TrCP (BTRC andFBXW11 in mammals).[15]
Neh3 may play a role in NRF2 protein stability and may act as a transactivation domain, interacting with component of the transcriptional apparatus.[18]
The "domains" of Nrf2 are regions of conservation, notprotein domains in the structual sense. Neh2, Neh7 and Neh1 are partially unstructured. Neh3 and Nah6 is predicted to be mainly unstructured. Neh4 and Neh5 are disordered, meaning they do not fold into a fixed shape.[19] Neh4 and Neh5 have been predicted as structured, but experimental data show otherwise.[20] The methods employed byInterPro, from curated domain patterns toAlphaFold, cover less than half of human Nrf2.[21]
Activating inputs and functional outputs of the NRF2 pathway
Under normal or unstressed conditions, NRF2 is kept in the cytoplasm by a cluster of proteins that degrade it quickly. Under oxidative stress, NRF2 is not degraded, but instead travels to the nucleus where it binds to a DNA promoter and initiates transcription of antioxidative genes and their proteins.
NRF2 is kept in the cytoplasm by Kelch like-ECH-associated protein 1 (KEAP1) andCullin 3, which degrade NRF2 byubiquitination.[22] Cullin 3 ubiquitinates NRF2, while Keap1 is a substrate adaptor protein that facilitates the reaction. Once NRF2 is ubiquitinated, it is transported to theproteasome, where it is degraded and its components recycled. Under normal conditions, NRF2 has a half-life of only 20 minutes.[23]Oxidative stress or electrophilic stress disrupts critical cysteine residues in Keap1, disrupting the Keap1-Cul3 ubiquitination system. When NRF2 is not ubiquitinated, it builds up in the cytoplasm,[24][25] and translocates into the nucleus. In the nucleus, it combines (forms a heterodimer) with one ofsmall Maf proteins (MAFF,MAFG,MAFK) and binds to the antioxidant response element (ARE) in the upstreampromoter region of many antioxidative genes, and initiates their transcription.[26]
Activation of NRF2 induces the transcription of genes encodingcytoprotective proteins. These include:
NAD(P)H quinone oxidoreductase 1 (Nqo1) is a prototypical NRF2 target protein which catalyzes the reduction and detoxification of highly reactivequinones that can causeredox cycling andoxidative stress.[27]
Glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase regulatory subunit (GCLM) form a heterodimer, which is the rate-limiting step in the synthesis ofglutathione (GSH), a very powerful endogenousantioxidant. Both Gclc and Gclm are characteristic NRF2 target genes, which establish NRF2 as a regulator of glutathione, one of the most important antioxidants in the body.[28]
Heme oxygenase-1 (HMOX1,HO-1) is an enzyme that catalyzes the breakdown ofheme into the antioxidantbiliverdin, the anti-inflammatory agentcarbon monoxide, and iron. HO-1 is a NRF2 target gene that has been shown to protect from a variety of pathologies, includingsepsis,hypertension,atherosclerosis, acute lung injury, kidney injury, and pain.[31] Conversely, induction of HO-1 has been shown to exacerbate early brain injury afterintracerebral hemorrhage.[32]
Theglutathione S-transferase (GST) family includes cytosolic,mitochondrial, andmicrosomal enzymes that catalyze the conjugation of GSH with endogenous andxenobioticelectrophiles. After detoxification byglutathione (GSH) conjugation catalyzed by GSTs, the body can eliminate potentially harmful and toxic compounds. GSTs are induced by NRF2 activation and represent an important route of detoxification.[33]
Multidrug resistance-associated proteins (Mrps) are importantmembrane transporters that efflux various compounds from various organs and intobile or plasma, with subsequent excretion in the feces or urine, respectively. Mrps have been shown to be upregulated by NRF2 and alteration in their expression can dramatically alter thepharmacokinetics and toxicity of compounds.[35][36]
Kelch-like ECH-associated protein 1 is also a primary target of NFE2L2. Several interesting studies have also identified this hidden circuit in NRF2 regulations. An AREs located on a negative strand of the murine Keap1 (INrf2) gene can subtly connect Nrf2 activation to Keap1 transcription.[37] Regarding NRF2 occupancies in human lymphocytes, an approximately 700 bp locus within the KEAP1 promoter region was consistently top rank enriched, even at the whole-genome scale.[38] These basic findings have depicted a mutually influenced pattern between NRF2 and KEAP1. NRF2-driven KEAP1 expression characterized in human cancer contexts, especially in human squamous cell cancers,[39] implicated a new perspective in understanding NRF2 signaling regulation.
Genetic activation of NRF2 has been implicated in the development ofde novo tumors,[40][41] as well as in the progression ofatherosclerosis by increasing plasma cholesterol levels and hepatic cholesterol content.[42] It has been suggested that these pro-atherogenic effects may outweigh the protective benefits of NRF2-mediated antioxidant induction.[43]
Activation of theNRF2 (nuclear factor erythroid 2–related factor 2) pathway has been explored as a therapeutic strategy due to its role in regulating antioxidant and cytoprotective responses. One of the most clinically advanced NRF2 activators isdimethyl fumarate, marketed as Tecfidera byBiogen Idec. It was approved by theFood and Drug Administration in March 2013 following a successfulPhase III clinical trial that demonstrated reduced relapse rates and delayed progression of disability in individuals withmultiple sclerosis.[6]
Although the precise mechanism of action of dimethyl fumarate is not fully understood, it is known to activate the NRF2 signaling pathway. Both dimethyl fumarate and its active metabolite,monomethyl fumarate, promote NRF2 nuclear translocation and the subsequent transcription of antioxidant response element (ARE)-driven genes. In addition, they have been shown to act asnicotinic acid receptoragonists in vitro.[44]
Despite its clinical efficacy, dimethyl fumarate is associated with several adverse effects, including anaphylaxis, angioedema,progressive multifocal leukoencephalopathy (PML),lymphopenia, andliver damage. Common side effects include flushing and gastrointestinal symptoms such as diarrhea, nausea, and upper abdominal pain.[44]
Other NRF2 activators have also been investigated. The dithiolethiones are a class of organosulfur compounds known to induce NRF2 activity. Among them,oltipraz is the most extensively studied.[45] Oltipraz has been shown to suppress tumor formation in multiple rodent tissues, including the bladder, colon, liver, lung, and pancreas, by upregulating NRF2-dependent detoxification pathways.[46]
However, clinical trials of oltipraz have failed to demonstrate clear therapeutic benefit and have reported significant toxicities, includingneurotoxicity and gastrointestinal disturbances. Additionally, oltipraz has been found to generatesuperoxide radicals, which may offset its NRF2-mediated protective effects.[46][47]
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^abMotohashi H, Yamamoto M (November 2004). "Nrf2-Keap1 defines a physiologically important stress response mechanism".Trends in Molecular Medicine.10 (11):549–557.doi:10.1016/j.molmed.2004.09.003.PMID15519281.
^Kopacz A, Rojo AI, Patibandla C, Lastra-Martínez D, Piechota-Polanczyk A, Kloska D, et al. (November 2022). "Overlooked and valuable facts to know in the NRF2/KEAP1 field".Free Radical Biology & Medicine.192:37–49.doi:10.1016/j.freeradbiomed.2022.08.044.hdl:10261/289786.PMID36100148.
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^Hayes JD, Chanas SA, Henderson CJ, McMahon M, Sun C, Moffat GJ, et al. (February 2000). "The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin".Biochemical Society Transactions.28 (2):33–41.doi:10.1042/bst0280033.PMID10816095.
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