basal transcription occurs, mediated by Nfe2l2 molecules that can bypass the Keap1 system. Keap1, in association with the Cullin 3 (Cul3/Rbx1) complex, promotes Nrf2 ubiquitination, followed by its degradation by the proteasome. Reactive oxygen species (ROS) and electrophiles inhibit the activity of cysteine-rich Keap1, leading to the nuclear accumulation of Nrf2. In the nucleus, Nrf2 heterodimerizes with the small MAF (musculoaponeurotic fibrosarcoma) proteins (sMAFs), to prompt the inducible expression of genes it regulates (200+). While the redox sensor Keap1 is a major regulator of Nrf2, other systems also regulate Nfe2l2. Beta-transducin repeat-containing protein (Btrc), is the ubiquitin E3 ligase adaptor for the Cullin 1 (Skp1/Cul1/Rbx1) complex, whose activity is stimulated by phosphorylation by glycogen synthase kinase 3 (Gsk3). A direct interaction with retinoid X receptor alpha (Rxra) also exerts an inhibitory effect (not shown). Overall, Nfe2l2 is regulated at the transcriptional and translational levels, via posttranslational modifications and epigenetic mechanisms. Molecular and structural aspects of Nfe2l2 regulation and function are described in greater detail.

Nfe2l2 (Nrf2), Keap1, Btrc structural aspects Nfe2l2 is a member of the cap'n'collar (CNC) subfamily of basic leucine zipper (bZIP) transcription factors. Seven highly conserved regions known as Nrf2-ECH homology (Neh) domains carry out distinct functions. Neh1 contains the CNC-bZIP region involved in heterodimerization with sMAFs and DNA binding. Neh3, 4 and 5 are involved in transactivation: Neh3 recruits the chromodomain helicase Chd6, Neh4 and 5 recruit CREB binding protein Crebbp or Ncoa3 coactivator. Neh2, Neh6 and Neh7 are involved in the negative control of Nrf2. Neh2 contains the two motifs DLG and ETGE that together, each bind one molecule of redox sensor Keap1 dimer. Neh6, the redox-independent regulator, contains the DSGIS and DSAPGS motifs that independently bind Btrc. Neh7 mediates Nrf2 repression by Rxra; this system is less characterized (not shown). Neh domains involved in negative regulation are highlighted in red, activating domains are highlighted green.
Keap1 belongs to the BTB-Kelch family of proteins. The Broad, Tramtrack and Bric-à-Brac (BTB) N-terminal domain mediates homodimerization and contributes to the interaction with Cul3. The C-terminal Kelch domain, consisting of six Kelch repeats folding into a six-blade beta-propeller structure, is involved in the interaction with Nfe2l2. The intervening region (IVR), between BTB and Kelch, contains a '3-box' at its N-terminal end, which also contributes to Cul3 interaction. Multiple cysteine residues (27 in the human gene), ~twice the number found in other proteins, can be modified by ROS and electrophilic compounds.
Btrc has an N-terminal dimerization domain followed by an F-box involved in recruiting Skp1. The WD40 repeat domain is involved in the interaction with Nfe2l2.

Nfe2l2 (Nrf2)-Keap1 system The DLG and ETGE motifs in the Neh2 domain each bind one of the two molecules of Keap1 dimer, with affinities that differ by almost two orders of magnitude. The 'hinge and latch' model proposes that initial binding to the high affinity ETGE motif is followed by docking of the lower affinity DLG motif into the beta-propeller of the second Keap1 molecule; thus, providing an optimal positioning of the seven conserved lysine residues between the two motifs for ubiquitin conjugation. An alternative model - 'conformational cycling', proposes that the Nfe2l2-Keap1 complex alternates between 'open' and 'closed' conformations, with the 'closed' being optimal for Nfe2l2 ubiquitination. Modification of cysteine residues by Nfe2l2 inducers does not lead to dissociation of Nfe2l2, but promotes conformational changes within the Nfe2l2-Keap1-Cul3/Rbx1 complex that prevent ubiquitination and likely, trap the complex. Newly synthesized Nfe2l2 can now accumulate in the nucleus, heterodimerize with sMAF to bind the antioxidant response element (ARE) sequences and activate the transcription of target genes. All 27 cysteine residues in human Keap1 are reactive, to varying degrees; collectively they are modified by several classes of compounds/Nfe2l2 inducers. Entries on the diagram for inducer preferences show a small subset of inducers, known to specifically modify that/those cysteines. The distinctiveness of Cys reactivities, referred to as the 'cysteine code', underlies the ability of the Nrf2-Keap1 axis to respond to a broad array of inducers. Based on chemical structure, direct activators of Keap1 belong to eight classes [see for instance, PMID: 27192495].

Nfe2l2 (Nrf2)-Btrc system The DSGIS and DSAPGS motifs in Neh6 domain independently bind the Btrc dimer. The DSGIS motif can be phosphorylated by the constitutively active glycogen synthase kinase 3 (Gsk3) isoforms, markedly increasing the affinity for Btrc. The DSAPGS motif is not phosphorylated and binds Btrc in a Gsk3-independent manner. Gsk3 phosphorylation and targeting of the substrates for ubiquitination via Btrc adaptor are evidenced in other systems, such as the hedgehog (Gli proteins) and canonical Wnt (beta-catenin) signaling pathways. Cellular stresses and environmental cues can set in motion growth factor-mediated signaling, and downstream intracellular pathways and MAP kinase cascades. Some of these systems may produce ROS by activating NADPH oxidases. The intracellular phosphatidylinositol 3 kinase-Akt signaling pathway is a main regulator of Gsk3; phosphorylation of Gsk3 proteins by Akt kinases inhibits their function and rescues Nfe2l2 from proteasomal degradation. ROS/electrophilic compounds, in addition to inactivating Keap1, impact on the activity of other proteins whose reactive cysteines are important for their function. Many phosphatases contain a conserved reactive cysteine at their active site; its modification inhibits their function. Examples include protein tyrosine phosphatases (PTP), dual specificity phosphatases, and Pten. They participate as functional modulators of the signaling systems mentioned above, largely via negative regulation. Pten is a major regulator of the PI3K-Akt pathway; its substrates also include phosphorylated Gsk3. Thus, in the Nfe2l2-Btrc system, various cellular cues and ROS integrate, to directly phosphorylate and inhibit Gsk3, or indirectly, via Pten inhibition, respectively, promoting Nfe2l2 activity.

Other interactors
Several protein with ETGE motifs can interact with Keap1 and interfere with Keap1 mediated ubiquitination of Nfe2l2, promoting Nfe2l2 activity. These include dipeptidyl peptidase 3 (Dpp3), Ikbkb - a component of the IKK complex that activates both the canonical and non-canonical NF-kappaB pathway, Palb2, Pgam5 - with a role in the hypoxia inducible mitochondrial autophagy via mitophagy receptors. Sequestome 1 (Sqstm1/p62), an autophagy/mitophagy scaffold/adaptor protein, contains an STGE motif. When phosphorylated by the mTORC1 complex of the mTOR pathway, it becomes a very potent inhibitor of Keap1 via an increase in affinity. It is believed that electrophile modified and impaired Keap1 is targeted to autophagosomal degradation via Sqstm1. There is positive cross-regulation between mTOR and PI3K-Akt signaling, the latter pathway with a role in the in Nfe2l2-Btrc system.
Additional proteins interact with Nfe2l2 to either promote or inhibit its function. Proteins that promote Nfe2l2 activity by interfering with its recruitment to the Cul3/Rbx1 complex include Cdkn1a (known as p21) and Brca1. Several interactors repressing Nfe2l2 function are reported, although the molecular mechanisms are less well understood. They include Gadd45gip1 (known as Crif1), Siah2 - may mediate Nfe2l2 inhibition during hypoxic conditions, and Rnf4 - may mediate degradation of Nfe2l2 in the nucleus where it ubiquitinates a sumoylated form of the transcription factor.

Nfe2l2 pathway, alterations and diseases, therapeutic approaches
Nfe2l2 signaling is crucial for proper stress defense response mechanisms. Deregulation of the pathway is associated with numerous conditions, including cancer, Alzheimer's (AD) and Parkinson's (PD) neurodegenerative diseases, asthma, diabetes, atherosclerosis, and others. The development of inducers that could modify Keap1 and the development of inhibitors that could disrupt the Keap1-Cul3 or the Nfe2l2-Keap1 protein-protein interactions, are intended to promote Nfe2l2 signaling. On the other hand, sustained Nfe2l2 signaling promotes chemoresistance and cancer cell survival. Gain-of-function mutations of Nfe2l2 and loss-of-function mutations of Keap1 have been found in human cancer, and many are present in patients with lung cancer. A small number of Nfe2l2 inhibitors are known [see for instance, PMID: 27192495 for molecules targeting the cysteine residues of Keap1, peptides and compounds targeting New2l2-Keap1 interaction and a few inhibitors of Nfe2l2]. To see the ontology report for annotations, GViewer and download, click here...(less)