The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway

Abstract

The KEAP1-NRF2 pathway is the principal protective response to oxidative and electrophilic stresses. Under homeostatic conditions, KEAP1 forms part of an E3 ubiquitin ligase, which tightly regulates the activity of the transcription factor NRF2 by targeting it for ubiquitination and proteasome-dependent degradation. In response to stress, an intricate molecular mechanism facilitated by sensor cysteines within KEAP1 allows NRF2 to escape ubiquitination, accumulate within the cell, and translocate to the nucleus, where it can promote its antioxidant transcription program. Recent advances have revealed that KEAP1 contains multiple stress sensors and inactivation modalities, which together allow diverse cellular inputs, from oxidative stress and cellular metabolites to dysregulated autophagy, to regulate NRF2 activity. This integration of the KEAP1-NRF2 system into multiple cellular signaling and metabolic pathways places NRF2 activation as a critical regulatory node in many disease phenotypes and suggests that the pharmaceutical modulation of NRF2's cytoprotective activity will be beneficial for human health in a broad range of noncommunicable diseases.

Keywords: E3 ubiquitin ligase; KEAP1; NRF2; antioxidant; oxidative stress; stress response.

FIG 1

The domain architectures of the NRF2 and KEAP1 proteins. The frequency and spectrum of mutations in NRF2 in human tumors are shown above the domain scheme. For KEAP1, the locations of the stress sensors are shown, with a dashed line linking the three parts of the H₂O₂ sensor.

FIG 2

The KEAP1-NRF2 pathway integrates the sensing of a wide range of cellular stresses to the upregulation of cytoprotective gene expression. Endogenous and exogeneous stress molecules are able to directly bind to reactive cysteine residues within KEAP1, resulting in the stabilization of NRF2 and the upregulation of its cytoprotective transcription program.

FIG 3

The cycle of cullin-RING ligase activation. 1, the KEAP1-dependent E3 ubiquitin ligase consisting of KEAP1, CUL3, and RBX1 binds to both the substrate, NRF2, and an E2 ubiquitin-conjugating enzyme. 2, neddylation by Nedd8 (N8) of CUL3 induces a conformational change in the complex such that the ubiquitin-bound E2 is able to transfer the ubiquitin to an acceptor lysine within NRF2. 3, after multiple rounds of ubiquitination, the polyubiquitinated NRF2 becomes a substrate for degradation by the proteasome. In the absence of NRF2, the KEAP1-dependent E3 ubiquitin ligase becomes a target for deneddylation, and thus inactivation, by COP9. 4, the deneddylated CUL3 is then bound by CAND1, which promotes the formation of new E3 ubiquitin ligase complexes, and the cycle of ubiquitination can begin again.

FIG 4

The conservation of KEAP1’s four main stress sensors within vertebrates. The sensor cysteine residues are highlighted in red, with the adjacent positively charged amino acids underlined and in bold. Zebrafish contain two copies of KEAP1, with the stress sensors distributed between the paralogues.

FIG 5

The classification of NRF2 inducers based on their specificity for different stress sensors within KEAP1. Inducers of NRF2 activity can be divided into five categories based on their preference for the Cys151, Cys273, Cys288 or the Cys226, Cys613, Cys622/624 ROS sensor within KEAP1. The sixth class of inducers function independently of the stress sensors by directly inhibiting NRF2’s binding to KEAP1.

FIG 6

The mechanism of NRF2 activation by oxidative stress. In the basal nonstressed state, NRF2 is targeted for ubiquitination and proteasome-dependent degradation by the KEAP1-dependent E3 ubiquitin ligase. In response to oxidative stress, the direct binding of stressors to reactive cysteine residues results in a conformation change in KEAP1, which inhibits the ubiquitination of NRF2. As NRF2 is not release by KEAP1, it saturates all of KEAP1’s binding sites, allowing newly translated NRF2 to bypass KEAP1, translocate to the nucleus, and upregulate cytoprotective gene expression.