Evolutionary conserved N-terminal domain of Nrf2 is essential for the Keap1-mediated degradation of the protein by proteasome

Under homeostatic conditions, Nrf2 activity is constitutively repressed. This process is dependent on Keap1, to which Nrf2 binds through the Neh2 domain. Since the N-terminal subdomain of Neh2 (Neh2-NT) contains evolutionarily conserved motifs, we examined the roles they play in the degradation of Nrf2. In Neh2-NT, we defined a novel motif that is distinct from the previously characterized DIDLID motif and designated it DLG motif. Deletion of Neh2-NT or mutation of the DLG motif largely abolished the Keap1-mediated degradation of Nrf2. These mutations were found to enfeeble the binding affinity of Nrf2 to Keap1. The Neh2-NT subdomain directed DLG-dependent, Keap1-independent, degradation of a reporter protein in the nucleus. By contrast, mutation of DLG did not affect the half-life of native Nrf2 protein in the nucleus under oxidative stress conditions. These results thus demonstrate that DLG motif plays essential roles in the Keap1-mediated proteasomal degradation of Nrf2 in the cytoplasm.     

Oxidative and electrophilic stresses activate Nrf2 through inhibition of ubiquitination activity of Keap1

The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.     

Microsecond molecular dynamics simulations of intrinsically disordered proteins involved in the oxidative stress response

Intrinsically disordered proteins (IDPs) are abundant in cells and have central roles in protein-protein interaction networks. Interactions between the IDP Prothymosin alpha (ProTα) and the Neh2 domain of Nuclear factor erythroid 2-related factor 2 (Nrf2), with a common binding partner, Kelch-like ECH-associated protein 1(Keap1), are essential for regulating cellular response to oxidative stress. Misregulation of this pathway can lead to neurodegenerative diseases, premature aging and cancer. In order to understand the mechanisms these two disordered proteins employ to bind to Keap1, we performed extensive 0.5-1.0 microsecond atomistic molecular dynamics (MD) simulations and isothermal titration calorimetry experiments to investigate the structure/dynamics of free-state ProTα and Neh2 and their thermodynamics of bindings. The results show that in their free states, both ProTα and Neh2 have propensities to form bound-state-like β-turn structures but to different extents. We also found that, for both proteins, residues outside the Keap1-binding motifs may play important roles in stabilizing the bound-state-like structures. Based on our findings, we propose that the binding of disordered ProTα and Neh2 to Keap1 occurs synergistically via preformed structural elements (PSEs) and coupled folding and binding, with a heavy bias towards PSEs, particularly for Neh2. Our results provide insights into the molecular mechanisms Neh2 and ProTα bind to Keap1, information that is useful for developing therapeutics to enhance the oxidative stress response.