Different electrostatic potentials define ETGE and DLG motifs as hinge and latch in oxidative stress response

Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress.     

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.     

Structural and functional characterization of Nrf2 degradation by glycogen synthase kinase 3/β-TrCP

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of cellular homeostasis that controls the expression of more than 1% of human genes related to biotransformation reactions, redox homeostasis, energetic metabolism, DNA repair, and proteostasis. Its activity has a tremendous impact on physiology and pathology and therefore it is very tightly regulated, mainly at the level of protein stability. In addition to the very well established regulation by the ubiquitin E3 ligase adapter Keap1, recent advances have identified a novel mechanism based on signaling pathways that regulate glycogen synthase kinse-3 (GSK-3). This kinase phosphorylates specific serine residues in the Neh6 domain of Nrf2 to create a degradation domain that is then recognized by the ubiquitin ligase adapter β-TrCP and tagged for proteasome degradation by a Cullin1/Rbx1 complex. Here we review the mechanistic elements and the signaling pathways that participate in this regulation by GSK-3/β-TrCP. These pathways include those activated by ligands of tyrosine kinase, G protein-coupled, metabotropic, and ionotropic receptors that activate phosphatidyl inositol 3-kinase (PI3K)/ATK and by the canonical WNT signaling pathway, where a fraction of Nrf2 interacts with Axin1/GSK-3. Considering that free Nrf2 protein is localized in the nucleus, we propose a model termed “double flux controller” to explain how Keap1 and β-TrCP coordinate the stability of Nrf2 in several scenarios. The GSK-3/β-TrCP axis provides a novel therapeutic strategy to modulate Nrf2 activity.