Covalent Modification of Keap1 by the Key Metabolic Cofactor Coenzyme A Under Oxidative and Metabolic Stress

Kelch-like ECH-associated protein 1 (Keap1) acts as a repressor of nuclear factor-erythroid 2-related factor 2 (Nrf2), a major transcription factor regulating cellular antioxidant response. Keap1 is the substrate adaptor subunit of the cullin 3-RING E3 ubiquitin ligase complex that specifically facilitates Nrf2 ubiquitination and its proteasomal degradation. Keap1 is rich in cysteine residues, and several of them undergo various modifications, such as sulphydration, nitrosylation and glutathionylation under cellular stress conditions. Some of these modifications alter the conformation of Keap1, preventing Nrf2 from ubiquitination and subsequent proteasome-mediated degradation. As a result, newly synthesised Nrf2 translocates to the nucleus to induce the expression of diverse genes involved in protecting cells against oxidative stress. Protein CoAlation is a reversible redox-dependent post-translational modification (PTM) in which coenzyme A (CoA) forms disulphide bonds with oxidised cysteine residues under oxidative or metabolic stress. In this study, we demonstrate for the first time that disulphide Keap1 dimer undergoes CoAlation in cellular response to oxidative stress induced by various oxidising compounds. Furthermore, glucose deprivation also induces CoAlation of the disulphide Keap1 dimer in HEK293/Pank1β cells. We also demonstrate that the Keap111 Cys-less mutant is not CoAlated in response to diamide treatment or glucose deprivation. In summary, this study uncovers a novel PTM of Keap1 by the key metabolic integrator CoA, which provides new insights into the regulation of the Keap1-Nrf2 antioxidant pathway under oxidative and metabolic stress.