Abstract
Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology-like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1+/+ than in KEAP1-/- isogenic cell lines, whereas NRF2 is dramatically stabilized in KEAP1-/- cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.
Highlights
Nuclear factor erythroid-2–related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses
The CNC-bZIP transcription factors NRF1 and NRF2 are essential for maintaining redox homeostasis, metabolism, normal development, and proliferation and in coordinating other cellular stress responses
Gene duplication and diversion events led to the appearance of new proteins with different functionalities that gave rise to the mammalian CNC-bZIP family of which NRF1 and NRF2 are members [53]
Summary
Kelch-like ECH-associated protein 1 (KEAP1) differentially regulates nuclear factor erythroid-2–related factors 1 and 2 (NRF1 and NRF2). Dimerization with small MAF proteins and binding to the antioxidant-response element (ARE) in the regulatory regions of their target genes [7] Both NRF1 and NRF2 are expressed ubiquitously and are under very tight controls to regulate their protein levels and cellular localization and their transcriptional activity [8]. Because the Neh2L is conserved but not identical to the Neh, we sought to determine what are the critical amino acid residues necessary for effective KEAP1-mediated degradation This information is necessary to deepen our basic mechanistic understanding of KEAP1-mediated degradation and to correlate this with the high mutation rate of the NRF2 Neh domain in cancer that leads to constitute NRF2 activation and resistance to treatments [47, 48], as well as the emerging role of NRF1 in cancer
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