NFE2L1/Nrf1 forms a coactivator complex post-peptide: <i>N</i> -glycanase-mediated sequence editing and mitigates proteasome dysfunction

Nuclear factor erythroid 2-like 1 (NFE2L1/Nrf1), an endoplasmic reticulum (ER)-associated transcription factor, is responsible for the coordinated expression of proteasome subunit genes upon proteasomal dysfunction. <i>N</i>-glycosylated proteins undergo protein sequence editing by peptide:<i>N</i>-glycanase (NGLY1)-mediated conversion of <i>N</i>-glycosylated asparagine residues to aspartic acid. Nrf proteins are the only transcription factors that undergo sequence editing for transcriptional activation. However, the mechanism via which sequence editing regulates the transcriptional activity of Nrf1 has remained unclear. Here, we demonstrated that sequence editing of the ninth <i>N</i>-glycosylation site (Asn574) in human Nrf1 is required for proteasome gene expression in HeLa cells. Editing of Asn574 is essential for interaction with host cell factor C1 and <i>O</i>-GlcNAc transferase, which is required for Nrf1 chromatin binding and sufficient proteasome expression. Furthermore, sequence editing of <i>N</i>-glycosylation sites other than Asn574 is required for the interaction with the coactivator CREBBP/EP300, thereby enhancing Nrf1's transcriptional activity. Unexpectedly, the expression of Nrf1 mutants that mimic proteolytic processing by DNA-damage-inducible 1 homolog 2 and sequence editing by NGLY1 markedly diminished the growth rate in HeLa cells, suggesting that the constitutive activation of Nrf1 exhibits cytotoxicity. Collectively, our study explains the strategy of on-demand Nrf1 activation for survival benefits. Nrf1 is synthesized as a proteasome-targeting protein and is highly glycosylated in the ER. Nrf1 is activated via sequence editing-dependent coactivator complex formation only when the proteasome needs to be compensated for.