Regulation and Functions of the ER-Associated Nrf1 Transcription Factor

Abstract

Nrf1 is a member of the nuclear erythroid 2-like family of transcription factors that regulate stress-responsive gene expression in animals. Newly synthesized Nrf1 is targeted to the endoplasmic reticulum (ER) where it is N-glycosylated. N-glycosylated Nrf1 is trafficked to the cytosol by the ER-associated degradation (ERAD) machinery and is subject to rapid proteasomal degradation. When proteasome function is impaired, Nrf1 escapes degradation and undergoes proteolytic cleavage and deglycosylation. Deglycosylation results in deamidation of N-glycosylated asparagine residues to edit the protein sequence encoded by the genome. This truncated and "sequence-edited" form of Nrf1 enters the nucleus where it induces up-regulation of proteasome subunit genes. Thus, Nrf1 drives compensatory proteasome biogenesis in cells exposed to proteasome inhibitor drugs and other proteotoxic insults. In addition to its role in proteasome homeostasis, Nrf1 is implicated in responses to oxidative stress, and maintaining lipid and cholesterol homeostasis. Here, we describe the conserved and complex mechanism by which Nrf1 is regulated and highlight emerging evidence linking this unusual transcription factor to development, aging, and disease.

Figure 1.

Nrf family transcription factors of humans and Caenorhabditis elegans. The Human Nrfs are encoded by distinct genes, whereas the various SKN-1 proteins of C. elegans are generated by alternative transcription start sites and splicing at the skn-1 locus. The amino acid sequences of SKN-1A/C/B are identical aside from the amino-terminal 90 amino acids of SKN-1A, and the amino-terminal 77 amino acids of SKN-1B, which are each encoded by isoform-specific exons. Nrf1, Nrf3, and SKN-1A contain an amino-terminal transmembrane (TM) domain. The site of DDI-1/DDI2-dependent proteolysis of SKN-1A, Nrf1, and Nrf3 is marked in red. Nrf1 additionally contains a cholesterol recognition amino acid consensus (CRAC) domain. Nrf1 contains eight N-glycosylation sites within its N-X-S/T-rich (NST) domain, and one site further carboxy terminal. Nrf3 also contains a central NST domain and is N-glycosylated. SKN-1A contains four N-glycosylation sites within its NST domain. All three vertebrate Nrfs contain a highly homologous cap'n’collar (CNC) basic leucine zipper (bZIP) DNA-binding domain. All SKN-1 isoforms contain an identical DNA-binding domain that consists of a CNC domain followed by a carboxy-terminal basic region (BR). The DDI-1-dependent proteolysis sequence and NST domain are present in SKN-1C, but SKN-1C does not undergo N-glycosylation or proteolysis since endoplasmic reticulum (ER)-association is required for both these post-translational modifications. For ease of interpretation, the proteins and their domains are depicted to approximate scale.

Figure 2.

The mechanism of SKN-1A/Nrf1 activation under proteasome dysfunction. SKN-1A/Nrf1 is inserted into the endoplasmic reticulum (ER). The amino-terminal transmembrane domain is anchored within the ER membrane and the carboxyl terminus of the protein is extruded into the ER lumen. SKN-1A/Nrf1 is N-glycosylated in the ER. The SKN-1A/Nrf1 glycoprotein is extracted from the ER and ubiquitinated by the ER-resident ERAD factors SEL-11/HRD1 and SEL-1/HRD3. The extraction of SKN-1A/Nrf1 from the ER requires the p97 ATPase. In cells with sufficient proteasome capacity, cytosolic SKN-1A/Nrf1 is rapidly degraded. In cells with impaired proteasome function, SKN-1A/Nrf1 half-life may be increased to the point that it is cleaved by the protease DDI-1/DDI2 and deglycosylated by the peptide:N-glycanase PNG-1/NGLY1. In this model, deglycosylation precedes proteolysis, but the order of these events is not known. Proteolytic cleavage liberates the transmembrane domain-containing amino terminus of SKN-1A/Nrf1. Removal of N-linked glycans post-translationally converts N-glycosylated asparagine (N) residues into aspartates (D). Proteolytically processed and N-to-D edited SKN-1A/Nrf1 activates the expression of proteasome subunits and other target genes via binding to antioxidant response elements (AREs) within their promoters. Elevated proteasome subunit gene expression leads to increased proteasome biogenesis, thus restoring adequate proteasome function.