The selective post-translational processing of transcription factor Nrf1 yields distinct isoforms that dictate its ability to differentially regulate gene expression

Abstract

Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of Nrf1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where Nrf1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa Nrf1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa Nrf1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of Nrf1 by the proteasome and/or calpains to generate a cleaved active 85-kDa Nrf1 or a dominant-negative 36-kDa Nrf1γ. Production of Nrf1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa Nrf1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81–106). Importantly, Nrf1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors.

Synergism and antagonism of two distinct, but confused, Nrf1 factors in integral regulation of the nuclear-to-mitochondrial respiratory and antioxidant transcription networks

“…The ER-targeting NTD of Nfe2l1 enables it to be topologically anchored within and around the membranes (56)(57)(58), whilst its NST glycodomain facilitates its proper protein folding and subsequent processing (59)(60)(61). The membrane-topology of Nfe2l1 determines its selective proteolytic processing by proteasomes and/or other cytosolic proteases in close proximity to the ER, in order to yield distinct lengths of its isoforms, before its mature CNC-bZIP factor is translocated into the nucleus (44,61,62). Such the membrane-bound Nfe2l1 factor can also be activated in the unfolded protein response to the ER stress stimulated by tunicamycin (to inhibit its N-linked glycosylation) (38), in addition to its proteasomal 'bounce-back' response (63,64).…”

Transcription factor Nrf1 is negatively regulated by its O‐GlcNAcylation status

“…The plasmid pcDNA-Flag-OGT (#29760) and the pRL-CMV reporter were purchased from Addgene and Promega, respectively. Additional expression constructs for Nrf1 and its mutant that had been created on the base of pcDNA3.1/V5His, as well as pcDNA-3 Â HA-Ub and the ARE-driven reporter gene P SV40 6 Â ARE-GSTA2-Luc, were developed in our laboratory [34][35][36][37][38][39]. All these constructs were verified by DNA sequencing.…”

“…Conversely, a loss of the NST domain (aa 299-400, lanes #4 with 1.00/0.37), but not of the AD2 regions (aa 403-455, lanes #5 & 6), caused an apparent enhancement in OGT-mediated destabilization of the Nrf1 D299À440 mutant by $42%, when compared with the wild-type Nrf1 protein. Furthermore, our previous work has showed that the NST domain of Nrf1 is N-glycosylated and also enables it to be anchored within the ER lumen and, hitherto demonstrated that the process controls its topovectorial repartitioning out of the organelle [34][35][36][37][38][39]. Together, our findings suggest that the NST glycodomain of Nrf1 contributes to stabilization of its wild-type protein possibly through a mechanism that negatively monitors the repositioning of NST-flanking regions (aa 454-488 and 81-291) from the ER lumen into the cyto/nucleoplasmic compartments, such that both regions would be allowed for association with the OGT-mediated event for O-GlcNAcylation.…”

“…Subsequently, glycosylated NST-containing TADs are allowed for dynamic repartitioning out of the ER and repositioning into the cyto/nucloplasm, whereupon Nrf1 is deglycosylated to yield an active 95kDa factor because its TADs can gain access to the general transcriptional machinery, enabling target gene transactivation. The membranetopovectorial organization of Nrf1 also dictates selective proteolytic processing of the CNC-bZIP protein to generate distinct isoforms of between 85kDa and 25kDa that differentially regulate ARE-battery genes [37,[39][40][41]. In the present study, we have investigated whether and how the protein stability of Nrf1 is influenced by OGT-mediated O-GlcNAcylation.…”

“…The unique biological function of Nrf1 is dictated by its membrane-topovectorial processing within and around endoplasmic reticulum (ER) to produce several isoforms through different post-translational modifications [34][35][36][37][38][39]. Upon translation of Nrf1, its uncleavable N-terminal signal sequence (called NHB1) enables the nascent protein to be integrated in a specific topology within and around the ER.…”