Molecular mechanisms controlling the multistage post-translational processing of endogenous Nrf1α/TCF11 proteins to yield distinct proteoforms within the coupled positive and negative feedback circuits

Abstract

In an attempt to terminate the chaotic state of the literature on Nrf1/TCF11 with various confused molecular masses, we herein establish a generally acceptable criterion required for identification of its endogenous full-length proteins and derivative isoforms expressed differentially in distinct experimental cell lines. Further work has been focused on the molecular mechanisms that dictate the successive multistate post-translational modifications (i.e. glycosylation by OST, deglycosylation by NGLY, and ubiquitination by Hrd1) of this CNC-bZIP protein and its proteolytic processing to yield multiple isoforms. Several lines of experimental evidence have demonstrated that the nascent Nrf1/TCF11 polypeptide (non-glycosylated) is transiently translocated into the endoplasmic reticulum (ER), in which it becomes an inactive glycoprotein-A, and also folded in a proper topology within and around membranes. Thereafter, dynamic repositioning of the ER-resident domains in Nrf1 glycoprotein is driven by p97-fueled retrotranslocation into extra-ER compartments. Therein, glycoprotein of Nrf1 is allowed for digestion into a deglycoprotein-B and then its progressive proteolytic processing by cytosolic DDI-1/2 and proteasomes to yield distinct proteoforms (i.e. protein-C/D). The processing is accompanied by removal of a major N-terminal ~12.5-kDa polypeptide from Nrf1. Interestingly, our present study has further unraveled that coupled positive and negative feedback circuits exist between Nrf1 and its cognate target genes, including those encoding its regulators p97, Hrd1, DDI-1 and proteasomes. These key players are differentially or even oppositely involved in diverse cellular signalling responses to distinct extents of ER-derived proteotoxic and oxidative stresses induced by different concentrations of proteasomal inhibitors.