The NHB1 (N-terminal homology box 1) sequence in transcription factor Nrf1 is required to anchor it to the endoplasmic reticulum and also to enable its asparagine-glycosylation

Abstract

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is negatively controlled by its NTD (N-terminal domain) that lies between amino acids 1 and 124. This domain contains a leucine-rich sequence, called NHB1 (N-terminal homology box 1; residues 11-30), which tethers Nrf1 to the ER (endoplasmic reticulum). Electrophoresis resolved Nrf1 into two major bands of approx. 95 and 120 kDa. The 120-kDa Nrf1 form represents a glycosylated protein that was present exclusively in the ER and was converted into a substantially smaller polypeptide upon digestion with either peptide:N-glycosidase F or endoglycosidase H. By contrast, the 95-kDa Nrf1 form did not appear to be glycosylated and was present primarily in the nucleus. NHB1 and its adjacent residues conform to the classic tripartite signal peptide sequence, comprising n-, h- and c-regions. The h-region (residues 11-22), but neither the n-region (residues 1-10) nor the c-region (residues 23-30), is required to direct Nrf1 to the ER. Targeting Nrf1 to the ER is necessary to generate the 120-kDa glycosylated protein. The n-region and c-region are required for correct membrane orientation of Nrf1, as deletion of residues 2-10 or 23-30 greatly increased its association with the ER and the extent to which it was glycosylated. The NHB1 does not contain a signal peptidase cleavage site, indicating that it serves as an ER anchor sequence. Wild-type Nrf1 is glycosylated through its Asn/Ser/Thr-rich domain, between amino acids 296 and 403, and this modification was not observed in an Nrf1(Delta299-400) mutant. Glycosylation of Nrf1 was not necessary to retain it in the ER.

Basic Leucine Zipper Protein Cnc-C Is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome

“…The function of Keap1 binding domains present in Nrf1 is currently unknown. It has been shown that the Nrf1 N terminus is an important functional regulatory element that anchors the transcription factor to the membrane (57). The above analysis supports the idea that the various functions for the Nrf family were present before multiple duplications in modern vertebrates.…”

“…This classification is based on the ability of Cnc-C to promote transcription of antioxidant genes and the observation that Cnc-C can also be regulated by a homologous cytoplasmic binding repressor, Keap1, as found with the vertebrate Nrf2 transcription factor. The vertebrate Nrf1 transcription factor also has Keap1 binding domains; however, (38,57) and anchor the proteins to membranes. The Drosophila Cnc-C N terminus shows similarities to both mammalian Nrf1 and Nrf2 critical domains.…”

TCF11 at the crossroads of oxidative stress and the ubiquitin proteasome system

“…Thus glycosylation may direct the intracellular transport or regulates the activation of TCF11. 46,58 Zhang and colleagues discuss a cleavage-independent mechanism, requiring an importin (KPNA4-16)-mediated translocation of TCF11 in a membranebound state.…”

“…46,57,58 This process requires the p97-mediated extraction from the membrane and the ubiquitylation by the ER-resident E3-ubiquitin-ligase HRD1. In response to proteasome inhibition TCF11 is translocated from the ER to the nucleus, where it dimerizes with other bZIP-TFs and transactivates the transcription of UPS-genes (Fig.…”

“…However, Zhang and colleagues found no evidence for S1P and S2P cleavage of TCF11. 58 In addition to S2P (metalloprotease) other intramembrane proteases are the γ-secretase/presenilin (aspartyl proteases), the signal peptidase (aspartyl proteases) as well as the rhomboids (serine proteases). 64 The RIP-hypothesis in TCF11 activation needs further evaluation by treating cells with specific RIP in an inactive state via a transmembrane anchorage in the ER and active parts are released by RIP if required.…”