image: Ubiquitin is a small protein consisting of 76 amino acids that binds to lysine residues of substrates, usually by a combination of three enzymes: a ubiquitin-activating enzyme (E1, two types), a ubiquitin-conjugating enzyme (E2, 40 types), and a ubiquitin ligase (E3, over 600 types). This reaction is repeated to form ubiquitin chains. This study found that the ubiquitination that inactivates Nrf1 was performed by two E3s (SCFFBS2-ARIH1) and one E2 (UBE2L3), which bind to the hydroxy groups of Ser and Thr near N-glycans and GlcNAc generated by the removal of N-type sugar chains by ENGASE. The ubiquitin chains formed were complex and branched. These atypical ubiquitin chains were resistant to deubiquitinating enzymes. Thus, in the absence of NGLY1, Nrf1 is detected as a stable ubiquitinated protein in the cytosol, and this ubiquitination prevents Nrf1 from nuclear transport.
Credit: TMIMS
The proteasome is a major player in maintaining cellular homeostasis, and proteasome dysfunction leads to the synthesis of new proteasomes via the transcription factor Nrf1. Nrf1 is synthesized as a glycoprotein with N-glycans on the endoplasmic reticulum and is activated by NGLY1 and DDI2. NGLY1 is de-N-glycosylating enzyme, and mutations in the NGLY1 gene cause NGLY1 deficiency, a rare genetic disease. The Ngly1-KO mouse, an animal model of NGLY1 deficiency, is embryonically lethal, but its lethality is circumvented by the loss of FBS2, a ubiquitin ligase subunit that recognizes N-glycans. This suggests that ubiquitination by SCFFBS2 in the absence of NGLY1 leads to cellular abnormalities. We have therefore characterized the ubiquitination in detail.
This ubiquitination was triggered by ENGASE, another deglycosylating enzyme, which generates GlcNAc on the protein. Ubiquitin normally binds to lysine in proteins, but in this reaction, it bound to hydroxy groups such as serine and threonine near the sugar chains and GlcNAc of Nrf1. The ubiquitin chains formed were complex and resistant to deubiquitinating enzymes. Two E3 enzymes (SCFFBS2 and ARIH1) and one E2 enzyme (UBE2L3) were responsible for this ubiquitination process. Ubiquitinated Nrf1 cannot enter the nucleus and thus cannot function as a transcription factor. Thus, Nrf1 is inactivated by non-canonical ubiquitination by SCFFBS2- ARIH1.
Proteasome dysregulation is associated with many age-related neurodegenerative diseases. The accumulation of damaged proteins is thought to be the cause of age-related loss of function in organisms. Aberrant ubiquitination of Nrf1 is a mechanism that prevents Nrf1 from functioning properly to restore activity when the proteasome is dysfunctional. This finding paves the way for new strategies to treat diseases related to proteasome dysfunction as well as a therapeutic target for NGLY1 deficiency.
Journal
Molecular Cell
Method of Research
Experimental study
Subject of Research
Cells
Article Title
Sugar-mediated non-canonical ubiquitination impairs Nrf1/NFE2L1 activation
Article Publication Date
7-Aug-2024
COI Statement
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of the paper.