NGLY1 deficiency is a rare congenital disorder caused by loss-of-function mutations in the cytosolic peptide:N-glycanase NGLY1. Patients present with multisystem symptoms, underscoring the essential role of cytosolic N-glycan quality control in cellular homeostasis. While glycosylation has traditionally been associated with extracellular functions, the finding of NGLY1 deficiency has revealed a critical link between cytosolic deglycosylation and ubiquitin-mediated proteasome regulation.
Ngly1-knockout (KO) mice are embryonic lethal, but this lethality can be completely rescued by deleting Fbs2, which encodes a glycan-recognizing F-box protein in the SCF ubiquitin ligase complex. In NGLY1-KO cells, FBS2 expression induces aberrant ubiquitination of the proteasome regulator Nrf1/NFE2L1, leading to proteasome dysfunction and cell death. We found that Nrf1 is highly ubiquitinated by SCFFBS2, and this ubiquitination required a second E3 ligase, ARIH1, as well as endo-β-N-acetylglucosaminidase (ENGASE). SCFFBS2-ARIH1 cooperatively generate atypical ubiquitin chains via oxyester bonds to Ser/Thr residues adjacent to N-glycans or to GlcNAc residues generated by ENGASE.
Under normal conditions, Nrf1 is constantly ubiquitinated by Hrd1 and degraded via the ERAD pathway. When proteasome activity is impaired, retrotranslocated Nrf1 escapes degradation, is deglycosylated by NGLY1—converting glycosylated Asn to Asp—and activates transcription of proteasome genes. Our findings demonstrate that, in the absence of NGLY1, pathogenic SCFFBS2–ARIH1–mediated ubiquitination of Nrf1 disrupts this activation pathway, providing mechanistic insight into the ubiquitin-dependent pathology of NGLY1 deficiency. In this talk, the significance of Nrf1’s ER origin and the molecular mechanism by which NGLY1 regulates Nrf1 activity, providing insight into the pathogenesis of NGLY1 deficiency.