Abstract：
Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1 (Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-β-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.
摘要：
蛋白酶体是细胞存活所必需的,和蛋白酶体抑制通过激活的内质网相关转录因子核因子红系2样1（Nrf1/NFE2L1）诱导蛋白酶体基因转录。Nrf1活化需要通过DDI2的蛋白水解切割和通过NGLY1的N-聚糖去除。我们先前显示，通过SKP1-CUL1-F-box（SCF）FBS2/FBXO6（一种N-聚糖识别E3泛素连接酶）进行Nrf1泛素化，削弱了它的激活，尽管分子机制仍然难以捉摸。这里,我们显示SCFFBS2与RING-between-RING(RBR)型E3连接酶ARIH1协同作用,通过人细胞中的羟酯键使Nrf1泛素化。内切β-N-乙酰氨基葡萄糖苷酶（ENGASE）从N-聚糖中产生天冬酰胺连接的N-乙酰氨基葡萄糖苷酶（N-GlcNAc）残基，Nrf1上的N-GlcNAc残基充当SCFFBS2-ARIH1介导的泛素化的受体位点。我们重建了糖肽上N-GlcNAc和丝氨酸/苏氨酸残基的多泛素化，发现RBR特异性E2酶UBE2L3是Nrf1上非典型泛素链组装所必需的。非典型泛素链抑制DDI2介导的激活。本结果鉴定了抑制Nrf1活化的非常规泛素化途径。
