Yoshida Yukiko, Takahashi Tsuyoshi, Ishii Nozomi, Matsuo Ichiro, Takahashi Satoshi, Inoue Haruka, Endo Akinori, Tsuchiya Hikaru, Okada Meari, Ando Chikara, Suzuki Takehiro, Dohmae Naoshi, Saeki Yasushi, Tanaka Keiji, Suzuki Tadashi
Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, 2-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
Graduate School of Science and Technology, Gunma University, 1-5 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
Mol Cell. 2024 Aug 22;84(16):3115-3127.e11. doi: 10.1016/j.molcel.2024.07.013. Epub 2024 Aug 7.
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), an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCF 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 SCF-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.
蛋白酶体对细胞存活至关重要，蛋白酶体抑制通过激活的内质网相关转录因子核因子红细胞2样1（Nrf1/NFE2L1）诱导蛋白酶体基因转录。Nrf1的激活需要DDI2进行蛋白水解切割以及NGLY1去除N-聚糖。我们先前表明，SKP1-CUL1-F-box（SCF）（一种识别N-聚糖的E3泛素连接酶）对Nrf1进行泛素化会损害其激活，尽管分子机制仍不清楚。在这里，我们表明SCF与RING-between-RING（RBR）型E3连接酶ARIH1协同作用，通过人细胞中的氧酯键对Nrf1进行泛素化。内切β-N-乙酰氨基葡萄糖苷酶（ENGASE）从N-聚糖产生天冬酰胺连接的N-乙酰葡糖胺（N-GlcNAc）残基，Nrf1上的N-GlcNAc残基作为SCF-ARIH1介导的泛素化的受体位点。我们在糖肽上重建了N-GlcNAc和丝氨酸/苏氨酸残基的多聚泛素化，并发现RBR特异性E2酶UBE2L3是在Nrf1上组装非典型泛素链所必需的。非典型泛素链抑制了DDI2介导的激活。目前的结果确定了一种抑制Nrf1激活的非常规泛素化途径。
