Hwang Chi Hyun, Lee Minhong, Kim Ju Won, Nam Young Woo, Hwang Gyuho, Ryu Hyun Sung, Seo Jinho, Lee Eun-Woo, Ko Hyuk Wan, Song Jaewhan
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea.
Aging Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.
Nat Commun. 2025 Aug 9;16(1):7360. doi: 10.1038/s41467-025-62723-9.
Receptor-interacting protein kinase 3 (RIPK3), a key regulator of necroptosis, is modulated by ubiquitination through various E3 ligases and deubiquitinases. However, the effects of different polyubiquitination processes on RIPK3 and necroptosis remain unclear. Using a proteomic approach, we identify SMAD Ubiquitination Regulatory Factor 1 (SMURF1) and Ubiquitin-specific peptidase 5 (USP5) as crucial regulators of RIPK3 within the necrosome during necroptosis. SMURF1 facilitates K63 polyubiquitination of RIPK3 at lysine 55 and 363, inhibiting necrosome formation and necroptosis. SMURF1 depletion accelerates necroptosis, while the reintroduction of functional SMURF1 reverses this. Conversely, USP5 acts as a deubiquitinase, removing K63 ubiquitin chains and promoting necroptosis. Reducing SMURF1, using a RIPK3 mutant defective in SMURF1-mediated ubiquitination, or overexpressing USP5 enhances necroptosis in leukaemia cells, leading to reduced tumour growth in xenograft models treated with birinapant and emricasan. These findings highlight the opposing regulation of K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis.
受体相互作用蛋白激酶3（RIPK3）是坏死性凋亡的关键调节因子，可通过各种E3连接酶和去泛素化酶进行泛素化修饰。然而，不同的多聚泛素化过程对RIPK3和坏死性凋亡的影响仍不清楚。我们采用蛋白质组学方法，鉴定出SMAD泛素化调节因子1（SMURF1）和泛素特异性肽酶5（USP5）是坏死性凋亡过程中坏死小体中RIPK3的关键调节因子。SMURF1促进RIPK3在赖氨酸55和363处的K63多聚泛素化，抑制坏死小体形成和坏死性凋亡。SMURF1缺失会加速坏死性凋亡，而重新引入功能性SMURF1可逆转这一过程。相反，USP5作为一种去泛素化酶，去除K63泛素链并促进坏死性凋亡。使用在SMURF1介导的泛素化中存在缺陷的RIPK3突变体减少SMURF1或过表达USP5可增强白血病细胞中的坏死性凋亡，导致在用birinapant和emricasan治疗的异种移植模型中肿瘤生长减少。这些发现突出了SMURF1和USP5在坏死性凋亡中对RIPK3的K63连接多聚泛素化的相反调节作用。
