Opposing Regulation of the K63-linked Polyubiquitination of RIPK3 by SMURF1 and USP5 in Necroptosis

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（Receptor-interacting protein kinase 3, RIPK3）是坏死性凋亡（necroptosis）的关键调控因子，其泛素化修饰受到多种E3泛素连接酶（E3 ligases）与去泛素化酶（deubiquitinases）的调控。然而，不同多泛素化过程对RIPK3及坏死性凋亡的影响仍不明确。本研究通过蛋白质组学方法，鉴定出SMAD泛素化调节因子1（SMAD Ubiquitination Regulatory Factor 1, SMURF1）与泛素特异性肽酶5（Ubiquitin-specific peptidase 5, USP5）为坏死性凋亡过程中坏死小体（necrosome）内RIPK3的关键调控因子。SMURF1可促进RIPK3在赖氨酸55与363位点发生K63位多泛素化，从而抑制坏死小体形成与坏死性凋亡。敲低SMURF1会加速坏死性凋亡，而重新导入功能正常的SMURF1可逆转这一效应。与之相反，USP5作为去泛素化酶，可去除RIPK3的K63位泛素链并促进坏死性凋亡。在白血病细胞中，敲低SMURF1、使用SMURF1介导的泛素化缺陷型RIPK3突变体，或过表达USP5，均可增强坏死性凋亡；在经birinapant与emricasan处理的异种移植模型中，该效应可导致肿瘤生长受到抑制。上述研究结果揭示了SMURF1与USP5通过对RIPK3的K63位连接型多泛素化的反向调控，参与坏死性凋亡的调控过程。