The proteolytic activity of caspase-8 suppresses lethal RIPK1-, RIPK3- and MLKL-dependent necroptosis during mouse embryogenesis. Caspase-8 is reported to cleave RIPK3 in addition to the RIPK3-interacting kinase RIPK1, but whether cleavage of RIPK3 is crucial for necroptosis suppression is unclear. Here we show that caspase-8-driven cleavage of endogenous mouse RIPK3 after Asp³³³ is dependent on downstream caspase-3. Consistent with RIPK3 cleavage being a consequence of apoptosis rather than a critical brake on necroptosis, Ripk3^D333A/D333A knock-in mice lacking the Asp³³³ cleavage site are viable and develop normally. Moreover, in contrast to mice lacking caspase-8 in their intestinal epithelial cells, Ripk3^D333A/D333A mice do not exhibit increased sensitivity to high dose tumor necrosis factor (TNF). Ripk3^D333A/D333A macrophages died at the same rate as wild-type (WT) macrophages in response to TNF plus cycloheximide, TNF plus emricasan, or infection with murine cytomegalovirus (MCMV) lacking M36 and M45 to inhibit caspase-8 and RIPK3 activation, respectively. We conclude that caspase cleavage of RIPK3 is dispensable for mouse development, and that cleavage of caspase-8 substrates, including RIPK1, is sufficient to prevent necroptosis.

caspase-8 的蛋白水解活性抑制小鼠胚胎发生过程中致死的 RIPK1-、RIPK3 和 MLKL 依赖性坏死性凋亡。据报道，除了 RIPK3 相互作用激酶 RIPK1 外，Caspase-8 还可以切割 RIPK3，但 RIPK3 的切割是否对坏死性凋亡抑制至关重要尚不清楚。在这里，我们表明 Asp³³³ 后内源性小鼠 RIPK3 的 caspase-8 驱动的切割依赖于下游 caspase-3。与 RIPK3 切割是细胞凋亡的结果而不是坏死性凋亡的关键制动一致，缺乏 Asp³³³ 切割位点的 Ripk3^D333A/D333A 敲入小鼠是可行的并且发育正常。此外，与肠上皮细胞中缺乏 caspase-8 的小鼠相比，Ripk3^D333A/D333A 小鼠对高剂量肿瘤坏死因子 （TNF） 的敏感性没有增加。TNF 加放线菌酮、TNF 加 emricasan、TNF 加 emricasan，或感染缺乏 M36 和 M45 的小鼠巨细胞病毒 （MCMV） 分别抑制 caspase-8 和 RIPK3 活化，Ripk3^D333A/D333A 巨噬细胞的死亡率与野生型 （WT） 巨噬细胞的死亡率相同。我们得出结论，RIPK3 的 caspase 切割对于小鼠发育是可有可无的，并且 caspase-8 底物（包括 RIPK1）的切割足以预防坏死性凋亡。