Necroptosis is mediated by signaling complexes called necrosomes, which contain receptor-interacting protein 3 (RIP3) and upstream effectors, such as RIP1. In necrosomes, the RIP homotypic interaction motif (RHIM) of RIP3 and RIP1 forms amyloidal complex. But how the amyloidal necrosomes control RIP3 activation and cell necroptosis has not been determined. Here, we showed that RIP3 amyloid fibrils could further assemble into large fibrillar networks which presents as cellular puncta during necroptosis. A viral RHIM-containing necroptosis inhibitor M45 could form heteroamyloid with RIP3 in cells and prevent RIP3 puncta formation and cell necroptosis. We characterized mutual antagonism between RIP3–RHIM and M45–RHIM in necroptosis regulation, which was caused by distinct inter-filament interactions in RIP3, M45 amyloids revealed with atomic force microscopy. Moreover, double mutations Asn464 and Met468 in RIP3–RHIM to Asp disrupted RIP3 kinase-dependent necroptosis. While the mutant RIP3(N464D/M468D) could form amyloid as wild type upon necroptosis induction. Based on these results, we propose that RIP3 amyloid formation is required but not sufficient in necroptosis signaling, the ordered inter-filament assembly of RIP3 is critical in RIP3 amyloid mediated kinase activation and cell necroptosis.

坏死性凋亡由称为坏死体的信号复合体介导，坏死体包含受体相互作用蛋白 3 (RIP3) 和上游效应子，如 RIP1。在坏死体中，RIP3 和 RIP1 的 RIP 同型相互作用基序 (RHIM) 形成淀粉样复合物。但淀粉样坏死体如何控制 RIP3 激活和细胞坏死尚未确定。在这里，我们表明 RIP3 淀粉样蛋白原纤维可以进一步组装成大的原纤维网络，在坏死性凋亡过程中表现为细胞点。含有病毒 RHIM 的坏死性凋亡抑制剂 M45 可以在细胞中与 RIP3 形成异源淀粉样蛋白，并防止 RIP3 斑点形成和细胞坏死性凋亡。我们表征了 RIP3-RHIM 和 M45-RHIM 在坏死性凋亡调节中的相互拮抗作用，这是由 RIP3 中不同的丝间相互作用引起的，用原子力显微镜显示 M45 淀粉样蛋白。此外，RIP3-RHIM 中 Asn464 和 Met468 向 Asp 的双重突变破坏了 RIP3 激酶依赖性坏死性凋亡。而突变体 RIP3(N464D/M468D) 可以在坏死性凋亡诱导后形成野生型淀粉样蛋白。基于这些结果，我们提出 RIP3 淀粉样蛋白形成是必需的，但在坏死性凋亡信号传导中是不够的，RIP3 的有序丝间组装在 RIP3 淀粉样蛋白介导的激酶激活和细胞坏死性凋亡中至关重要。