Abstract
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.
Highlights
These authors contributed : Hong Hu, Xialian WuEdited by: H
The RIP homotypic interaction motif (RHIM) of RIP1/3 can form A-beta-like amyloid fibrils in vitro and amyloid fibril formation is required for necroptosis signaling [5,6,7, 19]
Our data showed that RHIM-mediated amyloid formation of receptor-interacting protein 3 (RIP3) and M45 is induced by an upstream necrosis signal, RIP3 kinase activation and cell necrosis are blocked by M45 (Fig. 5f)
Summary
When the M45–RHIM is changed to an ICP6-RHIM, the ICP6-RHIM-containing M45 gains the ability to induce necroptosis in mouse cells [23]. This suggests that these different RHIM domains perform distinct functions by either promoting or inhibiting cell necroptosis. RIP3 mutant which disrupted the proper inter-filament interactions could not induce necroptosis These data suggested the ordered inter-filament assembly of RIP3–RHIM into the high-ordered RIP3 amyloid networks as cellular puncta is a critical step of necroptosis signaling, which could be blocked by specific mutations of RIP3 or via the formation of neutralized M45–RIP3 hetero-RHIM amyloids. Cells were transfected using Lipofectamine 2000 (Invitrogen), Lipofectamine 3000 (Invitrogen), or EZ transfection (Shanghai Life-iLab Biotech Co., Ltd) according to the manufacturer’s instructions
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