RIP3-mediated necroptosis is regulated by inter-filament assembly of RIP homotypic interaction motif.

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.

ICP6 Prevents RIP1 Activation to Hinder Necroptosis Signaling.

Necroptosis is a type of programmed necrosis which depends on the activation of receptor-interacting protein kinase 3 (RIP3). Herpes simplex virus type 1 (HSV-1) is known to block necroptosis by the viral protein ICP6 in human cells, but its specific inhibitory mechanism is not fully understood. Here we reported that ICP6 could promote rather than suppress the formation of necrosome, the necroptosis signaling complex containing RIP3 and upstream regulator receptor-interacting protein kinase 1 (RIP1), but blocked RIP3 activation. Moreover, ICP6 could reduce the necroptosis-specific auto-phosphorylation of RIP1 regardless of the presence of RIP3. These results indicate that ICP6 block necroptosis through preventing RIP1 activation dependent signal transduction in necrosome.