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

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.

The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3

“…TRIF interacts with RIPK3 for TLR3/TLR4induced necroptosis in response to viral and bacterial infections (8). Infected cell polypeptides 6 (ICP6) from herpes simplex virus type 1 inhibits virus-induced RIPK3 necroptosis, and the ICP6 homolog M45 from murine cytomegalovirus inhibits the DAI-RIPK3 necroptosis pathway (11,20). Thus, RIPK3 may form new fibril structures in complex with other mediators, which may link to the divergent signal transduction and activation of different signal pathways for RIPK3-mediated necroptosis.…”

Optogenetic activators of apoptosis, necroptosis and pyroptosis for probing cell death dynamics and bystander cell responses

“…We show that optoCDEs allow the specific induction of these forms of cell death orthogonally to and with faster kinetics and higher efficiency than the endogenous cell death pathways, offering several advantages over other currently used methods of 'clean' cell death induction such as chemical activators or laser ablation. Compared to the forced dimerization and activation of caspases 9,35,36 and RIP3/MLKL [37][38][39][40] using modified FKBP domains, optoCDE not only excel in their rapid kinetics of activation and reversibility (inactivate within minutes of ceasing illumination), but above all in their use for single cell manipulation and imaging in 2D, 3D or in vivo settings. As we show, focused laser illumination yields superior spatiotemporal resolution that allows to rapidly and selectively kill individual cells without directly affecting the neighbors or the organism, even if these also express optoCDEs.…”

“…The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101/2021.08.31.458313 doi: bioRxiv preprint activation of caspases 9,35,36 and RIP3/MLKL [37][38][39][40] using modified FKBP domains, optoCDE not only excel in their rapid kinetics of activation and reversibility (inactivate within minutes of ceasing illumination), but above all in their use for single cell manipulation and imaging in 2D, 3D or in vivo settings. As we show, focused laser illumination yields superior spatiotemporal resolution that allows to rapidly and selectively kill individual cells without directly affecting the neighbors or the organism, even if these also express optoCDEs.…”

Methamphetamine exposure induces neuronal programmed necrosis by activating the receptor‐interacting protein kinase 3 ‐related signalling pathway

“…RIP3 plays a role in the convergence points of multiple necrotic cell death pathways. 24,[48][49][50][51][52][53][54] Normally, the activation of death receptors can trigger programmed necrosis, but it can also be activated through non-death receptor-dependent pathways. In the death receptor-dependent pathway, the receptor is recruited through the death domain to form complex I after ligand binding.…”

“…After death receptors are activated, programmed necrosis occurs when apoptosis is blocked, which is a highly regulated and genetically controlled process. RIP3 plays a role in the convergence points of multiple necrotic cell death pathways 24,48‐54 . Normally, the activation of death receptors can trigger programmed necrosis, but it can also be activated through non‐death receptor‐dependent pathways.…”