RESUMO
Phase separation is a vital mechanism that mediates the formation of biomolecular condensates and their functions. Necroptosis is a lytic form of programmed cell death mediated by RIPK1, RIPK3, and MLKL downstream of TNFR1 and has been implicated in mediating many human diseases. However, whether necroptosis is regulated by phase separation is not yet known. Here, we show that upon the induction of necroptosis and recruitment by the adaptor protein TAX1BP1, PARP5A and its binding partner RNF146 form liquid-like condensates by multivalent interactions to perform poly ADP-ribosylation (PARylation) and PARylation-dependent ubiquitination (PARdU) of activated RIPK1 in mouse embryonic fibroblasts. We show that PARdU predominantly occurs on the K376 residue of mouse RIPK1, which promotes proteasomal degradation of kinase-activated RIPK1 to restrain necroptosis. Our data demonstrate that PARdU on K376 of mouse RIPK1 provides an alternative cell death checkpoint mediated by phase separation-dependent control of necroptosis by PARP5A and RNF146.
Assuntos
Necroptose , Separação de Fases , Animais , Camundongos , Apoptose/fisiologia , Morte Celular , Fibroblastos/metabolismo , Necroptose/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Poly(ADP-ribose) (PAR) is an RNA-like polymer that regulates an increasing number of biological processes. Dysregulation of PAR is implicated in neurodegenerative diseases characterized by abnormal protein aggregation, including amyotrophic lateral sclerosis (ALS). PAR forms condensates with FUS, an RNA-binding protein linked with ALS, through an unknown mechanism. Here, we demonstrate that a strikingly low concentration of PAR (1 nM) is sufficient to trigger condensation of FUS near its physiological concentration (1 µM), which is three orders of magnitude lower than the concentration at which RNA induces condensation (1 µM). Unlike RNA, which associates with FUS stably, PAR interacts with FUS transiently, triggering FUS to oligomerize into condensates. Moreover, inhibition of a major PAR-synthesizing enzyme, PARP5a, diminishes FUS condensation in cells. Despite their structural similarity, PAR and RNA co-condense with FUS, driven by disparate modes of interaction with FUS. Thus, we uncover a mechanism by which PAR potently seeds FUS condensation.