Necroptosis is a lytic form of programmed cell death mediated by RIPK1, RIPK3 and MLKL. Activation of necroptosis has been linked to many human diseases such as neurodegenerative, cardiovascular, lung, renal, hepatic, and inflammatory diseases. Normally, necroptosis is suppressed by brakers known as cell death checkpoint. Activated caspase-8 is the only known checkpoint that prevents RIPK1-mediated necroptosis, and it is not clear how necroptosis is checked after caspase-8 inhibition. Phase separation is a vital and ubiquitous phenomenon underlying the formation of biomolecular condensates and their functions. However, whether necroptosis is regulated by LLPS is not yet known.Recently, a research group led by Prof. XU Daichao at the Interdisciplinary Research Center on Biology and Chemistry for Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, published an article titled "PARP5A and RNF146 phase separation restrains RIPK1-dependent necroptosis" in Molecular Cell. This study proposes a new mechanism for the regulation of necroptosis, involving the phase separation of PARP5A and RNF146 as an alternative cell death checkpoint for necroptosis.
The study by XU Daichao’s team demonstrate the poly ADP-ribosylation (PARylation) and PARylation-dependent ubiquitination (PARdU) of activated RIPK1 during necroptosis. They found that PARdU predominantly occurs on K376 residue of mouse RIPK1, which promotes proteasomal degradation of kinase-activated RIPK1, to restrain the formation of necrosome. RIPK1 K376 is a critical residue for K63-linked ubiquitination in TNFR1 signaling complex (TNF-RSC), which acts as an early checkpoint to promote the activation of pro-survival NF-κB signaling and counteract RIPK1 kinase-dependent and independent apoptosis in TNFα pathway. They found that if K376 was defective in ubiquitination in TNF-RSC, K376 further undergoes PARdU, which acts as another checkpoint for RIPK1-dependent necroptosis.
The team further found that the PARdU of RIPK1 is mediated by the complex of PARP5A and the E3 ubiquitin ligase RNF146, which is recruited to the RIPK1 by TAX1BP1. They discovered that PARP5A and RNF146 can form liquid-like condensates through phase separation during early stage of necroptosis. Importantly, this necroptosis activation-triggered PARP5A/RNF146 LLPS, which can increase their local concentration and dynamics, is critical for PARP5A-catalyzed PARylation and subsequent RNF146-catalyzed PARdU of RIPK1 in necroptosis.
Overall, this study represents a major conceptual advance in the field of necroptosis by revealing PARdU of kinase-activated RIPK1 as a novel cell death checkpoint mediated by phase separation-dependent control of necroptosis by PARP5A and RNF146. These findings provide evidence that necroptosis can be regulated by dynamic biomolecular condensates, which may open up a new phase of drug discovery for necroptosis-associated pathologies, focusing on the regulation of PARP5A/RNF146 phase separation.
Fig. 1 A model of phase separation-dependent control of necroptosis by PARP5A and RNF146 (Image by XU Daichao)
XU Daichao Ph.D., Professor
Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences
State Key Laboratory of Chemical Biology, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences
Haike Road 100, Shanghai 201210, China
Tel: 0086-21-68582337
Email: xudaichao@sioc.ac.cn 
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