RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes

Xiang Li,Changchuan Xie,Chuan-Qi Zhong,Jianwei Shuai,Zhang-Hua Yang,Xiurong Wu,Shaowei Cai,Yihua Yan,Fei Xu,Hong Qi,Rui Wu,Jiahuai Han,Xiaozheng Xu,Qingzu He,Zhiyong Yin,Xin Chen,Dianjie Li

doi:10.1007/s13238-020-00810-x

Xiang Li, Changchuan Xie + Show 15 more

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https://doi.org/10.1007/s13238-020-00810-x

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Journal: Protein & cell	Publication Date: Jan 2, 2021
Citations: 81	License type: open-access

Affiliation: Xiamen University, Shanxi University

Abstract

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

Highlights

Tumor necrosis factor (TNF) can induce apoptosis or necroptosis depending on cellular contexts, and apoptosis and necroptosis pathways can compete with and convert into each other (Han et al, 2011; Brenner et al, 2015)
We employed MS to systematically analyze the dynamic assembly of three critical complexes—TNF receptor 1 (TNFR1), receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) complexes of TNF signaling in L929 cell line, which is a well-established cellular model to study necroptosis
The responses of wildtype, Flag-RIP1, and Flag-RIP3 cell lines to TNF stimulation were similar in terms of activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and mixed lineage kinase domain-like protein (MLKL) (Fig. S1A)

Summary

Introduction

Tumor necrosis factor (TNF) can induce apoptosis or necroptosis depending on cellular contexts, and apoptosis and necroptosis pathways can compete with and convert into each other (Han et al, 2011; Brenner et al, 2015). Complex I mainly consists of TNFR1-associated death domain protein (TRADD), receptor-interacting protein kinase 1 (RIP1), TNF receptor-associated factor 2 (TRAF2), cellular inhibitor of apoptosis 1/2 (cIAP1/2) and some other proteins. Polyubiquitination of RIP1 occurs in complex I, leading to NF-κB activation. The non-ubiquitinated or de-ubiquitinated RIP1 recruits Fas-associated death domain protein (FADD) and caspase-8 to form complex II, which allows caspase-8 auto-. RIP1-dependent linear and nonlinear recruitments processing, resulting in apoptosis (Brenner et al, 2015). If there is RIP3 expression in cells, RIP3 is recruited to the RIP1-FADD-caspase-8 complex to form necrosome. Caspase-8 exhibits low level protease activity in necrosome which is responsible for the cleavage and inactivation of RIP1 and RIP3 to halt necroptotic process (Tummers et al, 2017; Newton et al, 2019a; Newton et al, 2019b)

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