A20 protects cells from TNF-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms

Dario Priem,Arne Martens,Sarah Druwé,Mathieu J M Bertrand,Michael Devos,Adrian T Ting,Karolina Slowicka,Geert Van Loo,Wim Declercq,Peter Vandenabeele,Manolis Pasparakis

doi:10.1038/s41419-019-1937-y

Abstract

The cytokine TNF promotes inflammation either directly by activating the MAPK and NF-κB signaling pathways, or indirectly by triggering cell death. A20 is a potent anti-inflammatory molecule, and mutations in the gene encoding A20 are associated with a wide panel of inflammatory pathologies, both in human and in the mouse. Binding of TNF to TNFR1 triggers the NF-κB-dependent expression of A20 as part of a negative feedback mechanism preventing sustained NF-κB activation. Apart from acting as an NF-κB inhibitor, A20 is also well-known for its ability to counteract the cytotoxic potential of TNF. However, the mechanism by which A20 mediates this function and the exact cell death modality that it represses have remained incompletely understood. In the present study, we provide in vitro and in vivo evidences that deletion of A20 induces RIPK1 kinase-dependent and -independent apoptosis upon single TNF stimulation. We show that constitutively expressed A20 is recruited to TNFR1 signaling complex (Complex I) via its seventh zinc finger (ZF7) domain, in a cIAP1/2-dependent manner, within minutes after TNF sensing. We demonstrate that Complex I-recruited A20 protects cells from apoptosis by stabilizing the linear (M1) ubiquitin network associated to Complex I, a process independent of its E3 ubiquitin ligase and deubiquitylase (DUB) activities and which is counteracted by the DUB CYLD, both in vitro and in vivo. In absence of linear ubiquitylation, A20 is still recruited to Complex I via its ZF4 and ZF7 domains, but this time protects the cells from death by deploying its DUB activity. Together, our results therefore demonstrate two distinct molecular mechanisms by which constitutively expressed A20 protect cells from TNF-induced apoptosis.

Highlights

TNF is a well-established inducer of inflammation, and a pharmacological target in several inflammatory disorders[1]
We found that A20 deficiency suffices to sensitize Mouse embryonic fibroblasts (MEFs) and intestinal organoids to TNF-induced death in the absence of any additional inhibitor of the TNFR1 pathway
We found that A20 deficiency results in the simultaneous occurrence of RIPK1 kinase-dependent and -independent apoptosis, but not in the spontaneous induction of necroptosis

Summary

Introduction

TNF is a well-established inducer of inflammation, and a pharmacological target in several inflammatory disorders[1]. The initial binding of TRADD and RIPK1 to the receptor allows the subsequent recruitment of TRAF2 and of the E3 ubiquitin ligases cIAP1/2 and LUBAC. Together, these E3s generate a dense network of ubiquitin chains resulting in the stabilization of Complex I and in the recruitment of the kinases that activate the MAPKs and NF-κB signaling pathways[2,3]. The K63ubiquitin chains generated by cIAP1/2 operate as binding stations for the adapter proteins TAB2/3 and for the recruitment of the kinase TAK1, which subsequently activates the MAPK pathway by phosphorylation[3,4,5].

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Results

Conclusion