Paxillin mediates ATP-induced activation of P2X7 receptor and NLRP3 inflammasome

Wenbiao Wang,Weiyong Liu,Yuqian Feng,Yingle Liu,Pan Pan,Mingfu Tian,Dingwen Hu,Feng Xiao,Yunting Song,Huiyao Lan,Pin Wan,Aixin Li,Weijie Chen,Caifeng Wu,Qi Zhang,Jianguo Wu,Yingchong Wang,Keli Chen,Kailang Wu

doi:10.1186/s12915-020-00918-w

Abstract

BackgroundExtracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets. ATP directly activates the plasma membrane channel P2X7 receptor (P2X7R), leading to an intracellular influx of K+, a key trigger inducing NLRP3 inflammasome activation. However, the mechanism underlying P2X7R-mediated activation of NLRP3 inflammasome is poorly understood, and additional molecular mediators have not been identified. Here, we demonstrate that Paxillin is the molecule connecting the P2X7 receptor and NLRP3 inflammasome through protein interactions.ResultsWe show a distinct mechanism by which Paxillin promotes ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and then facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K+ efflux. Moreover, we demonstrated that USP13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from the cytosol to the plasma membrane and facilitates P2X7R-Paxillin interaction and PaxillinNLRP3 association, resulting in the formation of the P2X7R-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as well as in human PBMCs and THP-1-differentiated macrophages.ConclusionsWe have identified paxillin as a mediator of NLRP3 inflammasome activation. Paxillin plays key roles in ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7R-Paxillin-NLRP3 complex.

Highlights

Extracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets
Paxillin functionally is essential for ATP- and Nigericin-induced NLRP3 inflammasome activation. These results demonstrate that Paxillin promotes ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7RPaxillin-NLRP3 complex
Glutathione S-transferase (GST) pull-down assays showed that GST-LRR was pulled down with Paxillin (Fig. 1d) and GST-Paxillin was pulled down with NLRP3 (Fig. 1e), suggesting that Paxillin directly binds to the NLRP3 LRR domain

Summary

Introduction

Extracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets. ATP directly activates the plasma membrane channel P2X7 receptor (P2X7R), leading to an intracellular influx of K+, a key trigger inducing NLRP3 inflammasome activation. The NACHT, LRR, and PYD domain-containing protein 3 (NLRP3), one of the host pattern recognition receptors (PRRs), recognizes pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). NLRP3 (the cytoplasmic sensor molecular) together with apoptosis-associated speck-like protein with the CARD domain (ASC) (the adaptor protein) promote the cleavage of the pro-Caspase-1 (the effector protein) to generate active subunits p20 and p10, which regulate the maturation of IL-1β [2]. Activated Caspase-1 cleaves pro-IL-1β into active IL-1β, which is secreted

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