NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly

Tingting Niu,Jeimin Wong,Roméo Ricci,Séverine Chautard,Thomas Henry,Bénédicte F Py,Maggy Hologne,Laurent Boyer,Virginie Pétrilli ,Olivier Walker,Danish Patoli,Charlotte De Rosny,Sabine Hacot,Marine Groslambert,Brice Lagrange,Camille Cosson,Ping Wang,Zhi Rong Zhang ,Amaury Rey,Orane Visvikis

doi:10.1038/s41467-021-26142-w

Abstract

NLRP3 controls the secretion of inflammatory cytokines IL-1β/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. Here we show that NEK7 recruitment to NLRP3 is controlled by the phosphorylation status of NLRP3 S803 located within the interaction surface, in which NLRP3 S803 is phosphorylated upon priming and later dephosphorylated upon activation. Phosphomimetic substitutions of S803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. In addition, NLRP3-NEK7 binding is also essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Finally, we identify CSNK1A1 as the kinase targeting NLRP3 S803. Our findings thus reveal NLRP3 S803 phosphorylation status as a druggable apical molecular mechanism controlling inflammasome assembly.

Highlights

NLRP3 controls the secretion of inflammatory cytokines IL-1β/18 and pyroptosis by assembling the inflammasome
Mediated NLRP3 LRR deubiquitination, which is required for the inflammasome assembly, NLRP3 LRR has been shown to be ubiquitinated and targeted to degradation by FBXL2 at K689 as well as by MARCH7 and RNF125
NLRP3 LRR deubiquitination by BRCC3 plays a key role for inflammasome assembly[4,18]

Summary

Introduction

NLRP3 controls the secretion of inflammatory cytokines IL-1β/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. NLRP3-NEK7 binding is essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Mediated NLRP3 LRR deubiquitination, which is required for the inflammasome assembly, NLRP3 LRR has been shown to be ubiquitinated and targeted to degradation by FBXL2 at K689 (in mouse NLRP3 corresponding to K691 in human NLRP3) as well as by MARCH7 and RNF125. The phosphorylation status of NLRP3 LRR domain regulates the inflammasome as PTPN22-mediated dephosphorylation at Y859 (in mouse NLRP3 corresponding to Y861 in human NLRP3) promotes its activation[9]. We analyzed the post-translational modifications of the NLRP3 LRR domain by mass spectrometry and identified serine

Methods

Results

Conclusion