Identification And Functional Characterization Of Post-translational Modifications Of The Leucin Rich Repeat Domain (LRR) Of NLRP3

Innate immune response is the first line of defense against infections and damages and is triggered by pattern-recognition receptors(PRRs)activated by pathogenassociated molecular patterns(PAMPs)and/or damage-associated molecular patterns(DAMPs).Inflammation contributes to the recruit of immune cells and the elimination of pathogens and the repair of damages.The canonical inflammasome is a signaling inflammatory complex which plays a key role in inflammation process,it is assembled by the PRR sensors,the adaptor protein apoptosis-associated speck-like protein containing a caspase activating and recruitment domain(ASC)and effector proteins,pro-caspase-1.Among these PRRs,NACHT,leucine-rich repeat(LRR),and pyrin domain(PYD)-containing 3(NLRP3)inflammasome is the most extensively studied.The NLRP3 inflammasome senses a wide range of PAMPs and DAMPs and contributes to defense against infections and protection of the organisms.Adversely,dysregulation of NLRP3 inflammasome is involved in many diseases.“Gain-offunction” mutations in the NLRP3 gene lead to the development of auto-inflammatory diseases named CAPS.In addition,dysregulation of NLRP3 activation is involved in the development or progression of many multifactorial diseases(neurodegenerative diseases,type II diabetes,atherosclerosis…).The post-translational modifications(PTMs)are essential for NLRP3 inflammasome activation,in particular the PTMs of NLRP3 LRR domain is critical for its activation.However,the mechanisms are still elusive.Our goal is to better understand the regulation mechanism of PTMs in NLRP3 LRR domain.Using mass spectrometry,we identified 3 ubiquitinated lysines(K878 in human corresponding to K875 in mouse,K927 in human corresponding to T924 in mouse and K973 in human corresponding to K970 in mouse)and 3 phosphorylated serines(S735in human corresponding to S733 in mouse,S806 in human corresponding to S803 in mouse and S1035 in human corresponding to S1032 in mouse).In addition,we identified by site-targeted mutagenesis one more lysine K823(corresponding to K820 in mouse)which has effect on NLRP3 ubiquitination.The IL-1β and IL-18 secretion were significantly decreased in reconstituted human and mouse cell lines expressing NLRP3 mutants bearing substitution of these lysines to arginines(KR),which cannot be ubiquitinated,suggesting that these sites are important for NLRP3 inflammasome activation.The impact of these ubiquitinations need to be confirmed in primary cells and in vivo.Similarly,one of the phosphorylated serines S806 is critical for inflammasome assembly in reconstituted cell lines,as well as in primary bone marrow derived macrophages from in-house generated knock-in mice bearing a phosphomimetic substitution of this serine.Mechanistically,S806 D substitution suppresses NLRP3 inflammasome activity via impairing BRCC3 recruitment,leading to NLRP3 hyperubiquitination.Therefore,we discovered an additional checkpoint upstream of BRCC3 recruitment.Moreover,we identified 4 kinases as candidates to target this serine.Finally,we identified a serine/threonine specific phosphatase PPM1 A as a new regulator of the NLRP3 pathway by siRNA screen.Our studies could provide novel therapeutic targets for anti-inflammatory drugs in the future.