Function And Mechanism Of Deubiquitinase USP5 In NLRP3 Inflammasome Signaling Pathway

A variety of highly prevalent human diseases are exacerbated by NLR family pyrin domain containing 3(NLRP3)inflammasome,including diabetes,neurodegenerative diseases,and cardiovascular disease,so its activation needs to be tightly controlled.Although NLRP3 inflammasome signaling is involved in the pathological process of various diseases,little is known about the biochemical and molecular basis of inflammasome signaling mechanism.Post-translational modifications(PTMs),especially ubiquitination,are thought to be key molecular switches regulating inflammasome activation,but the specific mechanism in the regulation of inflammasome activation is still not clear.In recent years,it has become increasingly clear that the ubiquitin system is essential to activating the NLRP3 inflammasome,and it’s mainly E3 ubiquitin ligases and deubiquitinases(DUBs)that maintain protein ubiquitination.Ubiquitin can be transferred to substrate proteins by E3 ubiquitin ligases,and DUBs can cleave ubiquitin chains from substrate proteins and process ubiquitin precursors to maintain the dynamic balance of protein expression.E3 ubiquitin ligases can regulate NLRP3 expression via the proteasome or autophagy-lysosome pathways,while,although it has been reported that some DUBs can regulate NLRP3 expression via the proteasome pathway,however,whether DUBs can regulate the expression of NLRP3 through autophagy-lysosome pathway has not been reported.In this study,we found that Ubiquitin specific peptidase 5(USP5),a member of DUBs,has a clear lysosomal localization.Given that the NLRP3 inflammasome can be transferred to autophagosomes and lysosomes after activation,we speculate that USP5 may be involved in NLRP3 autophagic degradation.To investigate whether USP5 is involved in the inflammasome signaling,we first stimulated mouse peritoneal macrophages(PMs)with lipopolysaccharide(LPS)and interleukin-1β(IL-1β).The levels of USP5 mRNA and protein were significantly elevated,indicating that USP5 may be involved in activating inflammasome signaling.The NLRP3 inflammasome is mainly composed of NLRP3,Apoptosis associated specklike protein containing a CARD(ASC)and Caspase-1 and the activation of it can be divided into two stages.The first stage activates the NF-κB pathway and releases tumor necrosis factor(TNFα)and interleukin-6(IL-6),the second stage releases mature IL-1β.To further clarify the function of USP5 in this pathway,we synthesized small interfering RNA of Usp5,stimulated PMs with various inflammasome activators,and detected the secretion of TNFα,IL-6,and IL1β.The results showed that after Usp5 knockdown,the secretion of TNFα,IL-6 and IL-1β did not change significantly after stimulation with Absent in melanoma 2(AIM2)and NLR-family CARD-containing protein 4(NLRC4)inflammasome activators;in spite of the fact that TNFa and IL-6 secretion remained unchanged,the secretion of IL-1β was significantly increased after stimulation with NLRP3 inflammasome activators.These results suggested that knockdown of Usp5 can specifically promote the activation of the second stage of NLRP3 inflammasome.To determine the target of USP5,we performed co-immunoprecipitation(Co-IP)and immunofluorescence experiments.The results showed that USP5 only specifically binds and co-localizes with NLRP3.In order to find the interaction domain between the two,we constructed a series of truncated plasmids of USP5 and NLRP3,and detected that the USP domain of USP5 has obvious binding effect with the PYD domain of NLRP3.Next,we examined the effect of USP5 on NLRP3 protein stability.Firstly,exogenous overexpression and lentiviral infection experiments demonstrated that USP5 could specifically degrade NLRP3 protein,but it did not significantly affect the expression of ASC,Caspase-1,NLRC4 and AIM2 proteins.We then transfected the small interfering RNA of Usp5 in mouse PMs,and the results showed that the expression of endogenous NLRP3 protein was significantly increased after Usp5 knockdown.Together,these results indicated that USP5 can target NLRP3 and promote its degradation.To determine the pathway by which USP5 degrades NLRP3,we used inhibitors of the proteasome pathway and the autophagy-lysosome pathway,respectively.The results showed that only autophagy inhibitors could rescue the degradation of NLRP3 by USP5.In addition,ATG5 is a key gene of the canonical autophagy pathway,and the degradation of NLRP3 by USP5 was also significantly inhibited in ATG5-knockout HEK293T cells.Meanwhile,we found that after inflammasome activation,co-localization of NLRP3 and USP5 was detected in both autophagosomes and lysosomes.Taken together,these results suggested that USP5 degrades NLRP3 through the autophagy-lysosome pathway.Next,we discuss the effect of USP5 on the ubiquitin level of NLRP3.Both exogenous and endogenous ubiquitin experiments confirmed that USP5 could promote the ubiquitin modification of NLRP3.Through the screening of ubiquitin types,we found that USP5 can promote the K48-linked ubiquitination of NLRP3.Since USP5 is a DUB,but it can effectively increase the polyubiquitin level of NLRP3,we constructed the enzyme activity mutant USP5C335A of USP5.Exogenous overexpression and lentivirus infection experiments showed that USP5C335A could still promote the degradation and ubiquitination of NLRP3,which proved that USP5 did not depend on enzyme activity to degrade NLRP3.Further studies showed that USP5 recruited E3 ubiquitin ligase MembraneassociatedRING-CH-typefinger7(MARCH7)in an enzyme-independent manner.This is reflected in the obvious binding effect of USP5 with NLRP3 and MARCH7;after the addition of NLRP3 inflammasome activator to the cells knocking down Usp5,the secretion of IL-1β increased significantly,while after knocking down March7,the significant difference was not found in the secretion of IL-1β compared with the group that only knocked down Usp5.In addition,the expression of NLRP3 protein and K48 ubiquitin modification no longer changed significantly.To sum up,these results suggested that USP5 selectively promotes the K48 polyubiquitination of NLRP3 through MARCH7 and mediates its degradation through the autophagy-lysosomal pathway.Overall,in this paper,we first reported the function of USP5 in the inflammasome signaling pathway.After inflammasome activation,overexpression of USP5 inhibited the expression of NLRP3,whereas knockdown of Usp5 stabilized the expression of NLRP3 and promoted the subsequent secretion of IL-1β.By recruiting the E3 ligase MARCH7,USP5 selectively promoted K48-linked polyubiquitination of NLRP3 and mediated its degradation through autophagy-lysosomal pathway.Furthermore,animal models confirmed that overexpression of USP5 in vivo reduced IL-1β secretion and polymorphonuclear(PMN)infiltration in alum-induced peritonitis.Taken together,we identified the first DUB that targets NLRP3 and negatively regulates the NLRP3 inflammasome in the autophagy-lysosome system.In biomedical research,NLRP3 inflammasomes are a hotspot.Targeting the USP5-NLRP3 interaction may be a new therapeutic strategy against NLRP3-related inflammatory diseases.Innovative:1.Numerous studies have reported USP5’s role in the P53 pathway,Wnt-β-catenin pathway,type I interferon pathway,and Notch and RTK pathways,our study reports the effect of USP5 in the NLRP3 inflammasome pathway for the first time.2.At present,no DUB targeting NLRP3 and mediating its autophagy degradation has been reported.For the first time,we found that USP5 can directly target NLRP3 and promote its autophagic degradation,meanwhile,our results provide more details on the role of the E3 ubiquitin ligase MARCH7 in the regulation of NLRP3 inflammasome activation.3.This study demonstrated that USP5 negatively regulates the activation of NLRP3 inflammasome.Therefore,further studies on USP5,such as the development of various activators,may provide new therapeutic targets and strategies for NLRP3 inflammasome mediated hyperinflammatory diseases.