Molecular Mechanism By Which APC10 Activates NLRP3 Inflammasome

The innate immune system is the first line of host defense that senses signals,including pathogenic microbes and cell stresses.Pattern-recognition receptors(PRRs)initiate the innate immune system by detecting pathogen-associated molecular patterns and damage-associated molecular patterns.Several types of PRRs have been identified,including the Toll-like receptor,RIG-I-like receptor,NOD-like receptor(NLR),AIM2-like receptor,C-type lectin receptor,and intracellular molecular receptor.One of the best-characterized inflammasomes consists of an NLR family protein,the PYRIN domain containing-3(NLRP3).Stimulated by diverse stimulis,NLRP3 recruits ASC and pro-Caspase-1 to promote the cleavage of pro-Caspase-1,which in turn regulates the maturation of IL-1?and IL-18 and pyroptosis.The NLRP3 inflammasome has been reported to be related to several inflammatory diseases such as cryopyrin-associated periodic syndromes(CAPS),for example,Muckle-Wells syndrome,gout,and Alzheimer's disease.The anaphase-promoting complex/cyclosome(APC/C)is an E3 ubiquitin ligase that controls cell cycle progression by combining specific cell cycle-related proteins,such as Securin and Cyclin B.APC/C activity is strictly dependent on its coactivators Cdc20 and Cdh1,which are encoded by all known eukaryotic genomes.The APC/C^Cdc20activates the APC/C during early mitosis to promote the cell cycle from metaphase to anaphase,whereas Cdh1 activity is low because of its Cdk-dependent phosphorylation.APC/C^Cdh1regulates exit from mitosis.Anaphase promoting complex subunit 10(APC10)is the processivity factor of the APC/C,which promotes substrate recognition by the destruction box.Research has suggested that APC10 stimulates processivity by limiting substrate dissociation,which enhances ubiquitination stability.In this study,we firstly confirmed that APC10 interacted with NLRP3.We used NLRP3 PYD domain as the bait protein to screen the human normolized c DNA library by yeast-two hybrid assays.The results showed that the N-terminal of APC10 directly interacted with NLRP3 PYD domain.The co-IP results showed that APC10 interacted with NLRP3 LRR,NAD,and PYD domains.It was also found that the interaction between APC10 and NLRP3 was independent of APC/C complex.Moreover,confocal microscopy showed that APC10 and NLRP3 potentially co-localized to cytoplasm,and NLRP3 might influence the localization of APC10.In the HEK293T cells where the NLRP3 inflammasome model was reconstituted,it was found that overexpression of APC10 promoted NLRP3 inflammasome activation,whereas knockdown of APC10specifically attenuated NLRP3 inflammasome activation.Knockdown of APC10 also inhibited the activation of NLRP3 inflammasome in mouse J774A.1cells and TPA-differentiated THP-1 macrophages,and knockdown of APC10 attenuated the activation of NLRP3 inflammasome stimulated by several viruses,like EV7I,VSV,and Se V.Next,we found that APC10 interacted with NLRP3,ASC,and pro-Caspase-1.In HEK293T cells,overexpression of APC10 promoted the interaction between NLRP3 and ASC.In APC10 knockdown THP-1 cells,the induction of ASC oligomers was attenuated after Nigericin stimulation.Moreover,during interphase,APC10 interacts with NLRP3 to promote NLRP3 inflammasome activation,whereas during mitosis,APC10 disassociates from the NLRP3 inflammasome,thereby repressing inflammatory responses.In conclusion,we discovered a distinct mechanism controlling the NLRP3inflammasome which involves APC10 as a switch in both cell cycle and inflammatory responses.In interphase cells,APC10 interacts with NLRP3 and enhances its assembly to promote NLRP3 inflammasome activation.When the cell start to divide,APC10disassociates from the NLRP3 inflammasome to repress its activation for cell maintenance.This study provides a new thought about regulation of NLRP3inflammasome and cell cycle,also provides a new reference for subsequent prevention and treatment of those NLRP3 related diseases.