Molecular Mechanism Of Cullin1 And ASB17 Inhibiting NLRP3 Inflammasome

The innate immune system is a host that relies on pattern recognition receptors to detect cellular stress responses and foreign pathogens,where inflammasome is an important component of the innate immune system.NLRP3 inflammasome are multiprotein complexes that recognize pathogen-associated molecular patterns(PAMP)and danger signals associated molecular patterns(DAMP).The NLRP3 inflammasome is the most fully characterized inflammasome and consists of NLRP3,ASC and pro-Caspase-1.NLRP3 inflammasome regulates the maturation and secretion of the cytokines IL-1β and IL-18,which play important roles in inflammatory responses.The uncontrolled regulation of NRLP3 inflammasome is involved in diverse inflammatory diseases,such as gout,autoimmune diseases,atherosclerosis,type 2 diabetes,obesity,etc.,so the activation of NLRP3 inflammasome should be tightly controlled in the host.At present,there are many studies on the regulation of NLRP3 inflammasome activation.However,the negatively regulatory mechanisms of NLRP3 inflammasome are still unclear.In this paper,PYD,the first domain of NLRP3,as a bait protein was used to screen the prey proteins by yeast two-hybrid assay.It was first found that Cullin1 and ASB17 can interact with NLRP3,and Cullin1 and ASB17 can negatively regulate NLRP3 inflammasome.In this study,we confirmed that Cullin1 can interact with NLRP3.It was also found that the interaction between Cullin1 and NLRP3 was independent of Skp1 and ROC1.By overexpressing Cullin1 and interfering with Cullin1,it was found that Cullin1 specifically inhibited NLRP3 inflammasome in the HEK293 T cells where the NLRP3 inflammasome model was reconstituted.Cullin1 also inhibits the activation of NLRP3 inflammasome in TPA-differentiated THP-1 macrophages.Next,we found that Cullin1 promotes ubiquitination of NLRP3,but does not degrade NLRP3.In the mouse peritonitis model,Cullin1 was found to inhibit NLRP3 inflammasome activation and immune cell accumulation.At the same time,we also confirmed that ASB17 can interact with NLRP3.ASB17 can inhibit the activation of NLRP3 inflammasome in the HEK293 T where the NLRP3 inflammasome model was reconstituted,and ASB17 can degrade NLRP3.ASB17 significantly inhibited the activation of NLRP3 inflammasome in TPA-differentiated THP-1 macrophages where ASB17 was overexpressed.In four classical inflammasomes(NLRP3,NLRP1,NLRC4,and AIM2),ASB17 not only degrades NLRP3 but also degrades NLRC4.Furthermore,it was found that ASB17 caused the proteasome to degrade NLRP3 by ubiquitination.At the same time,it was found that the site of NLRP3 ubiquitination by ASB17 was K689 by constructing a series of ubiquitination site mutations of NLRP3.In conclusion,our results indicate that Cullin1 and ASB17 are able to inhibit the activation of NLRP3 inflammasome,which provides a deeper understanding of the molecular mechanisms underlying the negative regulation of NLRP3 inflammasome.