The Mechanism Of NLRP1 Inflammasome Activation

The NLRP1,one of the most important NOD-like receptor members,can form NLRP1 inflammasome with ASC and pro-caspase-1 to induce the secretion of mature proinflammatory cytokines interleukin 1β(IL-1β)and interleukin 18(IL-18),thus removing the PAMPs and DAMPs to maintain homeostasis.PYD and CARD domains ofNLRP1 belong to the death domain superfamily(DDS)members,and DDS members have three conserved asymmetric homotype interactions,namely Type Ⅰ interaction,Type Ⅱ interaction and Type Ⅲ interaction,playing a critical role in signal transduction.And current studies have found that the overactivation of NLRP1 inflammasome can induce a variety of autoimmune diseases,including multiple self-healing palmoplantar carcinoma(MSPC)and familial keratosis lichenoides chronica(FKLC).Therefore,it is very important to study the signal transduction amplification mechanism and regulation mechanism of NLRP1 inflammasome in deep and detail,in order to provide novel therapy strategy for these diseases caused by the overactivation of NLRP1 inflammasome.Our study focuses on exploration the signal transduction amplification mechanism and regulation mechanism of NLRP1 inflammasome,which contains two following major parts:Ⅰ.Homotypic CARD-CARD interaction is critical for the activation of NLRP1 inflammasomeFirstly,we used yeast two hybrid and mammalian two hybrid,combined with the results of size-exclusion chromatograph of purified NLRP1CARD and ASCCARD protein in vitro to verify that NLRP1CARD directly interacts with ASCCARD.Furthermore,we determined the crystal structure of ASCCARD and screened the key sites of the interaction between NLRP1CARD and ASCCARD by structure-directed mutagenesis.And we further discovered that the three asymmeytic and conservative interaction types of the DD superfamily(Type Ⅰ,Type Ⅱ and Type Ⅲ)play critical roles in the interaction between NLRP1CARD and ASCCARD.Based on the NLRP1 inflammasome activation system in the HEK 293T cells,we demonstrated that each of three asymmetric interaction interfaces is critical for the signal transduction and activation of NLRP1 inflammasome.In conclusion,this study not only revealed that NLRP1CARD and ASCCARD have a direct interaction,but also elucidated the interaction between NLRP1CARD and ASCCARD through three asymmetric and conserved interaction types of death domain superfamily,which is named as Mosaic model,is critical for the signal transduction and activation of NLRP1 inflammasome.Ⅱ.CARD8 negatively regulates NLRP1 Inflammasome activation level by interaction with NLRP1We found that CARD8-T48 isoform can bind to NLRP1 in HEK 293T cells.In the reconstituted NLRP1 inflammasome system,the results suggest that the T60 isoform of CARD8,but not the canonical T48 isoform,negatively regulates NLRP1 inflammasome activation level.Pull Down analysis have showed that CARD8-T60 isoform directly binds NLRP1 in vitro,and CARD8-T60 isoform interacts with NLRP1 through ZU5 subdomain,while NLRP1 interacts with CARD8 through the NACHT,LRR and FIIND domains.During the investigation into the effect of CARD8 on NLRP1 inflammasome,the data show that the assembly of human CARD8 inflammasome do not rely on the participation of ASC to induce the secretion of mature IL-1β and IL-18,while the NLRP1 inflammasome must require the presence of ASC.Based on protein structural biology and different molecular chimeras,the dependence of ASC is mainly caused by the CARD domain,but not the UPA domain.Furthermore,we screened the important amino acid sites of NLRP1CARD with sites-directed mutagenesis and structural biology,and have illuminated that the activation of NLRP 1 inflammasome rely on multiple interaction surfaces instead of a single interaction surface.Finally,we also proved that disease-causing mutations in NLRP1 can obviously trugger the oligomerization of NLRP 1 and weaken the interaction between NLRP1 and CARD8,lending to overactivation of NLRP 1 inflammasome and causing autoimmune diseases.Our results have partly explained the mechanism of NLRP 1 inflammasome overactivation-causing diseases.In summary,we not only identify that CARD8 negatively regulats NLRP 1 inflammasome activation level by interaction with NLRP 1,but also found the structural basis for the specifically relying on ASC to transducte signal in NLRP1 inflammasome.