Investigation of homotypic pyd and card domain interactions in ASC speck assembly

Apoptosis-associated speck-like protein containing a CARD (ASC) is a 22 kDa protein containing conserved PYD and CARD domains that belong to death-fold superfamily. Homotypic interactions between the domains occur via providing at least three certain surfaces of contact (Type I, II and III). ASC has an adaptor role between receptor and effector proteins in the inflammation process having the ability to form a supramolecular globular complex called ASC speck through PYD and CARD homotypic interactions. When expressed in truncated form as PYD and CARD separately, these domains form filamentous structures. Apparently, these fibers compact onto each other during the wild type ASC polymerization. In this study, our aim was to elucidate the importance of specific locations on PYD-PYD and CARD-CARD interaction surfaces during the polymerization process. Effects of 19 single and 22 double mutations were observed introducing them on important residues such as E13A, D48A, Y60A, E130A, Y146A, R150A and M159A to hit one and/or two interaction surfaces at the same time. Effects of the mutations were visualized using fluorescence and confocal microscopy to qualify the change in the level of organization of the phenotype from filamentous to soluble. Next, we analyzed rate of homooligomerizations in mutant sets using FRET technique to quantify the interaction efficiencies in presence of disruptive mutations. Our results showed that PYD mutants, which've been known to disrupt homooligomerization, are able to provide multimeric filamentous structures when expressed together with their wild type counterparts although CARD mutants have less tolerance to mutations in the interaction surfaces. We identified certain mutations that increase, decrease or block FRET signal. Our study provides better explanation and new insights about the oligomerization dynamics of the inflammasome complex.