Study of the polymerization dynamics of ASC and its domains

Abstract

Apoptosis-associated speck-like protein containing a CARD (ASC) is a 22 kDa protein composed of two conserved death-fold domains, PYD and CARD, that act as an adaptor protein in in ammasomes, multimolecular complexes that promote the maturation of pro-in ammatory cytokines IL-1 and IL-18. Death-fold domains are known to mediate the formation of higher order complexes through three distinct type of interactions. The importance of these interaction types are distinguished through ASC's ability to form a high molecular globular complex called the ASC speck. It has been shown that PYD-PYD and CARD-CARD homotypic interactions between ASC molecules is necessary to form this structure. Our group has reported the signi cance of homotypic PYD interactions, as well as of the residues on interaction surfaces. In this study, we have revisited the dynamics of homotypic PYD interactions by observing the formation of lamentous structures between mutant and wildtype variants. It was observed that PYD mutant variants previously known to disrupt homooligomerization such as E13A, K21A, K26A and D51A, were still able to contribute to multimeric structures along with their wildtype counterparts. Then, we have focused on the importance of homotypic CARD interactions, and investigated the signi cance of certain residues in CARD homooligomerization through Forster Resonance Energy Transfer (FRET) analysis. We have observed that the stability of interaction surfaces is more important in CARD, and mutant variants D134A, M159A and R160A showed a complete lack of lament formation. Lastly, we have selected wildtype CARD and CARD E130A mutant variant and done Single Molecule Force Spectroscopy (SMFS) to measure the physical strength of homotypic CARD interactions, and how disruption of interaction surfaces a ect said strength.