Structural aspects of ASC speck formation and its potential novel functions

Inflammasomes are cytoplasmic sensors that detect presence of intracellular patho-gens or cellular damage. Activation of NLRP3, NLRC4 and AIM2 inflammasomes is ac-companied by formation of a micrometer sized perinuclear structure called the ASC speck which is thought to be the platform for inflammasome activity. We carried out a mutational screen on the EGFP-ASC fusion protein in to determine ASC speck disrupting mutations. The ASC protein has two domains, namely pyrin domain (PYD) and caspase recruitment domain (CARD) which form filament structures when overexpressed in HEK293T cells as seperate domains. A subset of mutations we introduced in the full length ASC protein changed the globular ASC speck structure into filaments, which were similar to the fila-ments formed by the individual domains of ASC. We repeated a parallel mutational screen on PYD or CARD as seperate domains and found that mutations disrupting the ASC speck in the full length ASC construct also disrupt the filaments in the domain only constructs. The filaments formed by PYD was proposed to be formed by type I inter-actions. By using the type I binding mode deficient D48A mutant PYD, we showed that D48A-PYD is able to interact with wt PYD filaments, which indicates that PYD has alternative binding modes. Our results suggest that the ASC speck is an organized structure, as single mutations introduced in ASC changes the ultimate structure completely. The second part of the thesis is based on the observation that the ASC speck is able to co-aggregate a wide range of cytosolic proteins on itself. We postulate that antigen co-aggregation of the ASC speck might be important in antigen presentation. The ASC protein has already been implicated in the antigen presentation pathway via inflammasome dependent and independent mechanisms. Co-aggregation of cytosolic proteins on the ASC speck has potential implications in antigen presentation pathway which could be exploited to build better vaccines in the future.