Self-assembly and interdomain dynamics of natural and designed inflammasome adaptor isoforms of ASC: Implications for inflammasome regulation.
Inflammation is governed by the inflammasome: a large multiprotein complex necessary to trigger an inflammatory response. The inflammasome adaptor ASC is responsible for mediating inflammasome formation. ASC exists as 4 isoforms: ASC, ASCb, ASCc, and ASCd. Of the four isoforms ASC and ASCb can elicit an inflammatory response, however ASCn produces a lower inflammatory response compared to ASC. Structurally, ASC and ASCb are similar containing a PYD and CARD domain necessary for homotypic interactions. ASC contains a 23 amino acid linker separating the two domains while ASCb contains a significantly shorter linker, 4 amino acids. Self-association capabilities of ASC and ASCb as a result of linker length can impact inflammasome formation. To further study the effects of linker length, an engineered the isoform ASC3X with identical PYD and CARD connected by a 69-amino acid long linker (i.e., three-times longer than ASC’s linker) was used to test the influence of linker length on the self-association capabilities of ASC3X. To understand the self-association capabilities of each isoform: real-time NMR (RT-NMR) was used to determine differences in their self-association ability. Dynamic light scattering (DLS) and size-exclusion chromatography were performed to monitor their oligomer size distribution. Microstructures formed by each isoform were images using transmission electron microscopy (TEM). DLS data indicate ASC can form uniform structures in solution compared to ASCb and ASC3X. Interdomain dynamics of each isoforms reveal the effect of linker length on domain flexibility. The shorter linker of ASCb restricts the PYD and CARD domain causing ASCb to tumbles as a rod. Increasing the length of linker allowed the PYD and CARD of ASC and ASC3X to tumble semi-independently of each other. Altogether, our data suggest that ASC’s linker length is optimized by for participation in inflammasome self-assembly. In addition, our results help explain at the molecular level the differences in inflammatory response by ASC isoforms.