Natural and engineered inflammasome adapter proteins reveal optimum linker length for self-assembly.

The inflammasome is a multiprotein complex that triggers the activation of proinflammatory cytokines. The adapter ASC and its isoform ASCb mediate inflammasome assembly via self-association and oligomerization with other inflammasome proteins by homotypic interactions of their two identical Death Domains, PYD and CARD, connected by a linker of different length: 23 (ASC) and 4 (ASCb) amino acids long. However, ASC is a more potent inflammasome activator compared to ASCb. Thus, adapter isoforms might be involved in the regulation of the inflammatory response. As previously reported, ASC's faster and less polydisperse self-association compared to ASCb points to interdomain flexibility resulting from the linker length as a key factor in inflammasome regulation. To test the influence of linker length in self-association, we have 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). Real-time NMR and dynamic light scattering data indicate that ASC3X polymerization is less effective and more polydisperse compared to ASC or ASCb. However, transmission electron micrographs show that ASC3X can polymerize into filaments. Comparative interdomain dynamics of the three isoforms obtained from NMR relaxation data reveal that ASCb tumbles as a rod, whereas the PYD and CARD of ASC and ASC3X tumble independently with marginally higher interdomain flexibility in ASC3X. Altogether, our data suggest that ASC's linker length is optimized for self-association by allowing enough flexibility to favor intermolecular homotypic interactions but simultaneously keeping both domains sufficiently close for essential participation in filament formation.

Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS.

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRASG12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRASG12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRASG12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).