PROJECT SUMMARY/ABSTRACT. Inflammation is our primary response from the innate immune system to
fight infection and self-protect from damage. However, dysfunctional regulation of inflammation results in
disease, including certain types of cancer, autoimmune, cardiovascular and neurological disorders, rheumatoid
arthritis, and even depression. The onset of inflammation depends on the assembly of a multiprotein complex
known as the inflammasome. The main players in inflammasome assembly are; - sensor proteins that react upon
danger signals derived from pathogens or damaged tissue; - procaspase-1 that activates inflammatory cytokines
as a result of inflammasome assembly; - the adapter ASC that functions like a molecular glue by connecting
sensor and procaspase-1 molecules. ASC exists in two inflammasome activating isoforms; canonical ASC and
ASC_short (both subject of study of the parent R15), and has two more isoforms, one that inhibits inflammasome
formation (ASCc) and one isoform with unknown function (ASCd). The presence of protein isoforms is a well-
known, natural mechanism for the regulation of protein function. However, little is known on the factors controlling
the formation of the inflammasome at the level of the adapter and its isoforms, or how these isoforms impact
inflammation. In the parent R15 grant we proposed to study the regulation of the inflammasome via its two
activating isoforms (canonical ASC and ASC_short). Both bimodular proteins have two Death Domains
connected by a linker, and their amino acid sequences differ solely in the linker length. Our recent findings
resulting from the parent R15 indicate that the two isoforms have significantly different kinetics of oligomerization,
which in turn correlate with their different dynamic behavior. Our results provide the first explanation at the
molecular level of why these isoforms activate the inflammasome to different extent based on their respective
oligomerizing capabilities. This Diversity Supplement proposal aims at addressing a knowledge gap in the role
of the other two ASC isoforms (ASCc and ASCd) on inflammasome formation and thus in the inflammatory
response. The objective of this proposal is innovative because it will decipher the unknown molecular bases for
inflammasome regulation mediated by ASCc and ASCd. Our hypotheses are that; 1) ASCc, with an intact Death
Domain, inhibits inflammasome formation by competing with ASC self-association, which can account for the
observed decreased in the inflammatory response, 2) Based on our preliminary studies, ASCd is an intrinsically
disordered protein (IDP), thus we propose that ASCd can fold upon binding to ASC, a typical mechanism of
IDPs, resulting in a more moderate inhibition. To test these hypotheses we propose to; 1) study at the atomic
level the structural characteristic of ASCc and ASCd with NMR, and 2) quantitatively determine their potential
interactions with ASC (identifying the interacting regions and measuring the affinity of the complexes) and 3)
discern with Transmission Electron Microscopy the potentially different characteristics of the macrostructures
formed by canonical ASC in the presence of ASCc and ASCd.
