PROJECT SUMMARY Excessive intravascular hemolysis of red blood cells (RBCs) leads to numerous complications associated with uncontrolled inflammation including pain, vaso-occlusion, and organ damage that underlie the pathophysiology of several disease states including sepsis, intravascular hemorrhage, and sickle cell disease (SCD). The primary mediator of this inflammation is heme released from damaged RBCs. The applicant’s goup recently reported that heme activates the inflammatory caspases, caspase-1, caspase-4, and caspase-5 to coordinate the cleavage and release of the apical pro-inflammatory cytokine IL-1β. Importantly, unlike caspase-1, caspase-4 and caspase-5 are activated independently of inflammasome assembly. Thus, heme is one of the first examples of a DAMP (damage associated molecular pattern) that activates the non-canonical inflammasome pathway. The applicant has shown that heme binds caspase-4 and caspase-5 and induces oligomerization of these caspases. The central hypothesis is that heme directly binds to caspase-4 and caspase-5 to induce oligomerization and activation. The hypothesis will be tested by the following Aims: 1) to determine the biophysical composition of heme-induced inflammatory caspase complexes and 2) to identify the protein components required for heme-induced inflammatory caspase activation. Under the first Aim full length recombinant caspase proteins will be produced and the ability of heme to bind caspases and to induce caspase oligomerization and cleavage will be assessed. Under the second aim the amino acid residues required for heme binding, oligomerization and cleavage will be identified as well as the type of cytosolic factors, if any, contribute heme-induced caspase activation. This work will provide an in vitro assay that will be the basis for future rational drug design approaches and a source of purified caspases that can be used for future studies to determine the structures of the heme-induced caspase complexes. Understanding the mechanisms of inflammatory caspase activation with provide justification for targeting these proteins to improve clinical outcomes in hemolytic conditions.

PROJECT NARRATIVE Correct regulation of inflammatory caspases is critical for preventing uncontrolled inflammation in response to hemolysis. The proposed research is relevant to public health because it will provide important mechanistic insights into how inflammatory caspases are regulated by extracellular heme, a key pathophysiological feature of several disease states. Thus, the proposed research is relevant to part of the mission of the National Institute of Allergy and Infectious Diseases (NIAID) that pertains to understanding, treating, and preventing infectious and immunologic diseases.