Project 3 Extensive research has provided strong support for the role of the multi-ligand receptor for advanced glycation endproducts (RAGE) in the pathogenesis of diabetes complications, such as accelerated cardiovascular (CVD) and peripheral arterial disease (PAD) and their common complication, ischemia-reperfusion (IR) injury. Due to its central role in various disease states, antagonism of RAGE signaling pathways is considered to be a promising therapeutic approach. Our Program Project team's research identified DIAPH1 as an intracellular effector of RAGE signaling and demonstrated a direct interaction between the intracellular domain (?tail?) of RAGE, or ctRAGE, and the FH1 (formin homology 1) domain of DIAPH1, in cell types that are relevant to RAGE- dependent pathologies using both in vitro and in vivo approaches. In macrophages, cardiomyocytes and endothelial cells, endoplasmic reticulum (ER) and mitochondrial stress, which are part of RAGE-dependent pathologies, have been linked to the interaction of DIAPH1 with Mitofusin2 (MFN2). Preliminary results from our labs strongly support the notion that small molecules can inhibit the RAGE-DIAPH1-MFN2 interaction. The critical question is how this inhibition affects physiologically important interactions of RAGE. The goal of this Project is to establish the biophysical and structural biology basis for RAGE-DIAPH1 signal transduction and identify modes of inhibition. We hypothesize that the in vitro mode of RAGE-DIAPH1 inhibition, as revealed by a combination of biophysical and structural biology tools, such as NMR spectroscopy, will provide a base for understanding cellular mechanisms of RAGE signaling inhibition and thus can be effectively used to optimize small molecule inhibitors of RAGE-DIAPH1-MFN2 interactions. NMR spectroscopy will be used as a main structural tool since ctRAGE and the FH1 domain of DIAPH1, as well as large segments of MFN2, are flexible and may not be amenable to crystallization or electron cryomicroscopy. We will probe the structural biology of novel small molecule antagonists of RAGE-DIAPH1 and use in-cell fluorescence techniques to meticulously characterize the RAGE-DIAPH1-MFN2 interaction. Our Project will work closely with Projects 1 and 2 to explore the mechanistic basis of this interaction and to identify novel and potent therapeutic strategies and agents for diabetes, CVD and PAD.