Amyloid beta-peptide Binding Alcohol Dehydrogenase (ABAD) is a novel intracellular enzyme localized in endoplasmic reticulum (ER) and mitochondria with properties of a beta-hydroxyacyl-Co-enzyme A dehydrogenase and a generalized alcohol dehydrogenase. ABAD interacts with amyloid beta-peptide (Abeta), a product of cellular processing of the beta-amyloid precursor protein (APP). Recombinant human ABAD binds Abeta (1-40-42) specifically, via the N-terminus (residues 1-20). ABAD functions as a cofactor in Abeta-induced cell stress; co-transfection of cells with constructs driving over-expression of APP(V717G) and wild- type (wt) ABAD caused generation of reactive aldehydes and induction of DNA fragmentation in COS and neuroblastoma cells. In contrast, similar co-transfection studies with constructs encoding APP(V717G) and mutationally-inactivated ABAD(168/K17G) did not result in cytotoxicity. Furthermore, levels of ABAD are increased in neurons in AD brain, especially those near deposits of Abeta. We propose that enzymatically active ABAD is a critical intracellular target potentiating Abeta-mediated cellular perturbation at the earliest stages of AD; in the endoplasmic reticulum, ABAD interacts with newly formed Abeta(1-42), and the resulting ABAD-Abeta complex generates cellular oxidant resulting in reactive aldehyde formation and induction of DNA fragmentation. The first specific aim is to analyze ABAD-Abeta interaction by structural techniques, exploiting our observation that Abeta(1-20), the latter not prone to aggregation and suitable for structural studies, bind ABAD similarly to full-length Abeta. Co-crystallization of ABAD and Abeta(1- 20) has already produced diffractable crystals. Our second aim is to determine mechanisms underlying ABAD-Abeta interaction in a cellular context based on insights from the structural studies. We hypothesize that two salient features of ABAD facilitate of ABAD facilitate potentiation of Abeta-mediated cell stress: localization of ABAD to the ER, where Abeta(1-42) is formed, at least in part; and determinants in ABAD mediating the binding to Abeta. Project 4 will work closely with Projects 1-3, and will, and will obtain technical assistance from Core B. Collaborative interactions we will include: sharing of reagents for analysis of ABAD (Project 3), mechanisms for evaluating for evaluating cell stress (Projects 1-3), and assistance in characterization of mutant ABAD molecules (Core B).