This work examines the evolution of two proteins that are widely distributed in metazoan animals and underlie Alzheimer's disease (AD) in humans - the beta-secretase enzyme (BACE1) and the amyloid beta (A-beta) sequence. The main goal is to understand when and how protein molecules evolve new functions over time. Broader outcomes of this work include expanded access to STEM research for underrepresented minorities and first generation undergraduate students, and outreach to the broader community via a high school teaching module and a popular science article. <br/><br/>Gene duplications are believed to be major facilitators of the evolution of novel protein function. BACE1 evolved from a gene duplication event just after the origin of animals. At a later but as yet unknown point in time, an APP gene duplicate evolved a novel BACE1 substrate motif (A-beta). This project traces back the functional evolutionary history of the origin and interaction of these molecules and resolves, to the most precise degree possible, when BACE1 and APP/A-beta evolved. Two specific aims make up this research: (1) determining the functional origin of BACE1 activity, via a cell culture-based functional test and ELISA, to investigate which species retain BACE1 enzymatic activity, and (2) determining the functional origin of the APP beta cleavage site to determine which taxa retain bona fide BACE1 substrates, using a cell culture-based functional test and mass spectrometry.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.