Project 1 - Molecular structure and function

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PROJECT 1 SUMMARY Molecular structure and function. The ectopic accumulation of mineralized calcium-phosphate deposits in the eye and brain is a hallmark of Age-Related Macular Degeneration (AMD) and Alzheimer’s Disease (AD). In addition to inorganic components, such as hydroxyapatite (HAP), these deposits also contain lipids and proteins of both blood and intracellular origin. The process of calcification is poorly understood, and it remains unclear why some deposits progress to the disease state while others do not. It is also not clear if the associated proteins are involved in the mineralization process, or if they deposit and accumulate on the already pre-formed hydroxyapatite surface. Here, we address these important questions by comprehensive and systematic investigation of the chemical, physical and structural properties of the molecular components associated with calcified deposits, with particular focus on three validated constituents: the proteins vitronectin (Vn), amyloid-b (Ab) and Tau. Vn is a blood glycoprotein with diverse functions in hemostasis, cell adhesion and migration, innate immunity, tissue and bone remodeling. Recent studies have indicated that Vn binds both soluble Ca2+ and solid HAP with chemical specificity and the T400M Vn variant is a major risk factor for AMD. Ab and Tau are well known components of amyloid plaques in AD, but they are also found in calcification deposits in the eye and the brain. First, we will investigate the role of these proteins in the calcification process through fluorescence-based experiments and binding studies. Then, we will reconstitute proto-aggregates of HAP, lipids and proteins to model pathological deposit formation in vitro. These aggregates will provide a platform for assaying the functional roles of proteins and lipids in calcification and will aid the discovery of small molecule and biomolecular inhibitors of calcification. And finally, we will use solid-state nuclear magnetic resonance (NMR) spectroscopy to determine the structures of Vn, Tau and Ab within calcified deposits and to describe the protein-mineral interactions that drive their assembly. This information will be essential for understanding the molecular mechanism of calcification and, ultimately, for the development of new diagnostic and therapeutic agents.