Molecular mechanisms of transthyretin amyloidosis

Many debilitating human diseases are associated with the extracellular misfolding and aggregation of globular proteins to form fibrillar deposits that are rich in -structure. There is considerable evidence that the process is initiated by local unfolding of the native structure to form aggregation- prone amyloidogenic intermediates. Aggregation then proceeds via nucleation-growth or downhill polymerization mechanisms. Transthyretin amyloidosis is associated with numerous neurodegenerative diseases and cardiomyopathies. Misfolding and aggregation of transthyretin leads to fibrous deposits in the peripheral nerves and heart. Deposition of wild type protein is age related, whereas the familial diseases associated with genetic mutations that destabilize the quaternary and/or tertiary structure are early onset. Aggregation is extremely slow at physiological pH and temperature and all in vitro studies of the aggregation pathway to date have used non- physiological conditions (acidic pH) to accelerate the process. Recent clinical studies suggest a causal relationship between arterial and aortic valve stenosis, which results in turbulent blood flow, and transthyretin cardiac amyloidosis. The proposed research will provide novel insights into the fundamental molecular mechanisms by which shear forces associated with fluid flow promote aggregation of wild type transthyretin and pathogenic variants under physiological conditions. A comprehensive suite of biophysical tools (light scattering, fluorescence, NMR, and optical and electron microscopy) will be used to elucidate the mechanistic pathway by which transthyretin aggregates under laminar and turbulent shear stress. NMR will be used to map the kinetic aggregation landscape of wild type and pathogenic variant transthyretin, characterize and quantify the population of intermediates that accumulate on the aggregation pathway, and elucidate the mechanism by which shear forces promote proteolysis and fibril formation. Morphological changes during aggregation and fibril growth will be monitored by optical and electron microscopy. Real-time NMR experiments using a novel rheological NMR device will be used to probe shear-induced changes in the conformation and dynamics of wild type and pathogenic variant TTR that initiate misfolding and aggregation. This research will advance our understanding of the fundamental molecular events that initiate transthyretin aggregation at physiological pH and temperature and promote entry into and progression down the aggregation cascade that leads to amyloid formation and human disease.