Protein Misfolding and Aggregation

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In a study to determine how membrane remodeling by α-syn affects the fibril formation process, we discovered that the protein is fully capable of transforming vesicles with no net surface charge (phosphatidylcholine, PC) into tubules while remaining unstructured as detected by circular dichroism spectroscopy. This result contradicts recent data that suggest membrane remodeling by α-syn requires both the presence of anionic phospholipids and helical structure. Notably, membrane remodeling inhibits α-syn amyloid formation, retarding both lag and growth phases. Using five single-tryptophan variants and time-resolved fluorescence anisotropy measurements, we determined that α-syn influences bilayer structure with surprisingly weak interaction (dissociation constant mM) and no site specificity. As cellular membranes are enriched in PC lipids, these results support possible biological consequences for α-syn induced membrane remodeling related to both function and pathogenesis. In addition, this work suggests that membrane bending by α-syn may be more complicated than the currently proposed amphipathic helix insertion model. Ongoing work aims to study the dynamics of membrane deformation to evaluate whether other mechanisms are at play. In efforts to discover new proteins that form amyloid and motivated by the fact that atherosclerotic plaques contain significant amounts of amyloid with poorly understood composition, we have begun investigations on apolipoproteins (Apo) and their propensity to form amyloid. We explored the possibility that ApoCIII is amyloidogenic in vitro, as has been shown for other apolipoproteins including ApoAI and ApoCII. We have obtained evidence that recombinant ApoCIII forms amyloid, albeit a rather unusual looped structure, which is stable at physiologically relevant 5-20 &#956M concentrations and blood pH. Numerous features of amyloids have been characterized: ThT and CR binding, β-sheet structure determined by CD and electron diffraction data, and protease resistance. TEM and AFM demonstrate that ApoCIII forms ribbon-like amyloid loops and we postulate that they are related to those reported for two other apolipoproteins, ApoCII and ApoAI. Apart from raising the possibility of a biological role for ApoCIII amyloid, we have identified novel loops in the shapes of triangles and squares. Future work is needed to determine how these structures play a role in the mechanism of amyloid assembly and to address whether these conformations are also related to potentially toxic oligomers (intermediates) characterized for other amyloid forming proteins.