Protein misfolding and fibrillar aggregation play a critical role in the pathology of some of the most serious diseases facing society today. Diseases long known to have such a relationship include Alzheimer's and Parkinson's. More recently, diabetes, cancer and HIV/AIDS have also been shown to have proteins for which self association of misfolded states into ?-sheet rich fibers is associated with disease progression. It is also long known, however, that disease severity does not correlate with the amount of aggregated protein deposited as plaques (termed amyloid). Numerous in vitro and in vivo studies in recent years have drawn attention instead to the role of non-fibrillar, pre-amyloid species as the relevant agents of pathology. Specifically, cellular dysfunction appears to be initiated by interactions between specific oligomeric species and cellular membranes, resulting in disruption of cellular homeostasis. These membrane-associated oligomers need not have amyloid- like structure and indeed in several systems are characterized by ?-helical structure. The challenge we propose to address here stems from observations that toxic gains-of-function emanate from subsets of a dynamic and heterogeneous oligomeric ensemble. By investigating the molecular mechanisms by which these ensembles form, we will determine the molecular basis of toxicity and illuminate a pathway towards relevant therapeutic targets.
The aims of this proposal are designed to elucidate the mechanism by which amyloid-associated oligomers form, undergo conformational change, and disrupt and translocate across lipid bilayers. We focus on two, ~40 residue peptides, A? and IAPP, implicated in Alzheimer's and Type II diabetes respectively. These peptides share many biophysical and biochemical properties. Key to this proposal is that they both undergo disorder to ?-helical transitions upon binding to membranes and that each forms heterogeneous arrays of membrane- associated oligomers that are capable of disrupting lipid bilayers in vitro and cause cell death when added to cultured cell lines. For both systems, targeted nonspecific disruption of their ?-helical states has been demonstrated to rescue cellular toxicity. By studying these peptides in tandem, we will elucidate a common molecular basis for cellular gains of toxic function, as well as pinpoint the basis for disease-specific differences. We propose to dissect mechanism through the use of complimentary ensemble and single molecule assays designed to elucidate structures, kinetics and energies associated with membrane bound conformational conversions. Molecular level insights will be achieved by using coarse grain simulations informed by experimentally determined constraints. Finally, we will ensure biological relevance in our findings by conducting parallel efforts on synthetic bilayers, purified sub-cellular organelles and whole cells.
Type II diabetes and Alzheimer's disease each have a small, disease-specific protein which shows almost no structure in solution, but can bind cell membranes. Once bound, a common structure emerges that is associated with pathology as they cause the death of insulin secreting cells and neurons respectively. The aim of this proposal is to elucidate the membrane bound molecular changes that these two systems have in common, as well as to determine what makes them distinct, in order to identify which structures are actually responsible for cell death.
|Ducas, Vanessa C; Rhoades, Elizabeth (2014) Investigation of intramolecular dynamics and conformations of ?-, ?- and ?-synuclein. PLoS One 9:e86983|
|Braun, Anthony R; Lacy, Michael M; Ducas, Vanessa C et al. (2014) ?-Synuclein-induced membrane remodeling is driven by binding affinity, partition depth, and interleaflet order asymmetry. J Am Chem Soc 136:9962-72|