Islet amyloid polypeptide or amylin is a recently discovered 37 amino acid residue signaling protein (hormone) that is produced and co-secreted along with insulin by pancreatic beta-cells. In late onset of diabetes, amylin readily aggregates forming protein deposits or plaques that are toxic to beta-cells, resulting in beta-cell apoptosis. Given the well-known role of cholesterol and lipids in etiology of diabetes, I explored whether these two essential PM components regulate amylin assembly and aggregation on artificial (synthetic) membranes. Using high resolution imaging and spectroscopic approaches, I demonstrated that amylin undergoes facilitated aggregation and conformational changes in the presence of membranes composed of anionic lipids such as phosphatidylserine (PS). The presence of cholesterol on the other hand inhibited lipid-induced aggregation of amylin in solution and on model planar membranes. However, the patho-physiological consequence of cholesterol-regulated amylin polymerization on membranes, and biochemical mechanisms that protect beta-cells from amylin toxicity are poorly understood. Hence, in my subsequent study, I reported that PM cholesterol plays a key role in molecular recognition, sorting and internalization of toxic amylin oligomers but not monomers in pancreatic rat insulinoma and human islet cells. Depletion of PM cholesterol or the disruption of the cytoskeleton network inhibited internalization of amylin oligomers, which in turn enhanced extracellular oligomer accumulation and potentiated amylin toxicity. In contrast to oligomers, amylin monomers followed clathrin-dependent endocytosis, which was not sensitive to cholesterol depletion. Our studies identified an actin-mediated and cholesterol-dependent mechanism for selective uptake and clearance of amylin oligomers, impairment of which greatly potentiated amylin toxicity.
However, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Hence, in my further study, I observed that pancreatic cells employed different strategies to eliminate amylin's toxic and non-toxic molecular forms. My study also revealed that macropinocytosis serves a major cyto-protective role in these cells, by clearing of amylin molecular forms. The overreaching goal was to fully elucidate the internalization and trafficking pathways of human amylin monomers and toxic oligomers in pancreatic cells.
|Commitee:||Donaldson, Robert P., Eleftherianos, Ioannis, O'Halloran, Damien, Reeves, Mark|
|School:||The George Washington University|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 74/12(E), Dissertation Abstracts International|
|Subjects:||Biology, Cellular biology, Biophysics|
|Keywords:||Binding, Human amylin, Islets, Pancreatic cells, Toxicity, Turnover|
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