| Protein misfolding is a common motif in a number of human diseases, including Alzheimer disease, Parkinson disease and type II diabetes mellitus (TTDM). In TTDM, over 90% of patients are found to have pancreatic amyloid deposits upon autopsy. These deposits are primarily composed of a 37-residue human islet amyloid polypeptide (hIAPP). Evidence suggests an association between these amyloid plaques and pancreatic beta-cell dysfunction. Elucidating the structure of these deposits and the effects modifiers have on the misfolding pathway can help further the understanding of its toxicity and could be of use in the design of drug inhibitors for amyloid diseases. I used site-directed spin-labeling and electron paramagnetic resonance (EPR) spectroscopy to analyze spin-labeled derivatives of hIAPP to determine structural features of the peptide in its fibrillar form. Using continuous-wave EPR and four-pulse DEER coupled with computational modeling, I determined a detailed structural model of hIAPP fibrils. The N-terminal and C-terminal regions are less ordered and more mobile and there is a turn region between residues 19-30 located between two beta-strands (13-18 and 31-36). My findings also show that the beta1 and beta2 strands from one molecule of hIAPP are staggered in relation to one another. The staggered peptide stacks directly on another staggered peptide, forming a protofilament with a twist that I aptly call a beta-spiral motif. In addition to these structural findings, I have studied the effects of modifiers, such as curcumin and annexin proteins, to the misfolding of amyloid fibrils. Using EPR, ThT and EM, I found that these molecules or proteins seem to alter hIAPP, Abeta and alpha-synuclein fibril formation and fibril morphology. Additionally, studies of these modifiers in tissue culture and animal models showed reduced toxicity and protein aggregation. Both the identification of hIAPP structure and the role of amyloid modifiers can give way to therapeutic intervention of amyloid diseases. |
