Identification and Characterization of an Insulin Based Inhibitor of Amylin and Aβ40 Amyloid Formation
The Graduate School, Stony Brook University: Stony Brook, NY.
The formation of amyloid fibrils plays a role in a wide range of diseases collectively known as amyloidosis. Amyloid fibrils are formed when peptides or proteins misfold and deposit in tissues and organs. These deposits are a characteristic feature of a number of diseases including Type II diabetes, Alzheimer's disease, and Parkinson's disease. Our lab studies the amyloidogenic peptides Islet Amyloid Polypeptide (IAPP) from type II diabetes and the Aβ40 and Aβ42 peptides from Alzheimer's disease. There is intriguing, albeit indirect, evidence that suggests that inhibitors of IAPP amyloid formation may also inhibit amyloid formation by Aβ. If true, this suggests a new strategy for designing inhibitors; specifically inhibitors of amyloid formation by one peptide can be used against the other.Insulin is known to be an inhibitor of IAPP. It is made up of an A and B chain and the B chain displays potent inhibitory effects. In addition, a short segment located in the B chain has been showed to bind IAPP and thus might inhibit amyloid formation.Based on these findings, experiments were conducted to test the possible inhibitory effects of insulin, the insulin B chain, and a short fragment from the insulin B chain on Aβ40 and IAPP.Solid phase peptide synthesis was used to synthesize thee Aβ40 peptide and a 12-residue fragment of the insulin B chain (IBSF). A new protocol was developed and employed to obtain the kinetic curve of Aβ40 amyloid formation using Thioflavin-Tbinding fluorescence assays. Aβ40 was incubated with insulin, the insulin B chain, and IBSF. Transmission electron microscopy was used to observe what had occurred during the incubation and confirm the results of the fluorescence assays. Similar experiments with IAPP were done as well, however, the rate of fibrillization was found to be much slower. This suggested that there is indeed an interaction between these two peptides. Thus, concentration dependent studies and further experimentation were done to better observe and assess these interactions.