Synthesis and Analysis of Analogs of hIAPP (1-19), a Peptide Involved in Membrane Disruption

摘要

Human islet amyloid polypeptide (hIAPP) is co-secreted with insulin in the β-cells of the pancreas. Capable of forming amyloid fibers and causing membrane disruption, this 37 amino acid peptide is cytotoxic to β-cells in type II diabetic patients [1,2]. The N-terminus of hIAPP (residues 1-19, KCNTATCATQRLANFLVHS) has been shown to bind to membranes and induce damage despite lacking the amyloidogenic (20-29) region [3]. These results indicate that the N-terminal (1-19) region of hIAPP can be used as a model in drug target studies, in place of the expensive and synthetically challenging full length hIAPP. It has been proposed that aggregation of hIAPP is mediated by pi-stacking interactions between phenylalanine residues [4]. In this study, two analogs of hIAPP 1-19 were synthesized by standard solid phase methods in which Phe~(15) was replaced with Ala (to reduce aromaticity) and 1-naphthylalanine (1-Nal, to increase aromaticity), respectively. These analogs were then purified by reverse phase high performance liquid chromatography and tested in a model system that mimics the pancreatic Pcell membrane, namely vesicles comprised of a 7:3 ratio of the lipids l,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) and l,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS). The percent leakage of fluorescent carboxyfluorescein dye from the vesicles in the presence of the peptides was calculated as compared to a 100% Triton-X detergent-treated control. Assays were run in triplicate in a 96-well plate, and average values are reported. Fluorescence values were recorded by an FLx fluorescence microplate reader.