(157b) Synthetic YE8 Peptide Triggers Secondary Heterogeneous Nucleation of Insulin

Authors: 
Takor, G., University at Albany, State University of New York
Higashiya, S., University at Albany, State University of New York
Kurouski, D., University at Albany, State University of New York
Lednev, I. K., University at Albany, State University of New York
Welch, J. T., University at Albany, State University of New York
Gehan, T., Rensselaer Polytechnic Institute
Zhang, S., Rensselaer Polytechnic Institute
Belfort, G., Rensselaer Polytechnic Institute


Amyloid diseases are characterized by misfolding of normally functioning proteins, which then aggregate into ordered β-sheet rich oligomers, protofibrils and fibrils.  What triggers this process is still not completely understood.  Researchers have suggested that specific amino acid segments are responsible for protein fibrillation, while others propose that amyloidogenic oligomerization is a generic property of the polypeptide backbone.  To investigate this, GH6[(GA)3GY(GA)3GE]8GAH6 (YE8), a synthetic amyloid-like peptide, was used to seed solutions of native insulin, a model amyloid protein.  The aggregation was followed by both absorbance at 600 nm (A600) and Thioflavin-T (ThT) measurements.  YE8 addition increased the fibrillation magnitude (asymptotic values of A600 and ThT) and decreased the pre-fibril incubation period (lag-time) by 2- and 0.9-fold, respectively.  Fibril morphology was characterized using atomic force microscopy, deep UV resonance Raman (DUVRR) spectroscopy and fluorescent probes.  In particular, DUVRR spectroscopy showed that seeded fibrils have a chemical structural intermediate between the unseeded insulin and YE8 fibrils.  These results suggest the existence of a secondary heterogeneous nucleation pathway, in which already formed YE8 fibrils are able (i) to catalyze the formation of new insulin fibrils, and (ii) to influence its misfolding.