284304 The Use of Insulin Fragments to Elucidate the Kinetic Differences Between Fibrillation of Insulin From Different Species

Monday, October 29, 2012
Hall B (Convention Center )
Daniel Forciniti, Cbe, Missouri University of Science and Technology, Rolla, MO and Paulina Morales, Chemical Engineering, Missouri University of Science and Technology, Rolla, MO

Amyloid fibrils  are ordered aggregates of peptides or proteins that are associated with  many diseases (e.g. type 2 diabetes mellitus, Alzheimer’s disease, Parkinson’s disease, etc).  Amyloid diseases are apparently unconnected; however, intermolecular secondary structure (mostly β-sheets) is present in all amyloid aggregates.  One protein that forms amyloid fibrils is insulin.  We have found that bovine insulin forms amyloid fibrils faster than human insulin.  In addition, we have found that the lag time for fibril formation depends on the species and on the presence of a solid/liquid interface.

Some of the results may be explained by considering the (minute) differences in the polypeptide chains of both proteins. Bovine insulin differs from human insulin in three of its amino acids; alanine substitutes threonine twice, and valine substitutes isoleucine once.  It is known that Thr, Val and Ile are “beta sheet” forming amino acids but Ala is an alpha helix forming one.  Moreover, aromatic amino-acids, like Thr,  are found to have the highest amyloidogenic propensity.

We decided to explore those differences further.  We synthesized the following peptides: a) Leu-Ser-Cys-Ile-Ser-Thr-Cys-Cys-Gla-Glu and b) Leu-Ser-Cys-Val-Ser-Ala-Cys-Cys-Gla-Glu.  These segments include the regions where the differences between bovine and humane insulin are found. Because of the limited solubility of these peptides a Lys chain was added to the C-termini.  The peptides were exposed to conditions known to induce the formation of fibrils by the hormone (pH ~ 2 and 60 C) in the absence and in the presence of solid/liquid interfaces. The solid/liquid interfaces were created by including liposomes of various degrees of fluidity.  The peptides did not form fibrils at low concentrations in bulk but they did at higher concentrations.  The liposomes seem to inhibit the formation of fibrils. Differences in the kinetics of fibril formation were observed.


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