Explosive Fibrillation Kinetics Of Two-Chain Insulin Fragment Released Upon Partial Digestion With Pepsin

Proteases are recognized for their role in the emergence of highly aggregation-prone protein fragments in vivo. On the other hand, limited proteolysis in vitro is often used to probe different phases of amyloidogenic pathways. Here we show that moderate amounts of pepsin induce “explosive” fibrillation in acidified samples of bovine insulin. Biochemical analysis of the pepsin-induced fibrils reveals previously unreported two-chain peptide with potent amyloidogenic properties as the main building block. The peptide (named ‘H’) comprises of N-terminal fragments of insulin A- and B-chains linked by disulfide bond between Cys7A-Cys7B and conceals up to 8 additional pepsin-cleavage sites which become protected upon fast fibrillation unless concentration of the enzyme is increased leading to complete digestion of insulin. Fibrils built of H-peptides are similar in terms of morphology (as probed by AFM) and infrared features to typical bovine insulin fibrils, but they appear to lack the ability to seed fibrillation of intact insulin. Controlled re-association of these fragments leads to ‘explosive’ fibrillation only under non-reducing conditions implying the key role of the disulfide bonds in the amyloidogenicity of H-peptides. Our study highlights the role of dynamics of the disulfide-bonded N-terminal fragments of A- and B-chains in insulin amyloidogenesis.