Deciphering the Molecular Dance: Exploring the Dynamic Interplay Between Mouse Insulin B9-23 Peptides and Their Variants

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic insulin-producing β-cells, primarily targeted by autoreac-tive T cells that recognize insulin B9-23 peptides as antigens. Using drift tube ion mobility spectrometry-mass spectrometry (DT-IMS), transmission electron microscopy (TEM), and two-dimensional infrared spectroscopy (2D IR), we characterized mouse insulin 1 B9-23 (Ins1 B9-23), insulin 2 B9-23 (Ins2 B9-23), along with two of their mutants, Ins2 B9-23 Y16A and Ins2 B9-23 C19S. We focus on two competing factors that may affect the aggregation kinetics of B9-23 variants: (a) the formation of intermolecular disulfide bonds by C19 which stabilizes parallel β-sheet assembly, and (b) the aggregation of B12-17 (VEALYL) which favors anti-parallel sheets. Their interplay likely contributes to the observed variations in aggregation kinetics and morphologies. Our findings indicate that Ins1 B9-23 and the Ins2 Y16A mutant exhibit rapid fibril formation, whereas Ins2 B9-23 and the Ins2 C19S mutant show slower fibrilization and a structural rearrangement from globular protofibrils to fibrillar aggregates. The Y16A mutation decreases the amyloidogenicity of the VEALYL sequence and, thus, indirectly promotes parallel β-sheet arrangements. The weak or lack of disulfide bond formation in Ins2 B9-23 and C19S, respectively, weakens the overall propensity of parallel β-sheet assembly. These differences in aggregation behaviors also manifest in interactions with (-)epigallocatechin gallate (EGCG), a canonical amyloid inhibitor. EGCG effectively disrupts the fibrils formed by Ins1 B9-23 and the Y16A mutant. However, it proves ineffective in preventing fibril formation of Ins2 B9-23 and the C19S mutant. These results establish a strong correlation between the aggregation behaviors of these peptides and their divergent effects on anti-islet autoimmunity.