Cryo-ElectronMicroscopy Provides Mechanistic Insightsinto Solution-Dependent Polymorphism and Cross-Aggregation Phenomenaof the Human and Rat Islet Amyloid Polypeptides

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Inhibitors targeting amyloids formed by the human IsletAmyloidPolypeptide (hIAPP) are promising therapeutic candidates for type2 diabetes. Peptide formulations derived from the nonamyloidogenicrat IAPP (rIAPP) sequence are currently used as hIAPP mimetics tosupport insulin therapy. rIAPP itself acts as a peptide inhibitor;yet, the structural-level consequences of such inhibition, particularlyits impact on amyloid polymorphism, have not been studied in detail.Here, we conduct coaggregation experiments with varying rIAPP-to-hIAPPconcentration ratios and employ high-resolution cryo-electron microscopy(Cryo-EM) to elucidate the polymorphism of the resulting fibril structures.Our results demonstrate that the polymorphism of hIAPP amyloids ishighly sensitive to the electrostatic environment, which can be modulatedby buffer composition, the concentration of the inhibitor, and cosolventssuch as hexafluoroisopropanol (HFIP). Under native conditions, rIAPPassociates with hIAPP but does not cross-aggregate, resulting in fibrilsprimarily composed of hIAPP. Significant inhibition is observed atrelatively high concentrations of rIAPP. However, trace amounts ofHFIP disrupt this inhibition, leading to increased fibril concentrationsdue to the formation of cross-seeded products composed of both hIAPPand rIAPP, as evidenced by mass spectrometry and two-dimensional infrared(2D IR) spectroscopy. These findings highlight the critical role ofexperimental conditions, particularly the electrostatic environment,in modulating amyloid polymorphism, cross-seeding, and inhibition.By providing structural insights into these processes, this studyadvances our understanding of peptide aggregation and offers valuableguidance for the rational design of more effective therapeutic inhibitorstargeting hIAPP-related amyloidosis.