Phosphatidylserine (PS) in the plasma membrane plays an important role in cell signaling and apoptosis. Cell degeneration is also linked to numerous amyloid diseases, pathologies that are associated with aggregation of misfolded proteins. In this work, we examine the effect of both saturated PS (DMPS) and unsaturated PS (DOPS and POPS) on the aggregation properties of insulin, as well as the structure and toxicity of insulin aggregates formed in the presence of these phospholipids. We found that the degree of unsaturation of fatty acids in PS alters the rate of insulin aggregation. We also found that toxicity of insulin–DMPS aggregates is significantly lower than the toxicity of DOPS– and POPS–insulin fibrils, whereas all these lipid‐containing aggregates exert lower cell toxicity than insulin fibrils grown in a lipid‐free environment.

Lipid bilayers play an important role in the pathological assembly of amyloidogenic proteins and peptides. This assembly yields oligomers and fibrils, which are highly toxic protein aggregates. In this study, we investigated the role of saturation in fatty acids of two phospholipids that are present in cell membranes. We found that unsaturated cardiolipin (CL) drastically shortened the lag phase of insulin aggregation. Furthermore, structurally and morphologically different aggregates were formed in the presence of unsaturated CL vs saturated CL. These aggregates exerted drastically different cell toxicity. Both saturated and unsaturated phosphatidylcholine (PC) were able to inhibit insulin aggregation equally efficiently. Similar to CL, structurally different aggregates were formed in the presence of saturated and unsaturated PC. These aggregates exerted different cell toxicities. These results show that unsaturated phospholipids catalyze the formation of more toxic amyloid aggregates comparing to those formed in the presence of saturated lipids.

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time when the intensity of ThT fluorescence reached 50% of the maximum; tgrow, time when the intensity of ThT fluorescence reached 90% of the maximum; ThT, thioflavin T; tlag, time when the intensity of ThT fluorescence reached 10% of the maximum.

Abrupt aggregation of misfolded proteins is a hallmark of a large number of severe pathologies, including diabetes types 1 and 2, Alzheimer, and Parkinson diseases. A growing body of evidence suggests that lipids can uniquely change rates of amyloid-associated proteins as well as modify the structure of formed oligomers and fibrils. In this study, we utilize atomic force microscopy infrared (AFM-IR) spectroscopy, also known as nano-IR spectroscopy, to examine the structure of individual insulin oligomers, protofilaments, and fibrils grown in the presence of phospholipids. Our findings show that AFM-IR spectra of insulin oligomers have strong signals of C–H and PO2 – vibrations, which points on the presence of lipids in the oligomer structure. Furthermore, substantial shifts in lipid vibrations in AFM-IR spectra of the oligomers relative to the corresponding bands of pure lipids have been observed. This points on strong interactions between a lipid and a protein that are developed at the stage of the oligomer formation.