Tuning the surface charge of phospholipid bilayers inhibits insulin fibrilization

Abstract

The interactions between proteins and materials, in particular lipid bilayers, have been studied extensively for their relevance in diseases and for the formulation of protein-based therapeutics and vaccines. However, the precise rules by which material properties induce favorable or unfavorable structural states in biomolecules are incompletely understood, and as a result, the rational design of materials remains challenging. Here, we investigated the influence of lipid bilayers (in the form of small unilamellar vesicles) on the formation of insulin amyloid fibrils using a fibril-specific assay (thioflavin T), polyacrylamide gel electrophoresis, and circular dichroism spectroscopy. Lipid bilayers composed of equal mixtures of cationic and anionic lipids effectively inhibited fibril formation and stabilized insulin in its native conformation. However, other lipid bilayer compositions failed to inhibit fibril formation or even destabilized insulin, exacerbating fibrilization and/or non-amyloid aggregation. Our findings suggest that electrostatic interactions with lipid bilayers can play a critical role in stabilizing or destabilizing insulin, and preventing the conversion of insulin to its amyloidogenic, disease-associated state.

Keywords: Aggregation; Amyloid; Catanionic lipids; Insulin; Lipid bilayers; Protein misfolding.

Figure 1.

Zeta potential of mixed DOEPC/DOPG vesicles versus composition in 20 mM Tris/HCl (pH 7.4 with 50 mM NaCl). For comparison, the zeta potential of 100% DOPC vesicles is also shown (dashed red line). Error bars are the standard deviation among three technical replicates.

Figure 2.

Images showing the turbidity of insulin upon incubation with mixed DOEPC/DOPG vesicles for 72 h at 37 °C in 20 mM Tris/HCl (pH 7.4 with 50 mM NaCl). The sample tubes were overlayed on a background with text to highlight the difference in turbidity of the samples.

Figure 3.

Characterization of insulin aggregation in the presence of mixed DOEPC/DOPG vesicles via SDS-PAGE. Aggregation was followed as a function of incubation time from 0 to 72 h at 37 °C in 20 mM Tris/HCl (pH 7.4 with 50 mM NaCl). The molecular weight of native insulin is 5808 kDa.

Figure 4.

Characterization of insulin aggregation in the presence of DOEPC/DOPG vesicles by ThT fluorescence upon incubation for 72 h at 37 °C in 20 mM Tris/HCl (pH 7.4 with 50 mM NaCl). Insulin-only control (also after 72 h) is shown as a dashed red line for comparison. Error bars are the standard deviation among three technical replicates.

Figure 5.

Characterization of the impact of mixed DOEPC/DOPG vesicles on the retention of secondary structure of insulin as measured by CD. The θ_208/222 represents the ratio of the ellipticity of insulin at 208 nm and 222 nm upon incubation for 72 h at 37 °C in 20 mM Tris/HCl (pH 7.4 with 50 mM NaCl). The values for θ_208/222 for native insulin and insulin incubated at 72 h under the same conditions without vesicles are shown as dashed red lines. Error bars are the standard deviation among three technical replicates.