The degree of unsaturation of fatty acids in phosphatidylserine alters the rate of insulin aggregation and the structure and toxicity of amyloid aggregates

Abstract

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.

Keywords: AFM-IR; amyloid; insulin; phospholipids; toxicity.

Fig. 1.

PS with different degrees of unsaturation of FAs alters the rates of insulin aggregation. Averages of triplicates of ThT aggregation kinetics of insulin in the lipid-free environment [(Ins), red] and in the presence of DMPS (blue), POPS (black), and DOPS (green) at 1 : 1 molar ratio. For Ins, 400 μM of protein was dissolved in 1×PBS with 2 mM of ThT; pH adjusted to pH 3.0. For Ins: DMPS, Ins:POPS, and Ins:DOPS, 400 μM of insulin was mixed with an equivalent concentration of the corresponding lipid; pH was adjusted to pH 3.0. All samples were kept at 37 °C under 510 rpm for 24 h.

Fig. 2.

PS with different degrees of unsaturation of FAs alters morphology of protein aggregates. AFM images of insulin aggregates grown in the lipid-free environment (A), as well as in the presence of DMPS (B), POPS (C), and DOPS (D). After 24 h of incubation of insulin (400 μM) with and without lipids at 37 °C under 510 rpm, sample aliquots were diluted with 1×PBS pH 3.0 and deposited onto precleaned silicon wafer. AFM imaging was performed in tapping mode. Scale bars are 200 nm.

Fig. 3.

Structural analysis of insulin aggregates. ATR-FTIR spectra of insulin fibrils grown in the lipid-free environment (red), as well as in the presence of DMPS (blue), POPS (black), and DOPS (green). After 24 h of incubation of insulin (400 μM) with and without lipids at 37 °C under 510 rpm, triplicates of samples were diluted with 1×PBS pH 3.0 and directly deposited onto ATR crystal (ATR-FTIR) and dried under room temperature. For each of the presented traces, three independent ATR-FTIR measurements were averaged.

Fig. 4.

Nanoscale analysis of lipid content of insulin aggregates. Averaged AFM-IR spectra of insulin aggregates (Ins) and Ins:DMPS, Ins: POPS, and Ins:DOPS. Amide I and II bands represent protein secondary structure, whereas L1-L3 bands demonstrate phospholipid vibrations. After 24 h of incubation of insulin (400 μM) with and without lipids at 37 °C under 510 rpm, sample aliquots were diluted with 1×PBS pH 3.0 and deposited onto precleaned silicon wafer. AFM-IR analysis was performed in contact mode. At least 30–40 individual aggregates were analyzed for each sample.

Fig. 5.

Insulin aggregates grown in the presence of lipids possess lower cell toxicity comparing to the aggregates grown in the lipid-free environment. Histograms of ROS (top), JC-1 (middle), and LDH (bottom) toxicity assays of Ins, Ins:DOPS, Ins:POPS, and Ins:DMPS, as we as DOPS, POPS, and DMPS lipids. After 24 h of incubation of insulin (400 μM) with and without lipids at 37 °C under 510 rpm, sample triplicates were exposed to mice midbrain N27 cells for 48 h. For each of the presented results, three independent measurements were made.