HEWL interacts with dissipated oleic acid micelles, and decreases oleic acid cytotoxicity

Abstract

Senile plaques are well-known hallmarks of Alzheimer's Diseases (AD). However, drugs targeting tangles of the protein tau and plaques of β-amyloid have no significant effect on disease progression, and the studies on the underlying mechanism of AD remain in high demand. Growing evidence supports the protective role of senile plaques in local inflammation driven by S100A9. We herein demonstrate that oleic acid (OA) micelles interact with hen egg white lysozyme (HEWL) and promote its amyloid formation. Consequently, SH-SY5Y cell line and mouse neural stem cells are rescued from OA toxicity by co-aggregation of OA and HEWL. Using atomic force microscopy in combination with fluorescence microscopy, we revealed that HEWL forms round-shaped aggregates in the presence of OA micelles instead of protofibrils of HEWL alone. These HEWL amyloids act as a sink for toxic OA micelles and their co-aggregate form large clumps, suggesting a protective function in amyloid and OA cytotoxicity.

Fig 1

(A) AFM height image of 1.4 mM HEWL only (HEWL, top), 1.4 mM HEWL with 14 mM OA (OA10, middle), and 1.4 mM HEWL with 140 mM OA (OA100, down), after incubation for 1d. Scale bars denote 1000 nm in all images. (B) Height distribution of fibrils obtained by AFM measurement in samples containing only HEWL (HEWL, top), 1.4 mM HEWL with 14 mM OA (OA10, middle) and 1.4 mM HEWL with 140 mM OA (OA100, down). (C) Amyloid formation kinetics of 1.4 mM hen egg white lysozyme (HEWL) with 14mM and 140 mM OA (OA10 and OA100, respectively). (D) Far UV CD spectra of 1.4mM hen egg white lysozyme (HEWL) with OA. All samples are maintained in 20 mM glycine buffer under continuous shaking at 800 rpm, pH 2.3 and 57°C for up to7 days.

Fig 2

(A) Appearance of 1.4 mM HEWL (left), 1.4 mM HEWL with 14 mM OA (middle), and 1.4mM HEWL with 140 mM OA (right), after incubation in 20 mM glycine buffer under continuous shaking at 800 rpm, pH 2.3 and 57°C for 4 d. (B) Fluorescent microscopy images of pre-formed HEWL-OA clumps. Scale bares denote 100 μm in the left panel, and 15 μm in the right panel. (C) Optical images of SH-SY5Y Cells being maintained in DMEM culture medium (Control, left), culture medium containing 400 mM OA (OA,middle), and solution containing 4 μM HEWL and 400 μM OA (OA+HEWL, right). Scale bar denotes 100 μM. The clumps were formed by positioning HEWL and OA mixture (HEWL 1.4 mM and OA 140 mM) under continuous shaking at 800 rpm and 57°C for 4 d.

Fig 3

Measurements of SH-SY5Ycell viability by WST-1 assay in the presence of different concentrations of oleic acid (4 to 400 μM), and (B) HEWL- OA complex. In control experiments the cells were incubated in DMEM cell culture buffer, Tris-HCl buffer, sphingocine and 16 mM DMSO. In DMEM culture medium, cell viability is equal to 100%. In the mixture of OA and DMSO, the OA concentration is 400 μM and DMSO16 mM. All HEWL samples were prepared at HEWL concentration of 1.4 mM (OA is 14 and 140 mM) and aged 1 d in 20 mM glycine buffer under continuous shaking at800 rpm, pH 2.3 and 57°C.

Fig 4

Brightfield images of neural stem cells (NSCs) exposed to (A) culture medium; (B) environmental conditions containing 400 μM OA; (C) 1.4 mM HEWL with 140 mM OA. Scale bar denotes 100 μm.The clumps were formed by positioning HEWL and OA mixture (HEWL 1.4 mM and OA140 mM)) under continuous shaking at 800 rpm and 57°C for 4 d. (D) Measurements of NSCs viability by WST-1 assay in the presence of culture medium, OA and HEWL-OA complex.