Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation

Abstract

Apolipoprotein A-I (apoA-I) shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and intermolecularly, providing structural stabilization by self-association. We have reported that lipid-free apoA-I becomes amyloidogenic upon physiologically relevant (myeloperoxidase-mediated) Met oxidation. In this study, we established that Met oxidation promotes amyloidogenesis by reducing the stability of apoA-I monomers and irreversibly disrupting self-association. The oxidized apoA-I monomers also exhibited increased cellular cholesterol release capacity and stronger association with macrophages, compared to nonoxidized apoA-I. Of physiologic relevance, preformed oxidized apoA-I amyloid fibrils induced amyloid formation in nonoxidized apoA-I. This process was enhanced when self-association of nonoxidized apoA-I was disrupted by thermal treatment. Solid state NMR analysis revealed that aggregates formed by seeded nonoxidized apoA-I were structurally similar to those formed by the oxidized protein, featuring a β-structure-rich amyloid fold alongside α-helices retained from the native state. In atherosclerotic lesions, the conditions that promote apoA-I amyloid formation are readily available: myeloperoxidase, active oxygen species, low pH, and high concentration of lipid-free apoA-I. Our results suggest that even partial Met oxidation of apoA-I can nucleate amyloidogenesis, thus sequestering and inactivating otherwise antiatherogenic and HDL-forming apoA-I.-Witkowski, A., Chan, G. K. L., Boatz, J. C., Li, N. J., Inoue, A. P., Wong, J. C., van der Wel, P. C. A., Cavigiolio, G. Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation.

Keywords: cholesterol efflux; myeloperoxidase; protein structure; self-association; solid-state NMR.

Figure 1.

Kinetics of amyloid fibril formation measured by ThT fluorescence. Representative ThT kinetics curves for H₂O₂-wt-ApoA-I, MPO-3:1-H₂O₂-wt-ApoA-I, MPO-10:1-H₂O₂-ApoA-I, and nonoxidized wt-ApoA-I. A portion of fibrillation incubation mixture was combined with the ThT stock solution at the indicated time points and the fluorescence emission spectrum of the sample recorded. Solid lines are fitting of the experimental values by exponential (H₂O₂-wt-ApoA-I and MPO-10:1-H₂O₂-ApoA-I) or logarithmic (MPO-3:1-H₂O₂-wt-ApoA-I) curves. T_1/2 data (means ± sem) from ≥3 independent experiments are shown.

Figure 2.

Release of cholesterol from cells into cell culture medium during a 4 h incubation in the presence of 1.0 and 2.5 µg/ml ApoA-I samples. To induce expression of ABCA1, cells were preincubated overnight with cAMP, as described in the Supplemental Data. Data are normalized to the cholesterol levels released in the presence of nonoxidized wt-ApoA-I at the indicated concentration. Data are reported as means ± sem of 3 independent experiments with 3 replications per experiment. *P ≤ 0.01, probability that the reported means are not significantly different from the cholesterol release efficiency of nonoxidized wt-ApoA-I (which is normalized to 1). Remaining histograms represent values not significantly different from nonoxidized wt-ApoA-I. P > 0.01.

Figure 3.

Western blot analysis of BMDM cell extracts. Cell-association of apoA-I samples with BMDM from wild-type mice (A) and CD36-KO mice (B).

Figure 4.

SEC analysis of 100 µl apoA-I samples at 1.0 or 0.1 mg/ml in iPBS. A) 90C-wt-ApoA-I was injected into the column after incubation at 90°C for 1 h and 10 min cooling at room temperature. B) After oxidation, H₂O₂-wt-ApoA-I was dialyzed against iPBS for 2 d with 2 buffer exchanges at 4°C before SEC analysis. To facilitate the comparison of different chromatograms, the y-axes were manually adjusted to normalize the intensity of the most prominent peak in each chromatogram to 1. 60C-4WF-ApoA-I and the 0.1 mg/ml sample chromatograms were arbitrarily normalized to 0.6.

Figure 5.

NDGGE analysis of heated and oxidized 4WF-ApoA-I samples. Protein samples at 1.0 mg/ml were incubated for 1 h at the indicated temperatures. After cooling at room temperature for 10 min, the samples were loaded on gel (3 µg/lane) for NDGGE analysis or oxidized by 1000:1 molar excess of H₂O₂, dialyzed against iPBS for 2 d with 2 buffer exchanges and then analyzed. Left lane: molecular weight markers. Molecular weight marker diameters (nm) are indicated on the left. Labels on the right identify the different oligomeric species of nonoxidized 4WF-ApoA-I (19). Met-oxidized oligomers and monomers have slightly reduced electrophoretic mobilities compared to nonoxidized samples.

Figure 6.

SDS-PAGE analysis of wt-ApoA-I, 3ML-ApoA-I, and 4WF-ApoA-I (1.0 mg/ml) upon oxidation by different methods. At the end of oxidation incubations (overnight for H₂O₂ oxidation and 90 min for MPO-mediated oxidation), 3 μg/lane of samples were loaded on gel. Electrophoresis was executed as described in Supplemental Data. Molecular masses of protein standards are indicated on the right. Met-oxidized apoA-I samples have slightly reduced electrophoretic mobility vs. nonoxidized samples.

Figure 7.

Seeding of nonoxidized wt-ApoA-I with molar 10% and 1% of preformed H₂O₂-wt-ApoA-I aggregates. As a control, nonoxidized wt-ApoA-I was incubated in the absence of seeds. Total protein concentration in each sample was 1.0 mg/ml. The samples were incubated in fibrillation buffer (pH 6.0) at 37°C with continuous vortexing at 800 rpm. ThT fluorescence was measured at the indicated time points. Solid lines show fitting of the experimental values by sigmoidal curves. T_1/2 data (means ± sem) from ≥3 independent experiments are shown.

Figure 8.

FTIR spectra in the amide I region (1602–1708 cm⁻¹) of nonoxidized wt-ApoA-I after seeding with preformed H₂O₂-wt-ApoA-I aggregates. Control (A) and 10% (molar) seeded (B) mixtures before incubation under fibrillation conditions (T₀) and after 48 h and 8 d incubations.

Figure 9.

MAS ssNMR spectra of pelleted [U-¹³C,¹⁵N] wt-ApoA-I aggregates. A) Direct excitation ¹³C spectra of H₂O₂-wt-¹³C,¹⁵N-ApoA-I aggregates (black trace) and aggregates formed by nonoxidized wt-¹³C,¹⁵N-ApoA-I incubated for 8 d with 10% (molar) of unlabeled H₂O₂-wt-ApoA-I seeds (light gray trace). An overlay of the 2 spectra is shown at the bottom. Black and gray arrows: the ¹³C signals of oxidized and unoxidized Met side chains, respectively. B) ¹³C spectra obtained via ¹H-¹³C refocused INEPT, which shows only highly dynamic residues. C) ¹³C spectra of the same samples using ¹H-¹³C cross polarization, which is selective for the more rigid parts of the pelleted protein samples. A–C) Only aliphatic spectral regions are shown; full spectra are reported in Supplemental Fig. S6. D) ¹H-¹³C CP spectra in the carbonyl region of H₂O₂-wt-¹³C,¹⁵N-ApoA-I (black trace) and nonoxidized wt-¹³C,¹⁵N-ApoA-I after seeding (light gray trace). Dotted lines (bottom) show simulated spectra for hypothetical fully α-helical and β-sheet structural models of ApoA-I, to illustrate the secondary structure dependence. E) 2D ¹³C-¹³C spectrum of the same H₂O₂-wt-¹³C,¹⁵N-ApoA-I as in A–D, using 20 ms dipolar-assisted rotational resonance mixing. The amino acid type assignments of select cross peaks are indicated. Color-coded lines connect sets of peaks from coexisting α-helical and β-sheet Val and Leu. All spectra were acquired at 600 MHz (¹H frequency).

Figure 10.

Seeding of nonoxidized wt-ApoA-I and 90C-wt-ApoA-I with molar 10% of preformed H₂O₂-wt-ApoA-I aggregates. In control experiments, nonoxidized wt-ApoA-I and 90C-wt-ApoA-I were incubated in the absence of seeds. Total protein concentration was 1.0 mg/ml. The samples were incubated in fibrillation buffer (pH 6.0) at 37°C with continuous vortexing at 1100 rpm. ThT fluorescence was measured at the indicated time points. Solid lines show fitting of the experimental values by monomolecular growth exponential curves. T_1/2 data (means ± sem) from ≥3 independent experiments are shown.

Figure 11.

Summary of the presented results. ApoA-I native self-association is disrupted by thermal treatment or Met oxidation. Destabilized monomeric apoA-I associates with macrophages more strongly and extracts cholesterol more efficiently than native apoA-I. Destabilized monomeric Met(O)-ApoA-I is also amyloidogenic.