Effect of tetracyclines on the dynamics of formation and destructuration of beta2-microglobulin amyloid fibrils

Abstract

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of β2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits β2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble β2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC(50) is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.

Figures

FIGURE 1.

Inhibition of wild type β2-m fibrillogenesis by seven tetracycline congeners was followed by thioflavin fluorescence assay. Experiments were carried out in triplicate at different molar drug concentrations (50, 100, 200, 300 μm). Data were normalized and fitted using a standard Hill slope dose-response curve. Means ± S.D. of three experiments are shown for each compound. The structure of doxycycline, the most effective compound, is also reported.

FIGURE 2.

a, SDS-PAGE of the supernatant and protein pellet obtained after centrifugation of the fibrillogenesis mixtures containing 100 μm β2-m and 300 μm of each tetracycline congener. Samples were centrifuged after 48 h of incubation. Alongside the molecular mass standards, supernatant (s) and pellet (p) are shown for 4-epi-oxytetracycline (lane 1), anhydrochlortetracycline (lane 2), minocycline (lane 3), demeclocycline (lane 4), doxycycline (lane 5), and 4-epianhydrotetracycline (lane 6), respectively. Treated and non-treated β2-m are shown as controls. b, the percentage of protein in both supernatant and pellet of samples with and without tetracyclines. Proteins within each lane were quantified by densitometric analysis, and the percentage was determined as compared with control β2-m samples.

FIGURE 3.

Doxycycline dose-dependent inhibition of fibrillogenesis of the P32G β2-m variant. Experiments were carried out in triplicate in the same conditions reported in the legend for Fig. 1. Error bars indicate means ± S.D.

FIGURE 4.

Tapping mode atomic force microscopy image (amplitude data) of a β2-m sample incubated for 3 months at 37 °C in the presence of fibrillar collagen and heparin, in the absence (a) and in the presence (b) of 360 μm doxycycline. Scan size was 2.8 μm. Amplitude data are shown to better visualize the sample surface, with relatively thin amyloid fibrils (a, black arrows) lying on the surface of large corrugations corresponding to the collagen fibers (white arrows). The inset shows a topographic image (height data) of the texture of the background of b (corresponding to a flat, homogeneous portion of the sample) at a higher magnification; scan size 460 nm, Z range 20 nm.

FIGURE 5.

Solubilization of preformed β2-m fibrils. a, comparative analysis of the doxycycline effect on fibrillogenesis (filled circles) and on fibril destructuration (filled triangles). Error bars indicate means ± S.D. b and c, electron microscopic images of ex vivo fibrils taken before (b) and after (c) 48 h of incubation with doxycycline.

FIGURE 6.

a, two-dimensional diffusion-ordered spectroscopy map for β2-m in pure water in the presence of doxycycline at 1:0 molar ratio (black), at 1:1 molar ratio (blue), and at 1:3 molar ratio (red). The diffusion coefficients were estimated in the limit of a globular isotropic assumption for aqueous β2-m, using the trimeric EMILIN1 gC1q D value as a calibrator for a 51.2-kDa isotropic globule (41). b, β2-m interaction sites with doxycycline in pure water. These locations were obtained from the 15N-1H HSQC chemical shift (Δδ) and intensity change analysis upon doxycycline titration. The compensated Δδ values were calculated as described under “Experimental Procedures” after Mulder et al. (27). The color code is: yellow, the residues with Δδ ≤ Δδ + σ, where Δδ is the mean chemical shift change and σ is the chemical shift difference standard deviation; orange, residues with Δδ + σ < Δδ ≤ Δδ + 2σ; red, residues with Δδ > Δδ Δδ + 2σ.

FIGURE 7.

a and b, 1H-15N HSQC spectra of β2-m 0.65 mm in TFE 18% (phosphate 70 mm, NaCl 100 mm) at 37 °C recorded without (a) or with (b) doxycycline (1.96 mm). The spectra were obtained using the same acquisition conditions from two samples prepared using the same β2-m mother solution, with a 10.0-μl addition of 0.100 m doxycycline for b. The experimental data were treated with the same processing parameters and at the same absolute scaling (TOPSPIN, Bruker). The reported maps were drawn starting at the same lowest contour level and with the same level numbers and spacing.

FIGURE 8.

Effects of doxycycline on cytotoxicity of β2-m. a, β2-m (100 μm) was resolubilized in the absence or in the presence of different concentrations of doxycycline at 37 °C for 1.0 h. 10-μl aliquots of the samples were diluted in 90 μl of cell medium and added to wells coated with SH-SY5Y cells whose viability is reported as a function of the initial doxycycline concentration. b and c, preformed β2-m fibrils (100 μm, monomer concentration) were treated with doxycycline (Dox) for 72 h at the indicated concentrations (b) or at a fixed concentration (300 μm) for the indicated time lengths (c) and then added to the culture medium to estimate toxicity. Cell viability was measured by the MTT assay. The data are expressed as the percentage with respect to the control values obtained in three independent experiments, each value being the average of six trials. d, effect of the time of exposure to doxycycline of preformed β2-m fibrils on cell viability and ThT fluorescence quenching (same experiment as c). In all panels, error bars indicate means ± S.D.

FIGURE 9.

Tapping mode AFM images (amplitude data) showing the evolution of the morphology of preformed β2-m fibrils as a function of the incubation time with doxycycline (300 μm). a, immediately after drug addition. b, at 24 h, when partial fibril dissolution into smaller units becomes visible. c, at 72 h, corresponding to maximum oligomer release and minimum cell viability, with initial rearrangement of the oligomers into more complex structures. d, at 7 days, when the sample is mostly aggregated into structures with uniform height (1 nm). Scan size: 2 μm (a, c, and d) and 1 μm (b).