Effect of TPA-activated neutrophils and MMP-12 on apoA-I variants processing and amyloidogenicity.

<p>ApoA-I mutants (0.2 mg/mL) were incubated in the presence of TPA stimulated neutrophils at 37°C for 1 h. A) Western blotting of aliquots of the supernatant developed with rabbit polyclonal anti-apoA-I. Lanes 1, 3 and 5 show samples incubated in the absence of neutrophils, while lanes 2, 4 and 6 represent the same amount of protein applied to each lane after neutrophil treatment. B) Following the treatment with or without neutrophils samples were incubated at 37°C for 24 h. One µl of ThT (1 mM) was added to each well and ThT fluorescence was measured in a microplate reader at 480 nm (excitation at 430 nm). Each bar shows the ratio of the ThT fluorescence binding to each protein variant incubated in the presence versus the absence of activated PMNs and corresponds to means ± SE. In a different experiment apoA-I was incubated in the presence of MMP-12. C) Western blotting was developed as in A). Numbers at the bottom of each lane represent the same as in A) but in the absence or presence of MMP-12. D) After 3 h at 37°C, MMP-12 was inhibited and apoA-I incubated as in B) to determine ThT binding.</p

Morphology characterization of apoA-I mutants’ aggregates.

<p>Analysis of images observed under AFM. ApoA-I Wt (A), Gly26Arg (B) and Lys107-0 (C) respectively, were incubated for 24 h at 0.6 mg/mL and loaded onto mica. Small size oligomers covering the surface of the mica were predominant in each sample. The distribution of the oligomers’ height is shown as Histograms obtained from the measurement in the z-plane of 100 oligomers. Insets in the histograms’ plot represent isolated aggregates occasionally observed by negative stain under Transmission Electron Microscopy. Bars in each image show the scale used in each case.</p

Spectral Properties and Stabilities of Wt apoA-I and the Gly26Arg and Lys107-0 mutants at 0.1 mg/mL, pH 7.4.

a<p>Wavelength of maximum fluorescence of Trp residues, determined from the from the intrinsic fluorescence spectra as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043755#pone-0043755-g001" target="_blank">Figure 1</a>.</p>b<p>and ^c Free energy change of unfolding and GndHCl concentration at which half of the protein is unfolded, respectively, calculated from equilibrium unfolding curves as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043755#pone.0043755-Ramella1" target="_blank">[8]</a> and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043755#pone-0043755-g002" target="_blank">Figure 2</a> (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043755#s2" target="_blank">Methods</a>").</p>d<p>Stern-Volmer quenching constant (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043755#s2" target="_blank">Methods</a>").</p

Characterization of apoA-I variants’ conformation.

<p>A) Intrinsic Trp fluorescence emission spectra of apoA-I variants. Proteins at a final concentration of 0.1 mg/mL in citrate phosphate Mc Ilvaines buffer pH 7.4. Excitation was set at 295 nm and emission was recorded between 310 and 370 nm. Continuous line represents Wt protein; Dashed and dotted lines are fluorescence spectra corresponding to Gly26Arg and Lys107-0 respectively. B) Fluorescence analysis of bis-ANS binding to apoA-I. ApoA-I variants, at a final concentration of 0.1 mg/mL were titrated with bis-ANS to a final concentration of 16 µM. The probe was excited at 360 nm, and emission registered as the Wavelength of Maximum Fluorescence for this probe. Dark circles represent the experimental data for Wt. Gray and white symbols correspond to Lys107-0 and Gly26Arg respectively. Inset: normalized fluorescence spectra of bis-ANS at a molar ratio 1∶1 probe to protein. Continuous line corresponds to Wt spectrum; Dashed and dotted lines correspond to Lys107-0 and Gly26Arg respectively.</p

Chemical unfolding of apoA-I variants.

<p>Dark circles represent the experimental data for Wt. Gray and white symbols correspond to Lys107-0 and Gly26Arg respectively. A) Equilibrium unfolding of apoA-I variants as followed by intrinsic Trp fluorescence. Spectral centers of mass are plotted as a function of [GndHCl]. Final protein concentration was 0.1 mg/mL; excitation was set at 295 nm and emission recorded between 310 and 420 nm. Continuous lines are fits to the data, in the same order using a sigmoidal model. B) Dependence of bis-ANS fluorescence as a function of [GndHCl]. Proteins were diluted to 0.1 mg/mL and incubated with bis-ANS at a molar ration probe: protein 1∶5. GndHCl was added stepwise. Fluorescence was registered as the Wavelength of Maximum Fluorescence at each [GndHCl]. C) Overlap of GndHCl-mediated denaturation curves for Wt apoA-I as followed by Trp (panel A) and bis-ANS fluorescence (panel B).</p

Effect of apoA-I variants in the release of pro-inflammatory species from macrophages.

<p>One μg/mL of Wt, Gly26Arg or Lys107-0 were added to RAW 264.7 macrophages previously incubated for 1 h with 50 µg/mL of polymyxin B. Lane labeled as B, as a negative control, represents the medium untreated with proteins. LPS, as a test of positive response, represents cells treated with 1 µg/mL LPS, plus (+P) or without (-P) the addition of polymyxin B. A) ROS production was measured after 6 h incubation and cell lysis by fluorescence detection of DCF formation in a microplate reader (excitation and emission filters centered at 485 and 525 nm, respectively). TNF-α (B) and IL-β (C) production is quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043755#s2" target="_blank">Methods</a>. Bars correspond to means ± SE. Symbol # represents significant difference respect to control (in the absence of LPS) at p<0.05. * represents significant difference respect to cells incubated with the same amount of Wt protein at p<0.05.</p