Aterosclerosis y amiloidosis: ¿dos patologías crónicas interrelacionadas?

A fin de poder llevar a cabo las distintas funciones biológicas, es esencial que las proteínas conserven su conformación nativa. Algunas proteínas son estructuralmente inestables, y entonces pequeños cambios en el microambiente en el que se encuentran pueden ser clave para alterar el equilibrio hacia una conformación patológica. Las amiloidosis se caracterizan por la presencia de depósitos extracelulares de proteínas que adoptan estructura fibrilar. La apolipoproteína A-I humana no está normalmente asociada a esta patología, aunque fueron detectados agregados de la misma con la secuencia nativa en placas ateroscleróticas seniles. A pesar de ser frecuente, se conoce relativamente poco de la patogénesis y significancia de la agregación patológica de la apoA-I

Human Apolipoprotein A-I-Derived Amyloid: Its Association with Atherosclerosis

Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis

Human Apolipoprotein A-I-Derived Amyloid: Its Association with Atherosclerosis

Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis

Model of the apoA-I peptide probably involved in the heparin binding site.

<p>(A) A model was built by using the Swiss-model database (<a href="http://www.expasy.org/" target="_blank">www.expasy.org</a>). The sequence loaded involves residues 143–186 in the wt form (PLGEEMRDRARAHVDALRTHLAPYSDELRQ<b>R</b>LAARLEALKENGG). Ball-and-stick representation was obtained with the Avogadro’s software. Positively charged residues are as follows: Arg are in green, His, in yellow and Lys, in pink. (B) Same for the Arg173Pro variant (PLGEEMRDRARAHVDALRTHLAPYSDELRQ<b>P</b>LAARLEALKENGG). The Pro residue incorporated with the mutation is shown in light blue.</p

Protein binding to SDS.

<p>A) The wt and Arg173Pro variants (0.2 mg/mL) were incubated (either at pH 7.4 or 5.0) in the absence or in the presence of 0.2 mM SDS. After 48 h at 37°C ThT was added to a 1:1 molar ratio to protein. Fluorescence intensity was quantified in the microplate reader at 480 nm (excitation set at 430 nm). Statistically significant differences between experimental conditions were evaluated by ANOVA followed by Tukey’s test. Bars correspond to means ± SE. The symbol * denotes difference with respect to wt at p<0.01. B) The effect of salt concentration was tested by incubating Arg173Pro (0.2 mg/mL) at pH 7.4 in the absence or the presence of 0.2 mM SDS plus 0, 100 and 400 mM ClNa. Incubation was performed as in A). The symbol # denotes difference with respect to the protein in the absence of ClNa at p<0.01</p

Chemical denaturation as followed by GndHCl titration.

<p>Proteins (0.1 mg/mL in citrates phosphates Mc Ilvaine’s buffer, pH 7.4) were incubated with increasing concentrations of GndHCl. Trp fluorescence emission spectra were obtained by excitation at 295 nm, and scanning the emission between 310 and 420 nm. With these data, the center of mass was calculated for each sample. Filled and open circles represent the experimental data for the wt and Arg173Pro variants, respectively.</p

Characterization of heparin binding to apo A-I variants.

<p>Polyacrylamide gradient gel electrophoresis (PAGGE, 4–25%) under native conditions. wt (lane 1) and Arg173Pro variants (lane 2); lanes 3 and 4 correspond to wt and Arg173Pro variants plus heparin (added at a 2:1 heparin to protein molar ratio), after 48 h incubation at 37°C at pH 7.4. B) Characterization of the morphology of Arg173Pro aggre<b>g</b>ates with he<b>p</b>arin. Analysis of images observed under AFM. Proteins (0.5 mg/mL) were incubated for 24 h and loaded onto mica plates. The bar shows the scale indicated in the image. C) Distribution of the height of oligomers resulting from the measurement in the <i>z</i>-plane. D) Proteins (0.2 mg/mL in citrates phosphates McIlvaine’s buffer, pH 5.0) were incubated for 48 h at 37°C in the presence or in the absence of heparin at a 1:1 molar ratio. Binding of ThT was measured as described above. Bars correspond to means ± SE. Statistically significant differences between experimental conditions were evaluated by Student t-test. Bars correspond to means ± SE. The symbol * denotes the difference with the same protein without heparin at p<0.01.</p

Protein structure, stability and aggregation tendency at pH 5.0.

<p>A) Proteins were dissolved in citrates phosphates McIlvaine’s buffer, pH 5.0 and far-UV circular dichroism measured as describe above. Dark and light grey solid lines correspond to the wt and Arg173Pro variants, respectively. To facilitate comparison, dashed lines correspond to both proteins measured at pH 7.4 (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124946#pone.0124946.g001" target="_blank">Fig 1A</a>); B) Arg173Pro (0.1 mg/ml, pH 5.0) was incubated with increasing concentrations of GndHCl and the center of mass of the Trp fluorescence measured as described above (filled circles). Here again, to facilitate comparison, dashed lines correspond to the protein measured at pH 7.4 (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124946#pone.0124946.g002" target="_blank">Fig 2</a>). C) Analysis of energy transfer from Trp to bis-ANS. The Arg173Pro variant was dissolved in citrates phosphates McIlvaine’s buffer (0.1 mg/mL) and Trp fluorescence measured (with excitation set at 295 nm, continuous line). Bis-ANS was added next and spectra measured (with excitation set at 295 nm, dotted line). Direct excitation of bis-ANS was achieved at 395 nm (dashed line). Dashed and dotted line represents bis-ANS excitation at 295 nm in protein-free buffer. Efficiency was calculated by measuring the intensity of Trp fluorescence at the wavelength of maximum emission (335 nm). D) Binding of ThT to apoA-I. Proteins (0.2 mg/mL in citrates phosphates McIlvaine’s buffer, pH 5.0) were incubated for 48 h at 37°C and ThT added to a 1:1 molar ratio to protein. Fluorescence intensity was quantified in the microplate reader at 480 nm (excitation set at 430 nm). Bars correspond to means ± SE. Statistically significant differences between experimental conditions were evaluated by Student t-test. The symbol * denotes a difference with respect to wt at p<0.05.</p

Spectroscopical characterization of protein structure.

<p>Wt and Arg173Pro variants (0.1 mg/mL) were dissolved in citrates phosphates McIlvaine’s buffer, pH 7.4. A) Far-UV circular dichroism. Dark and light grey lines correspond to wt and Arg173Pro proteins, respectively. B) Proteins were incubated with increasing concentrations of bis-ANS. The probe was excited at 360 nm, and the emission recorded at the wavelength of maximum fluorescence for this probe (488 nm). Filled and open circles represent the experimental data points for wt and Arg173Pro, respectively.</p