Department of Structural and Functional Biology, University of Naples Federico II, Italy.
Eur Biophys J. 2010 Aug;39(9):1289-99. doi: 10.1007/s00249-010-0582-2. Epub 2010 Feb 25.
In amyloidosis associated with apolipoprotein A-I (ApoA-I), heart amyloid deposits are mainly constituted by the 93-residue ApoA-I N-terminal region. A recombinant form of the amyloidogenic polypeptide, named [1-93]ApoA-I, shares conformational properties and aggregation propensity with its natural counterpart. The polypeptide, predominantly in a random coil state at pH 8.0, following acidification to pH 4.0 adopts a helical/molten globule transient state, which leads to formation of aggregates. Here we provide evidence that fibrillogenesis occurs also in physiologic-like conditions. At pH 6.4, [1-93]ApoA-I was found to assume predominantly an alpha-helical state, which undergoes aggregation at 37 degrees C over time at a lower rate than at pH 4.0. After 7 days at pH 6.4, protofibrils were observed by atomic force microscopy (AFM). Using a multidisciplinary approach, including circular dichroism (CD), fluorescence, electrophoretic, and AFM analyses, we investigated the effects of a lipid environment on the conformational state and aggregation propensity of [1-93]ApoA-I. Following addition of the lipid-mimicking detergent Triton X-100, the polypeptide was found to be in a helical state at both pH 8.0 and 6.4, with no conformational transition occurring upon acidification. These helical conformers are stable and do not generate aggregated species, as observed by AFM after 21 days. Similarly, analyses of the effects of cholesterol demonstrated that this natural ApoA-I ligand induces formation of alpha-helix at physiological concentrations at both pH 8.0 and 6.4. Zwitterionic, positively charged, and negatively charged liposomes were found to affect [1-93]ApoA-I conformation, inducing helical species. Our data support the idea that lipids play a key role in [1-93]ApoA-I aggregation in vivo.
在与载脂蛋白 A-I(ApoA-I)相关的淀粉样变性中,心脏淀粉样沉积物主要由 93 个残基的 ApoA-I N 端区域组成。一种称为[1-93]ApoA-I 的淀粉样多肽的重组形式,具有与其天然对应物相同的构象特性和聚集倾向。该多肽在 pH 值为 8.0 时主要处于无规卷曲状态,酸化至 pH 值为 4.0 后,它会经历一个短暂的螺旋/无定形球蛋白状态,从而导致聚集。在这里,我们提供了证据表明,纤维原纤维的形成也发生在类似生理的条件下。在 pH 值为 6.4 时,[1-93]ApoA-I 被发现主要呈α-螺旋构象,随着时间的推移,在 37°C 下会发生聚集,但聚集速度比 pH 值为 4.0 时慢。在 pH 值为 6.4 下孵育 7 天后,原子力显微镜(AFM)观察到原纤维。我们采用包括圆二色性(CD)、荧光、电泳和 AFM 分析在内的多学科方法,研究了脂质环境对[1-93]ApoA-I 构象状态和聚集倾向的影响。在添加脂质模拟去污剂 Triton X-100 后,发现多肽在 pH 值为 8.0 和 6.4 时均处于螺旋构象,酸化时不会发生构象转变。这些螺旋构象稳定,不会产生聚集物,这在 21 天后通过 AFM 观察到。类似地,胆固醇作用的分析表明,这种天然的 ApoA-I 配体在生理浓度下诱导了 pH 值为 8.0 和 6.4 时的α-螺旋形成。两性离子、带正电荷和带负电荷的脂质体被发现会影响[1-93]ApoA-I 构象,诱导螺旋构象。我们的数据支持这样一种观点,即脂质在[1-93]ApoA-I 体内聚集中发挥关键作用。