Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):1977-82. doi: 10.1073/pnas.0910136107. Epub 2010 Jan 19.
Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (T(m)) from 58.7 degrees C for native apoA-I to 48.2 degrees C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 A and 9.92 A, indicating a cross-beta amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation.
载脂蛋白 A-I（apoA-I）是 HDL 的主要蛋白成分，在胆固醇转运中发挥重要作用。apoA-I 衍生的淀粉样蛋白沉积与各种遗传性系统性淀粉样变性和动脉粥样硬化有关；然而，关于 apoA-I 淀粉样蛋白形成的机制知之甚少。apoA-I 中的蛋氨酸残基通过体内的几种机制被氧化，形成蛋氨酸亚砜（MetO），并且在正常的人类血清中存在大量的蛋氨酸氧化 apoA-I（MetO-apoA-I）。我们研究了蛋氨酸氧化对全长、无脂 apoA-I 的结构、稳定性和聚集的影响。圆二色光谱表明，apoA-I 中三个蛋氨酸残基的氧化导致蛋白质部分展开，并降低其热稳定性，使熔融温度（T(m)）从天然 apoA-I 的 58.7°C 降低到 MetO-apoA-I 的 48.2°C。分析超速离心显示，蛋氨酸氧化抑制了 apoA-I 的天然自组装，形成二聚体和四聚体。MetO-apoA-I 的孵育时间延长会导致蛋白质聚集，这些聚集体结合硫黄素 T 和刚果红。电子显微镜检查显示，聚集物具有纤维状结构，具有带状形态，宽度约为 11nm，长度可达数微米。纤维衍射研究表明，纤维具有正交峰的衍射图案，间距为 4.64A 和 9.92A，表明存在交叉-β淀粉样结构。全长 apoA-I 纤维形成的系统研究首次表明，蛋氨酸氧化可以诱导淀粉样纤维形成。
