Understanding the amyloid fibril formation of β2-microglobulin on the basis of protein conformation

基于蛋白质构象了解 β2-微球蛋白淀粉样原纤维的形成

• 批准号: 13480219
• 负责人: GOTO Yuji
• 金额: $ 9.28万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480219/
• 关键词: Amyloid fibril; Protein folding; Stability of proteins; Dialysis-related amyloidosis; Fluorescence microscopy; H; D exchange; β2-microglobulin; Amyloid β-peptide; アミロイド病; 蛋白質; フォールディング; NMR; 一分子観察

β2-Microglobulin (β2-m)-related amyloidosis is a serious complication in patients receiving long-term hemodialysis. To understand the mechanism of amyloid fibril formation by β2-m, we have been studying the conformation and amyloid fibril formation of recombinant human β2-m.1.We established a novel procedure using H/D exchange of amide protons combined with NMR analysis for characterizing the conformational flexibility of β2-m amyloid fibrils at single-residue resolution. The results indicated that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid β-sheet core, while the N-and C-termini are not part of this core. The exchange time course deviated largely from a single exponential curve, consistent with the supramolecular structure of fibrils.2.On the other hand, real-time monitoring of fibril growth is essential to clarify the mechanism of fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. We show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy, amyloid fibrils of β2-m can be visualized without requiring covalent fluorescence labeling. This method was used to follow the kinetics of seed-dependent β2-m fibril extension, revealing the unidirectional extension. Since ThT binding is common to amyloid fibrils, this method will have general applicability as confirmed with the Alzheimer's amyloid β-peptide.

β2-微球蛋白（β2-m）相关性淀粉样变性是长期血液透析患者的严重并发症。为了解β2-m形成淀粉样纤维的机制，我们对重组人β2-m的构象和淀粉样纤维的形成进行了研究。1.建立了一种利用酰胺质子H/D交换结合NMR分析表征β2-m淀粉样纤维构象柔性的新方法。结果表明，在分子的中间区域中的大多数残基，包括天然结构中的环区域，形成刚性β-折叠核心，而N-和C-末端不是该核心的一部分。交换时间曲线偏离单指数曲线，与纤维的超分子结构一致。2.纤维生长的实时监测是阐明纤维形成机制的必要条件。硫磺素T（ThT）是已知在与淀粉样蛋白原纤维结合时变得强烈荧光的试剂。我们发现，通过全内反射荧光显微镜监测ThT荧光，β2-m的淀粉样纤维可以可视化，而不需要共价荧光标记。该方法用于跟踪种子依赖性β2-m原纤维延伸的动力学，揭示了单向延伸。由于ThT结合对于淀粉样蛋白原纤维是常见的，因此该方法将具有普遍适用性，如用阿尔茨海默氏淀粉样蛋白β-肽所证实的。

项目成果

Fernandez, Ariel: "Protein folding : could hydrophobic collapse be coupled with hydrogen-bond formation?"FEBS Letters. 536. 187-192 (2003)

Fernandez, Ariel：“蛋白质折叠：疏水性塌陷能否与氢键形成相结合？”FEBS Letters。

Katou, Hidenori: "The role of disulfide bond in the amyloidogenic state of β2-microglobulin studied by heteronuclear NMR."Protein Sci.. 11. 2218-2229 (2002)

Katou, Hidenori：“通过异核 NMR 研究二硫键在 β2-微球蛋白淀粉样蛋白形成状态中的作用。”Protein Sci.. 11. 2218-2229 (2002)

Szewczuk, Z.: "A two-process model describes the hydrogen exchange behavior of molten globule of cytochrome c with various extents of acetylation"Biochemistry. 40(32). 9623-9630 (2001)

Szewczuk, Z.：“双过程模型描述了具有不同乙酰化程度的细胞色素 c 熔球的氢交换行为”生物化学。

Gennady Kozhukh: "Investigation of a peptide responsible for amyloid fibril formation of β2-microglobulin by Acromobacter protease I."J. Biol. Chem.. 277(2). 1310-1315 (2002)

Gennady Kozhukh：“通过 Acromobacter 蛋白酶 I 来研究负责形成 β2-微球蛋白的淀粉样原纤维的肽。J. Biol. 1310-1315 (2002)。

Hong, Dong-P.: "Conformation of β2-microglobulin amyloid fibrils analyzed by reduction of the disulfide bond."J.Biol.Chem.. 277. 21554-21560 (2002)

Hong, Dong-P.：“通过二硫键还原分析 β2-微球蛋白淀粉样原纤维的构象。J.Biol.Chem.. 277. 21554-21560 (2002)