Kinetic and thermodynamic analysis of the molecular interaction on amyloid fibril formation

淀粉样蛋白是不溶于天然构象的蛋白质在细胞外的沉积，导致组织结构和功能的损害。我们已经在体外建立了几个淀粉样纤维形成系统，包括阿尔茨海默病的β-淀粉样蛋白(Aβ)和透析相关的淀粉样变性的β2-微球蛋白(β2-m)淀粉样蛋白(Aβ2m)。利用这些系统，各种生物分子或化合物在淀粉样纤维的形成和解离中的重要作用被发现。为了通过体外实验系统或数值模型模拟体内淀粉样原纤维的形成，我们选择了纤维形成或解离的几个阶段，并通过生物物理、动力学和热力学分析阐明了它们的机制。为了了解β2m淀粉样变性的分子发病机制，通过影响β2-m和淀粉样纤维的构象和稳定性而诱导淀粉样原纤维形成的生物分子需要是…更具体化了。我们发现，包括非酯化脂肪酸(NEFAs)和一些溶血磷脂在内的阴离子两亲性化合物在中性pH下诱导纤维伸展。此外，血液透析患者的血浆溶血磷脂浓度显著高于健康受试者。这些结果提示溶血磷脂和NEFAs可能在Aβ2M淀粉样变性的发生发展中起作用。采用分光光度法、荧光分光光度法和表面等离子体共振法，研究了5种黄酮类化合物对β-淀粉样原纤维(FAβ)的体外抗淀粉样变性作用。这些结果表明，黄酮类化合物，尤其是杨梅素，在体外通过优先和可逆地与FAβ的淀粉样原纤维结构结合而不是与Aβ单体结合而发挥抗淀粉样变的作用。我们建立了利用硫代黄素T荧光光谱高通量筛选Aβ2M和Aβ淀粉样原纤维成核的系统。利用该体系考察了蛋白多糖、糖胺多糖等多种生物分子的作用效果。作为本研究的结论，在阐明Aβ2M和Aβ淀粉样原纤维形成机制方面取得了显著进展。较少

Amyloid is extracellular deposit of insoluble fibrillar aggregate of proteins those are normally soluble in their native conformation, resulting in the impairment of tissue structure and function. We have established several amyloid fibril formation systems in vitro, including β-amyloid of Alzheimer's disease (Aβ) and β2-microglobulin (β2-m) amyloid (Aβ2M) of dialysis-related amyloidosis. Using these systems, important roles of various biological molecules or compounds on the formation and dissociation of amyloid fibrils were discovered. To model the amyloid fibril formation in vivo by in vitro experimental system or numerical model, we selected several phases of fibril formation or dissociation, and clarified their mechanism by biophysical, kinetic and thermodynamic analyses.1. To understand the molecular pathogenesis of Aβ2M amyloidosis, the biological molecules that induce amyloid fibril formation by affecting the conformation and stability of β2-m and amyloid fibrils need to be ide … More ntified. We showed that anionic amphipathic compounds including non-esterified fatty acids (NEFAs) and some lysophospholipids induce the fibril extension at a neutral pH. Furthermore, hemodialysis patients had significantly higher plasma concentrations of lysophospholipids than healthy subjects. These results suggest possible role of lysophospholipids and NEFAs in the development of Aβ2M amyloidosis.2. Using spectrophotometry, spectrofluorometry and surface plasmon resonance, we investigated the anti-amyloidogenic effects of five flavonoids on β-amyloid fibril (fAβ) in vitro. The results suggest that flavonoids, especially myricetin exert an anti-amyloidogenic effect in vitro by preferentially and reversibly binding to the amyloid fibril structure of fAβ, rather than to Aβ monomers.3. We developed a high-throughput screening system for the nucleation of Aβ2M and Aβ amyloid fibril using thioflavin T fluorescence spectroscopy. Using this system, effect of various biological molecules including proteoglycans, glycosaminoglycans, were investigated. As the conclusion of this study, the remarkable advance in the clarification of the mechanism of Aβ2M and Aβ amyloid fibril formation were obtained. Less