Insoluble Off-Pathway Aggregates as Crowding Agents during Amyloid Fibril Formation
Insoluble Off-Pathway Aggregates as Crowding Agents during Amyloid Fibril Formation
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DOI：
10.1021/acs.jpcb.7b01120
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发表时间：
2017-03-16
影响因子：
3.3
通讯作者：
Martins, Pedro M.
中科院分区：
文献类型：
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作者：
Crespo, Rosa;Villar-Alvarez, Eva;Martins, Pedro M.
用于治疗淀粉样变性和神经变性疾病的候选药物的研究经常涉及淀粉样蛋白原纤维形成的体外测量。大分子拥挤和非途径聚集（OPA）是影响淀粉样蛋白抑制剂的可扩展性及其在体内成功应用的两个重要现象。一方面，细胞环境中挤满了大分子，大大增加了淀粉样蛋白的有效（热力学）浓度。另一方面，非途径聚集体而不是淀粉样纤维越来越多地被指定为毒性的致病因子。目前的贡献表明，鸡蛋白溶菌酶（HEWL）的不溶性关闭途径聚集体是一种特殊类型的拥挤剂，不像经典的大分子拥挤，降低蛋白质的热力学浓度。说明这种效果，显示在恒定的可溶性HEWL浓度下降低不溶性聚集体的分数后恢复OPA。蛋白质消耗和硫磺素-T荧光进展曲线表明，OPA再生并不伴随着额外的淀粉样纤维的形成。扩展到考虑OPA和时间依赖性活性系数的结晶样模型能够使用描述淀粉样蛋白成核、自催化生长和非途径成核的单组三个参数来拟合多个动力学结果。拟合结果的列表特别包括聚集再生的情况和针对不同HEWL浓度测量的所有类型的进展曲线。大分子拥挤和OPA带来的定量挑战在这里找到了一个统一的反应，对通路上和通路外抑制剂的开发具有更广泛的意义。
The study of drug candidates for the treatment of amyloidosis and neurodegenerative diseases frequently involves in vitro measurements of amyloid fibril formation. Macromolecular crowding and off-pathway aggregation (OPA) are, by different reasons, two important phenomena affecting the scalability of amyloid inhibitors and their successful application in vivo. On the one hand, the cellular milieu is crowded with macromolecules that drastically increase the effective (thermodynamic) concentration of the amyloidogenic protein. On the other hand, off-pathway aggregates, rather than amyloid fibrils, are increasingly appointed as causative agents of toxicity. The present contribution reveals that insoluble off-pathway aggregates of hen egg-white lysozyme (HEWL) are a peculiar type of crowding agents that, unlike classical macromolecular crowders, decrease the thermodynamic concentration of protein. Illustrating this effect, OPA is shown to resume after lowering the fraction of insoluble aggregates at a constant soluble HEWL concentration. Protein depletion and thioflavin-T fluorescence progress curves indicate that OPA rebirth is not accompanied by additional amyloid fibril formation. The crystallization-like model extended to account for OPA and time-dependent activity coefficients is able to fit multiple kinetic results using a single set of three parameters describing amyloid nucleation, autocatalytic growth, and off-pathway nucleation. The list of fitted results notably includes the cases of aggregation rebirth and all types of progress curves measured for different HEWL concentrations. The quantitative challenges posed by macromolecular crowding and OPA find here a unified response with broader implications for the development of on- and off-pathway inhibitors.