Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3010, Australia.
Biochemistry. 2011 May 17;50(19):4046-57. doi: 10.1021/bi2002482. Epub 2011 Apr 22.
The misfolding and self-assembly of proteins into amyloid fibrils that occur in several debilitating diseases are affected by a variety of environmental factors, including mechanical factors associated with shear flow. We examined the effects of shear flow on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Shear fields (150, 300, and 500 s(-1)) accelerated the rate of apoC-II fibril formation (1 mg/mL) approximately 5-10-fold. Fibrils produced at shear rates of 150 and 300 s(-1) were similar to the twisted ribbon fibrils formed in the absence of shear, while at 500 s(-1), tangled ropelike structures were observed. The mechanism of the shear-induced acceleration of amyloid fibril formation was investigated at low apoC-II concentrations (50 μg/mL) where fibril formation does not occur. Circular dichroism and tryptophan fluorescence indicated that shear induced an irreversible change in apoC-II secondary structure. Fluorescence resonance energy transfer experiments using the single tryptophan residue in apoC-II as the donor and covalently attached acceptors showed that shear flow increased the distance between the donor and acceptor molecules. Shear-induced higher-order oligomeric species were identified by sedimentation velocity experiments using fluorescence detection, while fibril seeding experiments showed that species formed during shear flow are on the fibril formation pathway. These studies suggest that physiological shear flow conditions and conditions experienced during protein manufacturing can exert significant effects on protein conformation, leading to protein misfolding, aggregation, and amyloid fibril formation.
在几种使人衰弱的疾病中，蛋白质错误折叠并自组装成淀粉样纤维，这一过程受到多种环境因素的影响，包括与切变流相关的机械因素。我们研究了切变流对人载脂蛋白 C-II（apoC-II）形成淀粉样纤维的影响。切变场（150、300 和 500 s(-1)) 使 apoC-II 纤维形成（1mg/mL）的速率加快了约 5-10 倍。在 150 和 300 s(-1) 的剪切速率下产生的纤维与无剪切时形成的扭曲带状纤维相似，而在 500 s(-1) 时，观察到纠结的绳状结构。在 apoC-II 浓度较低（50μg/mL）的情况下，研究了剪切诱导淀粉样纤维形成加速的机制，在这种情况下，纤维形成不会发生。圆二色性和色氨酸荧光表明，剪切诱导 apoC-II 二级结构发生不可逆变化。使用 apoC-II 中的单个色氨酸残基作为供体和共价连接的受体的荧光共振能量转移实验表明，切变流增加了供体和受体分子之间的距离。使用荧光检测的沉降速度实验鉴定了剪切诱导的更高阶寡聚体物种，而纤维接种实验表明，在剪切流中形成的物种处于纤维形成途径上。这些研究表明，生理切变流条件和蛋白质制造过程中经历的条件可以对蛋白质构象产生显著影响，导致蛋白质错误折叠、聚集和淀粉样纤维形成。
