PDF(Pubmed)
Abstract：
Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Previous studies established that LECT2 fibrillogenesis is accelerated by the loss of its bound zinc ion and stirring/shaking. These forms of agitation create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress-shear generated by fluid flow through a network of narrow channels-drives LECT2 fibrillogenesis. To mimic blood flow through the kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 μm in width. Shear was particularly pronounced at the branch points and in the smallest capillaries. Aggregation was induced within 24 h by shear levels that were in the physiological range and well below those required to unfold globular proteins such as LECT2. EM images suggested the resulting fibril ultrastructures were different when generated by laminar flow shear versus shaking/stirring. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both the size and the density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.
摘要：
白细胞来源的趋化素2（LECT2）的聚集导致ALECT2，一种影响肾脏和肝脏的全身性淀粉样变性。先前的研究确定LECT2原纤维形成通过其结合的锌离子的损失和搅拌/摇动而加速。这些形式的搅拌产生非均相剪切条件，包括使蛋白质变性的气液界面，不存在于体内。这里,我们确定了通过狭窄通道网络的流体流动产生的更生理形式的机械应力剪切驱动LECT2原纤维形成的程度。为了模拟血液通过肾脏的流动，LECT2和其他蛋白质形成淀粉样沉积物，我们开发了一种微流体装置，该装置由宽度从5毫米缩小到20μm的逐渐分支的通道组成。剪切在分支点和最小的毛细血管中特别明显。在24小时内通过剪切水平诱导聚集,所述剪切水平在生理范围内并且远低于使球状蛋白如LECT2展开所需的水平。EM图像表明，层流剪切产生的原纤维超微结构与摇动/搅拌。重要的是,来自微流体装置的结果显示I40V突变加速原纤维形成并增加聚集体的大小和密度的第一个证据。这些发现表明，肾样血流剪切，结合锌损失，与I40V突变联合作用以触发LECT2淀粉样蛋白生成。这些微流体装置通常可用于揭示血流诱导循环蛋白质的错误折叠和淀粉样变性的机制。
