PDF(Pubmed)
Abstract：
Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Homozygosity of the I40V LECT2 mutation is believed to be necessary but not sufficient for the disease. Previous studies established that LECT2 fibrillogenesis is greatly accelerated by loss of its single bound zinc ion and stirring or shaking. These forms of agitation are often used to facilitate protein aggregation, but they 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 artery and capillary-sized channels-drives LECT2 fibrillogenesis. To mimic blood flow through the human 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. Flow shear was particularly pronounced at the branch points and in the smallest capillaries, and this induced LECT2 aggregation much more efficiently than conventional shaking methods. EM images suggested the resulting fibril structures were different in the two conditions. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both size and 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 the 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。认为I40VLECT2突变的纯合性对于该疾病是必需的,但不足以。先前的研究确定LECT2原纤维形成通过其单一结合的锌离子的损失和搅拌或摇动而大大加速。这些形式的搅拌通常用于促进蛋白质聚集，但是它们会产生非均质的剪切条件，包括使蛋白质变性的气液界面，不存在于体内。这里,我们确定了通过动脉和毛细血管大小的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.为了模拟人体肾脏的血流，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。在分支点和最小的毛细管中，流动剪切特别明显。这诱导LECT2聚集比传统的摇动方法有效得多。EM图像表明所得原纤维结构在两种条件下是不同的。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。
