Mechanism and detection of LECT2 amyloidosis

摘要修订 白细胞衍生趋化蛋白-2(LECT2)淀粉样变性(ALECT2)是美国第三常见的肾脏淀粉样变性。ALECT2是由于LECT2蛋白错误折叠成淀粉样蛋白纤维而导致的，淀粉样蛋白在肾脏中积聚，导致肾脏衰竭。ALECT2被认为是由LECT2的Ile40→Val(I40V)突变和第二种尚未确定的疾病共同引起的。初步数据表明，LECT2‘S单一结合锌离子的丢失，再加上剧烈的搅拌，足以使蛋白质自行形成淀粉样蛋白。该项目将解决两个主要问题：拟议的微流控剪切设备(旨在模拟通过肾脏血管系统的血液流动)是否可以诱导淀粉样蛋白的形成，以及LECT2纤维和可溶低聚物的结构是否可以通过低温EM确定。 Aim 1将测试两种新的微流控芯片设计，这两种芯片设计的目的是在最大限度地提高血流剪切和蛋白质聚集的同时，重述肾血管的分支和平行结构。原型芯片已经制造出来，这些芯片将在测试ALECT2(锌损失，二硫键部分还原)假设的“二次命中”条件的条件下使用LECT2样本进行评估。纤维化将在流动条件下通过显微镜进行监测，聚集的蛋白质将从芯片中回收并进行负染色EM分析。目标2将寻求获得WT和I40V LECT2原纤维以及可溶低聚物的高质量低温电子显微镜图像。这些物种最初将通过常规搅拌产生，如果可能，稍后将通过目标1中的微流控装置产生。由于现有的纤化方案往往会产生大团块，因此将努力获得单个原纤维，并将可溶低聚物提纯为单分散群体。总而言之，这些目标将填补将血流切变和I40V突变与ALECT2的发生联系起来的假设中的关键空白。

Abstract revised Leukocyte cell-derived chemotaxin-2 (LECT2) amyloidosis (ALECT2) is the third most common type of kidney amyloidosis in the United States. ALECT2 results from misfolding of the LECT2 protein into amyloid fibrils, which accumulate in the kidneys and lead to their failure. ALECT2 is believed to be caused by the Ile40→Val (I40V) mutation in LECT2 in combination with a second, as-yet unidentified condition. Preliminary data demonstrated that loss of LECT2’s single bound zinc ion, in combination with vigorous stirring, was sufficient to cause the protein to form amyloid on its own. This project will address two main concerns: whether the proposed microfluidic shear devices (designed to mimic blood flow through the vasculature of the kidney) can induce amyloid formation, and whether structures of LECT2 fibrils and soluble oligomers can be determined by cryoEM. Aim 1 will test two new microfluidic chip designs that have been engineered to maximize flow shear and protein aggregation while recapitulating the branched and parallel structures of kidney vessels. Prototype chips have been fabricated, and these will be evaluated using LECT2 samples in conditions that test the hypothesized ‘second-hit’ condition for ALECT2 (loss of zinc, partial reduction of disulfide bonds). Fibrillization will be monitored by microscopy under flow conditions, and aggregated proteins will be recovered from the chips and subjected to negative stain EM analysis. Aim 2 will seek to obtain high-quality cryoEM images of WT and I40V LECT2 fibrils, as well as soluble oligomers. These species will initially be generated by conventional stirring, and later by the microfluidic devices in Aim 1 if possible. Efforts will be directed toward obtaining individual fibrils, as existing fibrillization protocols tend to produce large clumps, and on purifying soluble oligomers to a monodisperse population. Together, these aims will fill in critical gaps in the hypotheses that link flow shear and the I40V mutation to the development of ALECT2.