Mechanism and detection of LECT2 amyloidosis

摘要修订 白细胞细胞源性趋化因子-2（LECT 2）淀粉样变性（ALECT 2）是美国第三种最常见的肾淀粉样变性。ALECT 2是由LECT 2蛋白错误折叠成淀粉样原纤维引起的，淀粉样原纤维在肾脏中积累并导致肾脏衰竭。ALECT 2被认为是由LECT 2中的Ile 40 →瓦尔（I40 V）突变与第二种尚未鉴定的疾病联合引起的。初步数据表明，LECT 2的单一结合锌离子的损失，结合剧烈搅拌，足以导致蛋白质自身形成淀粉样蛋白。该项目将解决两个主要问题：所提出的微流体剪切装置（旨在模拟通过肾脏血管系统的血流）是否可以诱导淀粉样蛋白形成，以及LECT 2原纤维和可溶性低聚物的结构是否可以通过cryoEM确定。 Aim 1将测试两种新的微流控芯片设计，这些设计已被设计为最大化流动剪切和蛋白质聚集，同时重现肾脏血管的分支和平行结构。原型芯片已经制造，这些将使用LECT 2样品在测试ALECT 2的假设“二次击中”条件（锌损失，二硫键部分还原）的条件下进行评估。在流动条件下通过显微镜监测纤维化，从芯片中回收聚集的蛋白质并进行负染色EM分析。目标2将寻求获得WT和I40 V LECT 2原纤维以及可溶性寡聚体的高质量cryoEM图像。这些物质最初将通过常规搅拌产生，并且如果可能的话，随后通过目标1中的微流体装置产生。由于现有的纤维化方案倾向于产生大的团块，因此将努力获得单个原纤维，并将可溶性低聚物纯化成单分散群体。总之，这些目标将填补将流动剪切和I40 V突变与ALECT 2的发展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.