Mechanism and detection of LECT2 amyloidosis

LECT2淀粉样变性的机制及检测

• 批准号: 10475334
• 负责人: STEWART N LOH
• 金额: $ 10万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-25 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475334
• 关键词: Address; Agitation; Agreement; Air; Amino Acids; Amyloid; Amyloid Fibrils; Amyloidosis; Antibodies; Biochemical; Blood; Blood flow; Caliber; Cells; Chemotactic Factors; Communities; Cryoelectron Microscopy; Data; Detection; Development; Device Designs; Diagnosis; Engineering; Failure; Fluorescence Microscopy; Hepatocyte; Human; Image; Individual; Interlobular Artery; Ions; Kidney; Kinetics; Lead; Leukocytes; Link; Liquid substance; Liver; Mechanics; Microfluidic Microchips; Microfluidics; Microscopy; Molecular; Monitor; Morphology; Mutation; Negative Staining; Organ failure; Outcome; Pathogenicity; Patients; Polymorph; Population; Proteins; Protocols documentation; Recombinants; Reporting; Research; Resolution; Sampling; Stress; Structure; Testing; Time; United States; Zinc; amyloid formation; arteriole; design; disulfide bond; disulfide bond reduction; drug discovery; experience; insight; lithography; protein aggregation; protein misfolding; prototype; renal artery; stressor; tool

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.

摘要修改 白细胞源性趋化因子-2 (LECT2) 淀粉样变性 (ALECT2) 是美国第三种最常见的肾脏淀粉样变性类型，ALECT2 是由于 LECT2 蛋白错误折叠成淀粉样原纤维而导致的，淀粉样原纤维在肾脏中积聚并导致肾脏衰竭。 ALECT2 被认为是由 LECT2 中的 Ile40→Val (I40V) 突变与第二种尚未鉴定的突变结合引起的。初步数据表明，LECT2 的单结合锌离子的损失，加上剧烈搅拌，足以导致蛋白质自行形成淀粉样蛋白。该项目将解决两个主要问题：所提出的微流体剪切装置（设计用于）。模拟流经肾脏脉管系统的血流）可以诱导淀粉样蛋白形成，并且是否可以通过冷冻电镜确定 LECT2 原纤维和可溶性寡聚体的结构。 目标 1 将测试两种新的微流控芯片设计，这些设计旨在最大限度地提高流动剪切和蛋白质聚集，同时重现已制造的肾血管的分支和平行结构，并且将使用 LECT2 样本在测试条件下对其进行评估。检测到 ALECT2 的“二次打击”状况（锌损失、二硫键部分还原），将在流动条件下通过显微镜监测原纤维化，并回收聚集的蛋白质。目标 2 将寻求获得 WT 和 I40V LECT2 原纤维以及可溶性低聚物的高质量冷冻电镜图像，这些物质最初通过常规搅拌生成，随后通过微流体生成。如果可能的话，目标 1 中的装置将致力于获得单个原纤维，因为现有的原纤维化方案往往会产生大的团块，并致力于纯化可溶性低聚物，以产生大的团块。这些目标共同将填补将流剪切和 I40V 突变与 ALECT2 发展联系起来的假设的关键空白。