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.

摘要修订 白细胞衍生的趋化趋化蛋白-2（LECT2）淀粉样变性（ALECT2）是美国第三大常见的肾脏淀粉样变性类型。 ALECT2是由于将Lect2蛋白折叠到淀粉样蛋白原纤维中的原因是，该蛋白会积聚在肾脏中并导致其衰竭。据信ALECT2是由ILE40→Val（I40V）突变引起的，与第二个统一条件结合使用。初步数据表明，LECT2的单一结合锌离子的损失与剧烈搅拌相结合，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：拟议的微流体剪切装置（旨在模仿血液流过肾脏的脉管系统）是否可以诱导淀粉样蛋白的形成，以及lect2原纤维和固体寡聚物的结构是否可以由冷冻剂确定。 AIM 1将测试已设计的两种新的微流体芯片设计，这些设计是为了最大程度地提高剪切和蛋白质聚集的，同时概括了肾脏视频的分支和平行结构。原型芯片已被制造，并将使用LECT2样品在测试ALECT2的假设“第二次打击”条件的条件下对其进行评估（锌的损失，二硫键的部分降低）。在流动条件下，将通过显微镜监测纤维化，并将从芯片中回收聚集的蛋白质并进行阴性染色EM分析。 AIM 2将寻求获得WT和I40V Lect2原纤维以及实心低聚物的高质量冷冻图像。这些物种最初将通过常规搅拌产生，后来由AIM 1中的微流体设备产生。由于现有的纤维化方案倾向于产生较大的簇，并将纯化的固体寡聚物纯化为单分散群体，因此将努力努力获得单个纤维。总之，这些目标将填补将流剪切和I40V突变与ALECT2的发展联系起来的假设中的临界空白。