Genetically Encoded Probes of Huntingtin Misfolding

亨廷顿蛋白错误折叠的基因编码探针

基本信息

批准号：
10522868
负责人：
Jeannie Chen
金额：
$ 66.97万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10522868
关键词：
Address Affect Affinity Alzheimer&apos s Disease Animal Model Animals Antibodies Binding Binding Sites Biochemical Biological Biological Markers Brain Cell Culture Techniques Cell model Cell physiology Cells Cellular Structures Cultured Cells Deposition Directed Molecular Evolution Disease Disease Progression Disease model Electron Spin Resonance Spectroscopy Engineering Fluorescence Fluorescence Microscopy Foundations Generations Goals Huntington Disease Huntington gene Huntington protein In Vitro Interphase Cell Lead Ligands Liquid substance Messenger RNA Methods Molecular N-terminal Neurons Oranges Parkinson Disease Pathway interactions Patients Peptides Play Process Proline-Rich Domain Proteins Role Seeds Site Specificity Testing Therapeutic Time Tissues Toxic effect Work age related neurodegeneration aggregation pathway base biophysical techniques design detection limit experimental study inhibitor insight interest monomer mouse model novel polyglutamine potential biomarker protein aggregation protein structure function specific biomarkers stoichiometry

项目摘要

Abstract Huntington’s disease is caused by polyglutamine expansions in the huntingtin protein. These polyQ expansions make the huntingtin protein, and its naturally occurring exon1 fragment (Httex1), more aggregation prone. Deposition of fibrillar Httex1 aggregates in the brain is a hallmark of the disease in patients and animal models. We have shown that Httex1 aggregation is a step-wise process wherein Httex1 gives rise to different misfolded species prior to formation of fibrils. Early species in the misfolding pathway are of particular interest as they can cause the formation of seeds, which cause further misfolding of monomeric Httex1. This process not only enhances toxicity in a given cell, but it can also cause spreading of misfolding throughout the brain. In addition, earlier misfolding intermediates could also directly contribute to disease, as their toxicity has been observed in cell culture experiments. Despite their importance, early misfolding intermediates cannot easily be detected in biological tissues and it has not been possible to interfere with their seeding ability or toxicity. In this project, we aim to address these fundamental problems by developing genetically encoded ligands (peptides) that bind early misfolding intermediates and by testing their potential biomarker or therapeutic utility. To accomplish these goals, we have assembled a team of three PIs with expertise in peptide ligand discovery (Roberts), huntingtin protein structure and function (Langen), and cell-based and animal-based disease models (Chen). The Langen lab has laid the biochemical foundation for the proposal by identifying and characterizing different forms of Httex1 aggregates. Working together, the Roberts lab has used directed evolution and mRNA display to generate Httex1 directed (HD) peptide ligands against protofibrils. The Langen and Chen lab have demonstrated that HD peptides inhibit Httex1 misfolding in vitro and in cultured cells. Importantly, HD peptides also protect from Httex1 toxicity in cultured cells. In Aim 1, we propose to extend this work by characterizing the interactions of HD peptides with protofibrils using biophysical methods. Specifically, we will determine the HD peptide’s affinity, specificity, molecular mechanism of interaction with protofibrils and we will evaluate their ability to inhibit misfolding. Moreover, we will use peptide multimerization and other optimizations to achieve ultra-high affinity binding. Aim 2 then uses these well characterized binders in animal and cell models to evaluate their utility as biomarkers and therapeutics in cell cultures and animal models. In aim 3, we will generate binders for the earliest misfolding intermediate, the a-helical oligomers, and test our prediction that binders to these species block the formation of seeds and protect from Httex1 misfolding and toxicity.

抽象的亨廷顿氏病是由亨廷顿蛋白中的聚谷氨酰胺扩展引起的。这些polyq 扩展使亨廷顿蛋白及其天然发生的外en1片段（HTTEX1），更多的聚集易于。纤维纤维HTTEX1在大脑中的沉积是患者和动物的疾病的标志型号。我们已经表明，HTTEX1聚集是一个逐步的过程，其中HTTEX1产生不同在形成原纤维之前，物种错误折叠。错误折叠途径中的早期物种特别感兴趣因为它们会导致种子的形成，这会导致单体HTTEX1的进一步错误折叠。这个过程不是只能增强给定细胞中的毒性，但也会导致整个大脑中折叠式折叠率的扩散。在此外，较早的错误折叠中间体也可能直接导致疾病，因为它们的毒性一直是在细胞培养实验中观察到。尽管它们的重要性，但早期错误折叠的中间体无法轻易在生物组织中检测到，不可能干扰其播种能力或毒性。在这个项目，我们旨在通过开发一般编码的配体（肽）来解决这些基本问题结合早期错误折叠的中间体，并通过测试其潜在的生物标志物或治疗效用。为了实现这些目标，我们组建了一支由三个PI的团队，在胡椒配体发现方面具有专业知识（罗伯茨），亨廷顿蛋白蛋白结构和功能（Langen）以及基于细胞和基于动物的疾病模型（陈）。 Langen Lab通过识别和表征该提案的生化基础不同形式的HTTEX1聚集体。罗伯特实验室一起使用了定向进化和mRNA 显示以生成针对原纤维的HTTEX1（HD）胡椒配体。 Langen和Chen Lab有证明HD肽在体外和培养细胞中抑制了HTTEX1不折叠。重要的是，高清肽还可以保护培养细胞中的HTTEX1毒性。在AIM 1中，我们建议通过表征来扩展这项工作使用生物物理方法的HD肽与原纤维的相互作用。具体来说，我们将确定高清肽的亲和力，特异性，与原纤维相互作用的分子机制，我们将评估它们的能力抑制错误折叠。此外，我们将使用辣椒多层化和其他优化来实现超高亲和力结合。 AIM 2然后在动物和细胞模型中使用这些良好特征的粘合剂来评估其用作细胞培养物和动物模型中的生物标志物和治疗。在AIM 3中，我们将生成绑定器最早的错误折叠中间体，A螺旋寡聚物，并测试我们对这些规格的粘合剂的预测阻止种子的形成，并防止HTTEX1错误折叠和毒性。