Genetically Encoded Probes of Huntingtin Misfolding

亨廷顿蛋白错误折叠的基因编码探针

• 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
• 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' 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

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.

摘要 亨廷顿氏病是由亨廷顿蛋白中的多聚谷氨酰胺扩增引起的。这些polyQ 扩展使亨廷顿蛋白及其天然存在的外显子1片段（Httex 1）更加聚集 俯卧。纤维状Httex 1聚集体在脑中的沉积是患者和动物中疾病的标志 模型我们已经表明，Httex 1聚集是一个逐步的过程，其中Httex 1引起不同的 在原纤维形成之前错误折叠的种类。错误折叠途径中的早期物种特别令人感兴趣 因为它们可以导致种子的形成，从而导致单体Httex 1的进一步错误折叠。这个过程不 它只会增强特定细胞的毒性，但也会导致错误折叠在整个大脑中扩散。在 此外，早期的错误折叠中间体也可能直接导致疾病，因为它们的毒性已经被证明是有害的。 在细胞培养实验中观察到。尽管它们很重要，但早期错误折叠的中间体不能轻易地 在生物组织中检测到，并且不可能干扰它们的播种能力或毒性。在这 项目，我们的目标是通过开发基因编码的配体（肽）来解决这些基本问题。 结合早期错误折叠中间体，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，我们组建了一个由三名具有肽配体发现专业知识的PI组成的团队 （Roberts）、亨廷顿蛋白质结构和功能（Langen）以及基于细胞和基于动物的疾病模型 （陈）。兰根实验室通过识别和表征为该提案奠定了生物化学基础 不同形式的Httex 1聚集体。通过合作，罗伯茨实验室利用定向进化和mRNA 展示以产生针对原纤维的Httex 1定向（HD）肽配体。兰根和陈实验室 证明HD肽在体外和培养细胞中抑制Httex 1错误折叠。重要的是，HD肽 也可以保护培养细胞免受Httex 1毒性。在目标1中，我们建议通过表征 使用生物物理方法研究HD肽与原纤维的相互作用。具体来说，我们将确定房屋署 肽的亲和力，特异性，与原纤维相互作用的分子机制，我们将评估他们的能力 以抑制错误折叠。此外，我们将使用肽多聚化和其他优化，以实现超高 亲和结合Aim 2然后在动物和细胞模型中使用这些良好表征的结合剂来评估它们的生物学特性。 在细胞培养物和动物模型中作为生物标志物和治疗剂的用途。在aim 3中，我们将生成绑定器， 最早的错误折叠中间体，a-螺旋低聚物，并测试我们的预测，结合剂，这些物种 阻止种子的形成，并防止Httex 1错误折叠和毒性。