Genetically Encoded Probes of Huntingtin Misfolding

摘要 亨廷顿病是由亨廷顿蛋白中的聚谷氨酰胺膨胀引起的。这些多面体 扩张使亨廷顿蛋白及其自然产生的外显子1片段(Httex 1)更具聚集性 俯卧着。在患者和动物中，脑内纤维状Httex 1聚集体的沉积是该病的一个特征 模特们。我们已经证明了Httex 1的聚集是一个逐步的过程，其中Httex 1产生了不同的 在纤维形成之前的错误折叠的物种。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。此过程不会 它只会增加特定细胞的毒性，但也会导致错误折叠在整个大脑中扩散。在……里面 此外，早期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 在细胞培养实验中观察到。尽管它们很重要，但早期的错误折叠中间体不容易 在生物组织中检测到，而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)来解决这些根本问题。 这结合了早期错误折叠的中间体，并测试了它们潜在的生物标记物或治疗效用。 为了实现这些目标，我们组建了一个由三名具有多肽配体发现专业知识的PI组成的团队 (Roberts)，Huntingtin Protein Structure and Function(兰根)，以及基于细胞和动物的疾病模型 (陈)。兰根实验室已经通过鉴定和表征为这项提议奠定了生化基础 不同形式的Httex 1聚合体。在共同努力下，罗伯茨实验室使用了定向进化和信使核糖核酸 展示以产生针对原纤维的Httex 1定向(HD)肽配体。兰根和陈的实验室 证明HD多肽在体外和培养细胞中抑制Httex 1的错误折叠。重要的是，HD多肽 还可以保护培养细胞免受Httex-1的毒性。在目标1中，我们建议通过描述 生物物理方法研究HD多肽与原纤维的相互作用。具体来说，我们将确定房屋署 多肽的亲和力、特异性、与原纤维相互作用的分子机制以及我们将对其能力进行评估 以抑制错误折叠。此外，我们还将使用多肽多聚体等优化方法来实现超高 亲和力结合。然后，Aim 2在动物和细胞模型中使用这些具有良好特性的结合剂来评估它们的 在细胞培养和动物模型中作为生物标志物和治疗学的用途。在目标3中，我们将为以下内容生成活页夹 最早的错误折叠中间体，a-螺旋低聚物，并测试我们的预测，即结合到这些物种 阻止种子形成，防止Httex 1错误折叠和毒性。

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.