Molecular mechanisms of huntingtin misfolding

 描述（由申请人提供）：亨廷顿病（HD）是所有多聚谷氨酰胺（polyQ）疾病中最常见的一种，其特征在于亨廷顿蛋白（htt）的错误折叠和聚集。对HD的病因学特别重要的是N-末端htt外显子1（HDx 1）区域，随着其polyQ束长度的增加，其逐渐变得更倾向于聚集和错误折叠。谷氨酰胺数量增加（通常>40）会导致各种细胞毒性淀粉样结构的形成，包括原纤维、原纤维和较小的寡聚结构（图1）。细胞和动物研究表明，抑制错误折叠是预防HD的一个有前途的途径。然而，缺乏这些物种的结构信息，阻碍了对HDx 1错误折叠机制的清晰理解，并阻碍了调节这种错误折叠过程作为治疗方法的努力。这个提议利用了Langen小组最近在生物化学和方法学方面取得的两项进展。首先，有可能产生含有46 Q的各种错误折叠形式的HDx 1的清洁制剂。这些包括两种不同的原纤维类型，一种是有毒的，一种是只有弱毒性的，以及高毒性HDx 1的原纤维形式。其次，与Siemer和Chiu教授一起，Langen小组已经开始应用一种强大的结构方法的协同组合，包括定点自旋标记（SDSL）与EPR，固态NMR（ssNMR）和cryo-EM一起研究HDx 1错误折叠。初步结果揭示了这种新方法令人兴奋的潜力。第一个目的是使用SDSL，ssNMR和cryo-EM比较和对比毒性和弱毒性HDx 1纤维的结构。这种比较将提供对区分HDx 1的毒性和无毒形式的结构特征的见解。这些信息很可能有助于今后防止有毒物种形成的努力。在具体目标2中，我们将研究高度toxc的结构，A11阳性的原纤维，在错误折叠过程的早期形成。这种早期错误折叠中间体被认为是许多淀粉样疾病中的主要毒性病原体，但其结构仍然知之甚少。具体目标3跟进的初步数据表明，带负电荷的膜，特别是那些模仿线粒体膜，有力地加速错误折叠，并促进有毒的A11阳性结构的形成。这个目标将研究膜如何加速HDx 1错误折叠，以及这种相互作用如何反过来破坏膜的完整性。拟议的研究可能解释Hdx 1与细胞膜的相互作用如何促进毒性，以及为什么HD患者的线粒体功能受到如此破坏。在位置13和16处的磷酸化已经显示出保护免受毒性。为了了解这种修饰如何具有保护作用，我们将测试它如何影响原纤维、有毒原纤维和膜介导的错误折叠的形成。

 DESCRIPTION (provided by applicant): Huntington's disease (HD), the most common of all polyglutamine (polyQ) diseases, is characterized by the misfolding and aggregation of huntingtin (htt). Of particular importance to the etiology of HD is the N- terminal htt exon 1 (HDx1) region, which becomes progressively more prone to aggregation and misfolding as the length of its polyQ tract increases. Elevated numbers of glutamines (typically >40) cause the formation of various cytotoxic amyloid structures including fibrils, protofibrils and smaller oligomeric structures (Fig. 1). Cell and animal studies suggest that inhibition of misfolding is a promising avenue for preventing HD. However, the lack of structural information on these species has prevented a clear understanding of the mechanisms of HDx1 misfolding and hampered efforts to modulate this misfolding process as an avenue for therapeutic treatment. This proposal exploits two recent biochemical and methodological advances made by the Langen group. First, it became possible to generate clean preparations of various misfolded forms of HDx1 containing 46Q. These include two different fibril types, one that is toxic and one that is only weakly toxic, as well as a protofibrillar form of highly toxic HDx1. Second, together with Professors Siemer and Chiu, the Langen group has begun to apply a powerful and synergistic combination of structural methods that include site-directed spin labeling (SDSL) together with EPR, solid state NMR (ssNMR) and cryo-EM for studying HDx1 misfolding. The preliminary results have revealed exciting potential for this novel approach. The first aim seeks to compare and contrast the structures of toxic and weakly toxic HDx1 fibrils using SDSL, ssNMR and cryo-EM. This comparison will provide insights into the structural features that distinguish toxic from non-toxic forms of HDx1. Such information is likely to facilitate future efforts aimed at preventin the formation of toxic species. In Specific Aim 2, we will investigate the structures of highly toxc, A11 positive protofibrils, which form early during the misfolding process. Such early misfolding intermediates have been suggested to be the primary toxic pathogens in many amyloid diseases, but their structures remain poorly understood. Specific Aim 3 follows up on preliminary data that show that negatively charged membranes, especially those mimicking mitochondrial membranes, potently accelerate misfolding and promote the formation of toxic, A11 positive structures. This aim will study how membranes accelerate HDx1 misfolding and how this interaction, in turn, may disrupt membrane integrity. The proposed studies might explain how interaction of Hdx1 with cellular membranes can promote toxicity and why mitochondrial function is so disrupted in HD patients. Phosphorylation at positions 13 and 16 has been shown to protect from toxicity. In order to understand how this modification might be protective, we will test how it affects the formation of fibrils, toxic protofibrils and membrane-mediated misfolding.