Molecular mechanisms of huntingtin misfolding

 描述(申请人提供)：亨廷顿病(HD)是所有多聚谷氨酰胺(PolyQ)疾病中最常见的疾病，其特征是亨廷顿蛋白(HTT)的错误折叠和聚集。对于HD的病因学来说，特别重要的是N-末端HTT外显子1(HDx1)区域，随着其多Q链长度的增加，该区域逐渐变得更容易聚集和错误折叠。数量增加的谷氨酸(通常是-gt；40)会导致各种细胞毒性淀粉样蛋白结构的形成，包括纤维、原纤维和较小的寡聚体结构(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，缺乏关于这些物种的结构信息，阻碍了对HDx1错误折叠机制的清楚了解，并阻碍了将这一错误折叠过程调节为治疗途径的努力。这项建议利用了兰根小组最近取得的两项生化和方法学进展。首先，可以生产含有46Q的各种错误折叠形式的HDx1的清洁制剂。其中包括两种不同的纤维类型，一种是有毒的，另一种是弱毒性的，以及一种高毒性的原纤维形式的HDx1。其次，与Siemer和Chiu教授一起，朗根团队已经开始应用强大和协同的结构方法组合，包括位点定向自旋标记(SDSL)和EPR、固态核磁共振(ss核磁共振)和冷冻EM来研究HDx1错误折叠。初步结果表明，这一新方法具有令人兴奋的潜力。第一个目的是利用SDSL、单核磁共振和低温电子显微镜来比较和对比毒性和弱毒性的HDx1纤维的结构。这种比较将提供对区分有毒和无毒形式的HDx1的结构特征的洞察。这些信息可能有助于今后旨在预防有毒物种形成的努力。在特定的目标2中，我们将研究在错误折叠过程中早期形成的高毒性的A11阳性原纤维的结构。这种早期错误折叠的中间产物被认为是许多淀粉样蛋白疾病的主要毒性病原体，但它们的结构仍然知之甚少。具体目标3跟进初步数据，这些数据表明，带负电的膜，特别是那些模仿线粒体膜的膜，有效地加速了错误折叠，并促进了有毒的A11正性结构的形成。这个目标将研究膜如何加速HDx1的错误折叠，以及这种相互作用如何反过来可能破坏膜的完整性。拟议的研究可能解释Hdx1与细胞膜的相互作用如何促进毒性，以及为什么HD患者的线粒体功能如此紊乱。第13位和第16位的磷酸化已被证明可以保护其免受毒性。为了了解这种修饰如何起到保护作用，我们将测试它如何影响纤维、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.