Effects of PolyQ Expansion on Full-length Huntingtin Protein in HD

描述(申请人提供)：亨廷顿病(HD)是一种遗传性神经退行性疾病，影响美国超过10万人的生活。亨廷顿病(HD)慢性症状和病理的复杂性长期以来一直困扰着研究人员，并阻碍了治疗干预的进展。然而，HD的主要原因在遗传上很简单；扩展的HD CAG重复编码Huntingtin蛋白中的一个扩展的PolyQ区域。因此，了解亨廷顿蛋白的结构和功能与疾病有关，可能会阐明HD病理的基本来源，并对开发治疗方法至关重要。由于我们已经开发了一系列全长重组人亨廷顿蛋白，作为结构功能研究的资源，这项拨款的真正目的是确定多聚Q扩展对HD全长亨廷顿蛋白的影响，并 产生新的靶点，发现直接与亨廷顿蛋白结合并改变其功能活性的治疗分子。目的1通过各种生化分析和高分辨结构研究(电子显微镜、原子力显微镜和结晶学)，确定PolyQ扩展对亨廷顿蛋白全长结构和功能的影响。目的2利用纯化的具有不同多聚Q长度的全长亨廷顿蛋白，系统地鉴定突变的亨廷顿蛋白的磷酸化修饰，因为突变的亨廷顿蛋白的磷酸化与HD的发病有关。我们将生成并验证磷酸化抗体试剂，并使用它们来鉴定与HD发病机制密切相关的磷酸化全长Huntingtin的特定亚型。目标3将确定修改多聚Q区域对Huntingtin结构和功能的影响的适配子。适体具有较高的特异性和较强的S亲和力，能与靶蛋白结合，并能引起靶蛋白与治疗分子相似的结构和功能变化，因此将被用作高通量药物筛选的通用试剂。我们的结果将使我们深入了解作为主要致病原因的全长Huntingtin的结构和功能特性，并导致识别治疗分子，这些分子将在两个小鼠模型(CAG Knoke-in&YAC128)和从HD患者成纤维细胞分化的人iPS细胞中得到验证。这些也将使旨在干预HD疾病过程的治疗方法能够在神经细胞开始屈服于其累积效应之前进行合理设计。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an inherited neurodegenerative disorder that affects the lives of more than 100,000 people in the US. The complexity of the chronic symptoms and pathology of Huntington's disease (HD) has long puzzled researchers and prevented the progress of therapeutic intervention. However, the primary cause of HD is genetically simple; expanded HD CAG repeats that encode an expanded polyQ region in the huntingtin protein. Thus, understanding the structure and function of the huntingtin protein as it relates to the disease will likely elucidate a fundamental source o HD pathology and be crucial to developing therapies. Since we have developed a series of full-length recombinant human huntingtin proteins, as a resource for structure-function studies, this grant really aims to identify the effects of polyQ expansion on full-length huntingtin in HD and to generate novel targets and discover therapeutic molecules that directly bind to the huntingtin protein and modify its functional activities. Aim 1 will define the impacts of polyQ expansion on the structure and function of the full-length huntingtin protein by using various biochemical assays and high resolution structural studies (electron microscopy, atomic force microscopy and crystallography). Aim 2 will systematically identify altered phosphorylation modifications of mutant huntingtin using the purified full-length huntingtins with different polyQ lengths because phosphorylation of mutant huntingtin has been implicated in HD pathogenesis. We will generate and validate the phospho-antibody reagents and use them to identify the specific isoforms of the phosphorylated full-length huntingtins strongly related to HD pathogenesis. Aim 3 will identify aptamers that modify the impact of the polyQ region on huntingtin structure and function. Aptamers will be used as versatile reagents for high-throughput drug screening because they bind to target proteins with a high specificity and s strong affinity and introduce structural and functional changes of target proteins as similar as by therapeutic molecules. Our results will provide a thorough understanding of the structural and functional properties of full- length huntingtin as a primal disease-cause and lead to identify therapeutic molecules which will be validated in two mice models (CAG knock-in & YAC128) and human neuronal cells differentiated from human iPS from HD patient fibroblast. These will also enable rational design of therapeutics aimed at interfering with the HD disease process before neuronal cells begin to succumb to its cumulative effects.