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

描述（由申请人提供）：亨廷顿病（HD）是一种遗传性神经退行性疾病，在美国影响超过10万人的生活。亨廷顿病（HD）慢性症状和病理的复杂性长期困扰着研究人员，阻碍了治疗干预的进展。然而，HD的主要原因是遗传上的简单;扩展的HD CAG重复序列编码亨廷顿蛋白中扩展的polyQ区域。因此，了解亨廷顿蛋白的结构和功能，因为它与疾病有关，将可能阐明HD病理学的基本来源，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，这项资助的真正目的是确定polyQ扩增对HD全长亨廷顿蛋白的影响， 产生新的靶点并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。目标1将通过使用各种生化测定和高分辨率结构研究（电子显微镜，原子力显微镜和晶体学）来定义polyQ扩增对全长亨廷顿蛋白的结构和功能的影响。目的2将使用具有不同polyQ长度的纯化的全长亨廷顿蛋白系统地鉴定突变亨廷顿蛋白的改变的磷酸化修饰，因为突变亨廷顿蛋白的磷酸化与HD发病机制有关。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亨廷顿蛋白的特异性同种型。目标3将鉴定修饰polyQ区域对亨廷顿蛋白结构和功能的影响的适体。核酸适体与靶蛋白结合特异性强、亲和力强，能引起靶蛋白结构和功能的改变，与治疗性分子相似，是高通量药物筛选的通用试剂。我们的结果将提供对全长亨廷顿蛋白作为主要疾病原因的结构和功能性质的透彻理解，并导致鉴定将在两种小鼠模型（CAG敲入和YAC 128）和从来自HD患者成纤维细胞的人iPS分化的人神经元细胞中验证的治疗性分子。这些也将使得能够合理设计旨在在神经元细胞开始屈服于其累积效应之前干扰HD疾病过程的治疗剂。

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.