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

PolyQ 扩增对 HD 中全长亨廷顿蛋白的影响

基本信息

批准号：
8659524
负责人：
Ihn Sik Seong
金额：
$ 37.27万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8659524
关键词：
Affect Affinity Age Amino Acids Antibodies Atomic Force Microscopy Binding Biochemical Biological Assay CAG repeat Cells Chronic Circular Dichroism Clinical Complex Crystallography Data Dictyostelium Dictyostelium discoideum Disease Electron Microscopy Electroporation Exhibits Fibroblasts Genes Goals Grant Human Huntington Disease In Vitro Inherited Insecta Intervention Knock-in Mouse Knowledge Lead Length Libraries Mass Spectrum Analysis Modification Molecular Probes Mus Mutation Neurodegenerative Disorders Neurons Pathogenesis Pathology Pathway interactions Patients Phospho-Specific Antibodies Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Polycomb Post-Translational Protein Processing Preclinical Drug Evaluation Process Property Protein Isoforms Proteins Reagent Recombinants Research Personnel Resolution Resources Series Site Source Specificity Structure Symptoms System Testing Therapeutic Therapeutic Intervention Tissues Trinucleotide Repeats aptamer base design gain of function histone methyltransferase human Huntingtin protein in vivo induced pluripotent stem cell liquid chromatography mass spectrometry member mouse model mutant nervous system disorder new therapeutic target novel polyglutamine polypeptide prevent small molecule therapy development

项目摘要

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.

描述（由申请人提供）：亨廷顿氏病（HD）是一种遗传神经退行性疾病，影响了美国超过100,000人的生活。亨廷顿氏病（HD）的慢性症状和病理学的复杂性长期困扰研究人员，并阻止了治疗干预的进展。但是，HD的主要原因在遗传上很简单。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，了解与该疾病有关的亨廷汀蛋白的结构和功能可能会阐明基本来源O HD病理学，并且对于开发疗法至关重要。由于我们已经开发了一系列全长重组人类亨廷汀蛋白作为结构功能研究的资源，因此该赠款确实旨在确定PolyQ扩展对HD中全长Huntingtin的影响以及产生新的靶标并发现直接与亨廷汀蛋白结合并修饰其功能活性的治疗分子。 AIM 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扩展对全长亨廷泰蛋白蛋白结构和功能的影响。 AIM 2将使用具有不同polyq长度的纯化的全长亨廷汀进行系统地识别突变体亨廷汀的磷酸化修饰，因为突变体亨廷顿蛋白的磷酸化已与HD发病机理有关。我们将生成和验证磷酸抗体试剂，并使用它们来识别与HD发病机理密切相关的磷酸化全长亨廷汀的特定同工型。 AIM 3将确定适体，以改变Polyq区域对亨廷汀结构和功能的影响。适体将用作高通量药物筛查的多功能试剂，因为它们与具有高特异性的靶蛋白结合，并且具有强度的亲和力，并引入靶蛋白的结构和功能变化与治疗分子相似。我们的结果将提供对全长亨廷汀作为原始疾病的结构和功能特性的透彻理解，并导致鉴定治疗分子，这些分子将在两种小鼠模型（CAG敲入和YAC128）和人类神经元细胞中进行验证。与HD患者成纤维细胞区分开。这些还将使旨在干扰HD疾病过程的治疗剂的理性设计在神经元细胞开始屈服于其累积作用之前。