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Huntingtin interacting proteins as modifiers of Huntington's disease

亨廷顿蛋白相互作用蛋白作为亨廷顿病的修饰剂

基本信息

批准号：
7213540
负责人：
ROBERT E HUGHES
金额：
$ 42.44万
依托单位：
BUCK INSTITUTE FOR RESEARCH ON AGING
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7213540
关键词：
Affect Behavior Behavioral Binding Biochemical Biological Assay Brain Caspase Cell Survival Cell model Cells Class Collaborations Collection Corpus striatum structure Data Analyses Disease Disease model Down-Regulation Drosophila genus Drug Delivery Systems Exons Family Fluorescence Microscopy Functional disorder GAG Gene Gene Expression Gene Proteins Genes Genetic Glutamine Goals Human Huntington Disease Inherited Injection of therapeutic agent Iowa Length Letters Localized Longevity Mammalian Cell Mass Spectrum Analysis Measures Mediating Metabolism Methods Modeling Modification Mus Nature Nerve Degeneration Neurodegenerative Disorders Neurons Orthologous Gene Pathogenesis Pathology Patients Phenotype Play Post-Translational Protein Processing Property Proteins Purpose Range Research Personnel Role Sampling Small Interfering RNA Staging Statistical Methods System Testing Therapeutic Tissues Toxic effect Transfection Transgenic Mice Transgenic Organisms Universities Validation Viral Yeasts base cellular pathology design disease phenotype drug development drug discovery fly genetic analysis human Huntingtin protein in vivo insight knock-down loss of function member mouse model mutant neuropathology novel numb protein polyglutamine protein protein interaction small hairpin RNA therapeutic target yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a progressive fatal neurodegenerative disease caused by expansion in a polyglutamine encoding CAG tract in the huntingtin gene. The normal function of the huntingtin protein is not well understood. The precise nature of polyglutamine toxicity and the key targets that it acts upon to cause cellular dysfunction also remain to be elucidated. In order to provide greater insight into the normal and pathogenic functions of huntingtin we undertook a large-scale screen to discover huntingtin interacting proteins (HIPs) using yeast two-hybrid and mass spectrometry-based methods. After data analysis using numerical and statistical methods, a high confidence group of 234 HIPs was identified. In order to test these for relevance to the HD pathology, genes encoding orthologs of 60 interacting proteins were tested for their ability to modify a toxic polyglutamine phenotype in a Drosophila model of HD. 80% of those genes tested acted as modifiers of the HD toxicity indicating that the ability of a protein to physically interact with huntingtin correlated with its ability to show genetic interaction in a phenotypic assay. Genetic analysis in Drosophila has thus far identified 25 loss-of-function suppressors of HD toxicity. These results demonstrate that the ensemble of HIPs identified in our study are enriched for proteins that play a direct role in the cellular pathology of HD. These results also suggest that HIPs may be similarly enriched for proteins that can modify HD in human. The primary goal of this study is to test HIPs identified in our high-throughput studies for their ability modify HD phenotypes in mammalian cells and HD mouse models. We will use siRNAs to knock-down expression of each of these genes in human and mouse neuronal cell models of mutant HD toxicity. In specific cases, gene expression knock-downs will also be evaluated for effects on the metabolism, localization and/or post-translational modification of the huntingtin protein. Candidate proteins showing effects in these assays will be prioritized for more extensive studies in mouse models of HD. HIPs that modify the toxicity and/or biochemical properties of huntingtin will be tested for co-localization with huntingtin in transgenic HD mouse brain. HIPs whose reduced expression can suppress toxicity in cell-based assays (and/or Drosophila) will be tested for suppression in mouse models of HD. This will be done by constructing the appropriate shRNA-expressing transgenic mouse lines, crossing these into HD mouse models, and studying effects on the mouse HD phenotypes. We will also use AAV-mediated viral transfection of HIP shRNA to study effects of HIP knock-down in HD mice. The ultimate purpose of these studies is to identify modifiers of HD phenotypes in cell-based and/or Drosophila assays, determine their mechanisms of action and validate these in mouse models of HD. We anticipate that these studies will provide useful insight into the nature of HD pathology and also provide novel candidate targets for therapeutic drug discovery in HD.

描述（由申请人提供）：亨廷顿病（HD）是一种进行性致死性神经退行性疾病，由亨廷顿基因中编码CAG的多聚谷氨酰胺片段扩增引起。亨廷顿蛋白的正常功能还不清楚。聚谷氨酰胺毒性的确切性质及其作用于引起细胞功能障碍的关键靶点也仍有待阐明。为了更深入地了解亨廷顿蛋白的正常和致病功能，我们进行了大规模的筛选，发现亨廷顿蛋白相互作用蛋白（HIPs）使用酵母双杂交和质谱为基础的方法。在使用数值和统计方法进行数据分析后，确定了234名HIP的高置信度组。为了测试这些与HD病理学的相关性，测试了编码60种相互作用蛋白质的直系同源物的基因在HD的果蝇模型中修饰毒性聚谷氨酰胺表型的能力。80%的测试基因作为HD毒性的修饰剂，表明蛋白质与亨廷顿蛋白物理相互作用的能力与其在表型测定中显示遗传相互作用的能力相关。迄今为止，果蝇中的遗传分析已经确定了25种HD毒性的功能丧失抑制因子。这些结果表明，在我们的研究中鉴定的HIP的集合富含在HD的细胞病理学中起直接作用的蛋白质。这些结果还表明，HIP可以类似地富集可以改变人类HD的蛋白质。本研究的主要目标是测试在我们的高通量研究中鉴定的HIP在哺乳动物细胞和HD小鼠模型中改变HD表型的能力。我们将使用siRNA敲低这些基因在突变HD毒性的人类和小鼠神经元细胞模型中的表达。在特定情况下，还将评估基因表达敲低对亨廷顿蛋白的代谢、定位和/或翻译后修饰的影响。在这些试验中显示效果的候选蛋白将优先用于HD小鼠模型中更广泛的研究。将测试改变亨廷顿蛋白的毒性和/或生化性质的HIP与亨廷顿蛋白在转基因HD小鼠脑中的共定位。将在HD小鼠模型中检测其表达降低可抑制基于细胞的测定（和/或果蝇）中毒性的HIP的抑制作用。这将通过构建适当的表达shRNA的转基因小鼠系，将其与HD小鼠模型杂交，并研究对小鼠HD表型的影响来完成。我们还将使用腺相关病毒介导的HIPshRNA病毒转染来研究HIP敲低对HD小鼠的影响。这些研究的最终目的是在基于细胞和/或果蝇的试验中鉴定HD表型的修饰剂，确定其作用机制，并在HD小鼠模型中验证这些机制。我们预计，这些研究将提供有用的洞察HD病理学的性质，也提供了新的候选目标治疗药物的发现在HD。