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

• 批准号: 8084223
• 负责人: ROBERT E HUGHES
• 金额: $ 41.59万
• 依托单位: BUCK INSTITUTE FOR RESEARCH ON AGING
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8084223
• 关键词: Affect; Behavior; Behavioral; Binding; Biochemical; Biological Assay; Brain; Caenorhabditis elegans; Caspase; Cell Survival; Cell model; Cells; 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; 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; 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; polyglutamine; protein protein interaction; small hairpin RNA; therapeutic target; yeast two hybrid system

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）：亨廷顿氏病（HD）是一种逐渐致命的神经退行性疾病，是由编码亨廷顿基因中CAG道的聚谷氨酸扩张引起的。亨廷顿蛋白的正常功能尚不清楚。聚谷氨酸毒性的确切性质及其引起细胞功能障碍的主要靶标也尚待阐明。为了更深入地了解亨廷顿的正常和致病功能，我们使用基于酵母的两杂化和基于质谱的方法进行了大规模筛选，以发现亨廷顿相互作用的蛋白质（臀部）。在使用数值和统计方法进行数据分析之后，确定了一个高置信度234个臀部。为了测试与HD病理相关的这些相关性，测试了编码60种相互作用蛋白的直系同源物的基因，其能够在HD的果蝇模型中修改有毒的多谷氨酰胺表型。在经过测试的基因中，有80％充当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毒性功能丧失抑制因子。这些结果表明，在我们的研究中鉴定出的臀部集合富含在HD细胞病理中发挥直接作用的蛋白质。这些结果还表明，臀部可能会类似地富含可以改变人类HD的蛋白质。这项研究的主要目的是测试我们的高通量研究中鉴定出的臀部，以使其能力修饰哺乳动物细胞和HD小鼠模型中的HD表型。我们将使用siRNA在突变HD毒性的人和小鼠神经元细胞模型中敲除这些基因的表达。在特定情况下，还将评估基因表达敲低的作用，以实现亨廷汀蛋白的代谢，定位和/或翻译后修饰的影响。在这些测定中显示效果的候选蛋白将优先考虑在HD小鼠模型中进行更广泛的研究。修改亨廷顿蛋白的毒性和/或生化特性的臀部将在转基因HD小鼠脑中与亨廷汀共定位。表达降低的臀部可以抑制基于细胞的测定（和/或果蝇）中的毒性，以在HD小鼠模型中进行抑制。这将通过构建适当的表达shRNA的转基因小鼠系，将它们跨入HD小鼠模型，并研究对小鼠HD表型的影响来完成。我们还将使用AAV介导的嘻哈shRNA的病毒转染来研究HIP敲低HD小鼠的作用。这些研究的最终目的是鉴定基于细胞和/或果蝇测定中HD表型的修饰符，确定其作用机理，并在HD小鼠模型中验证它们。我们预计，这些研究将为HD病理的性质提供有用的见解，并为HD中的治疗药物发现提供新颖的候选靶标。

项目成果

Natural Genetic Variation in Yeast Reveals That NEDD4 Is a Conserved Modifier of Mutant Polyglutamine Aggregation.

• DOI: 10.1534/g3.118.200289
• 发表时间: 2018-11-06
• 期刊: G3 (Bethesda, Md.)
• 作者: Peters TW;Nelson CS;Gerencser AA;Dumas KJ;Tavshanjian B;Chang KC;Lithgow GJ;Hughes RE
• 通讯作者: Hughes RE

A large scale Huntingtin protein interaction network implicates Rho GTPase signaling pathways in Huntington disease.

大规模亨廷顿蛋白相互作用网络暗示亨廷顿病中的 Rho GTPase 信号通路。

• DOI: 10.1074/jbc.m113.523696
• 发表时间: 2014
• 期刊: The Journal of biological chemistry
• 作者: Tourette,Cendrine;Li,Biao;Bell,Russell;O'Hare,Shannon;Kaltenbach,LindaS;Mooney,SeanD;Hughes,RobertE
• 通讯作者: Hughes,RobertE