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：
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
项目类别：
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

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）：亨廷顿氏病（HD）是一种进行性致命的神经退行性疾病，由亨廷顿基因中编码多聚谷氨酰胺的CAG区扩增引起。亨廷顿蛋白的正常功能尚不清楚。聚谷氨酰胺毒性的确切性质及其作用导致细胞功能障碍的关键靶点也有待阐明。为了更深入地了解亨廷顿蛋白的正常和致病功能，我们使用酵母双杂交和基于质谱的方法进行了大规模筛选，以发现亨廷顿蛋白相互作用蛋白（HIP）。使用数值和统计方法进行数据分析后，确定了 234 个 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 种相互作用蛋白的直向同源物的基因在 HD 果蝇模型中修饰有毒多谷氨酰胺表型的能力。 80% 的测试基因充当 HD 毒性的调节剂，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，果蝇的遗传分析已鉴定出 25 种 HD 毒性功能丧失的抑制因子。这些结果表明，我们研究中鉴定的 HIP 集合富含在 HD 细胞病理学中发挥直接作用的蛋白质。这些结果还表明，HIP 可能同样富含可改变人类 HD 的蛋白质。本研究的主要目标是测试我们在高通量研究中发现的 HIP 改变哺乳动物细胞和 HD 小鼠模型中 HD 表型的能力。我们将使用 siRNA 来敲低突变 HD 毒性的人类和小鼠神经元细胞模型中每个基因的表达。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。在这些检测中表现出效果的候选蛋白将优先在 HD 小鼠模型中进行更广泛的研究。将测试改变亨廷顿蛋白毒性和/或生化特性的 HIP 与转基因 HD 小鼠大脑中亨廷顿蛋白的共定位。 HIP 的表达减少可以抑制基于细胞的测定（和/或果蝇）的毒性，将在 HD 小鼠模型中进行抑制测试。这将通过构建适当的 shRNA 表达转基因小鼠系，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 AAV 介导的 HIP shRNA 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/或果蝇检测中鉴定 HD 表型的修饰因子，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为 HD 病理学的本质提供有用的见解，并为 HD 治疗药物的发现提供新的候选靶点。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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.)
影响因子：
0
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
影响因子：
0
作者：
Tourette,Cendrine;Li,Biao;Bell,Russell;O'Hare,Shannon;Kaltenbach,LindaS;Mooney,SeanD;Hughes,RobertE
通讯作者：
Hughes,RobertE