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Localization and Function of Huntingtin Associated protein 1 (Hap1) in C.elegans

亨廷顿蛋白相关蛋白 1 (Hap1) 在秀丽隐杆线虫中的定位和功能

基本信息

批准号：
7568247
负责人：
CLAIRE-ANNE N GUTEKUNST
金额：
$ 8.08万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2010-12-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7568247
关键词：
Affect Affinity Antibodies Antibody Formation Behavioral Binding Biological Assay Brain Caenorhabditis elegans Caenorhabditis elegans Proteins Chemotaxis Chimeric Proteins Defecation Extramural Activities Genes Growth Head Homologous Gene Human Huntington Disease Individual Injection of therapeutic agent Knock-out Larva Length Maps Metabolic Pathway Modeling Mutate Mutation Neurodegenerative Disorders Neurons Neuropathogenesis Oryctolagus cuniculus Partner in relationship Patients Phenotype Physiological Primates Protein Isoforms Proteins RNA Interference Reporter Rodent Role Sensory Specificity Stretching Tail Techniques Testing Time Transgenic Organisms Translating Trinucleotide Repeats Western Blotting base design disorder risk feeding gene conservation human Huntingtin protein immunocytochemistry mutant novel therapeutics overexpression polyglutamine promoter public health relevance research study response

项目摘要

DESCRIPTION (provided by applicant): Huntington-Associated-Protein 1 (HAP1) was identified through its interaction with huntingtin, the protein mutated in Huntington's Disease (HD). In HD, huntingtin contains an expanded polyglutamine stretch which affects its interaction with other proteins including an increase in its binding to HAP1. In rodents and primates, HAP1 is mostly found in the brain where it is expressed in neurons. Although several functions have been proposed for HAP1, its physiological role has not been established. To further understand the role of HAP1 we have started studying its C. elegans homologue called T27A3.1. To map out the expression of T27A3.1a-e isoforms we have generated several transgenic worm lines. We have found expression of a fluorescent reporter protein under the control of the promoter for T27A3.1 in a subset of neurons including chemosensory neurons in the head and tail. We have also found T27A3.1 isoforms to be expressed in a similar subcellular localization as of mammalian HAP1. To further understand the role of T27A3.1 we propose 1) to generate antibodies against T27A3.1 and use these antibodies for immunocytochemical localization of the various isoforms both at the cellular and subcellular level, 2) to identify behavioral phenotypes resulting from silencing small sets of T27A3.1 isoforms or from mutational knockout and evaluate the ability of mammalian HAP1 to rescue those phenotypes and 3) to characterize the interaction between T27A3.1 and huntingtin and to determine the effect of manipulating HAP1 expression levels on the behavioral phenotype of an HD C. elegans model. These studies will serve as a basis for a larger extramural proposal to study the 19 different huntingtin interactors in C. elegans. PUBLIC HEALTH RELEVANCE: The experiments proposed in this application are aimed at characterizing the localization and function of T27A3.1, a C. elegans protein with strong similarities to mammalian Huntingtin Associated Protein 1 (HAP1) which binds to huntingtin, the protein bearing the mutation that causes Huntington's Disease, a neurodegenerative disorder affecting 1 in 10 000 individuals in the US with about 4 times more people at risk for the disease. Because of the high conservation of genes and metabolic pathways between C. elegans and humans, information obtained from these studies will help further our understanding of HD neuropathogenesis and in the design of new therapeutics. For example, if silencing or overexpression of T27A3.1 can suppress the phenotype of HD worms, then a similar strategy for manipulating the levels of HAP1 may have some benefits for patients with Huntington's Disease.

描述(申请人提供)：亨廷顿相关蛋白1(HAP1)是通过与亨廷顿蛋白的相互作用而鉴定的，该蛋白在亨廷顿病(HD)中突变。在HD中，Huntingtin包含一个扩展的聚谷氨酰胺拉伸，这会影响它与其他蛋白质的相互作用，包括增加它与HAP1的结合。在啮齿动物和灵长类动物中，HAP1主要在大脑中发现，在那里它在神经元中表达。虽然已经提出了HAP1的几种功能，但其生理作用尚未确定。为了进一步了解HAP1的作用，我们已经开始研究它的线虫同源物T27A3.1。为了定位T27A3.1a-e异构体的表达，我们建立了几个转基因蠕虫系。我们发现在T27A3.1启动子的控制下，一个荧光报告蛋白在包括头部和尾部的化学感受性神经元在内的一组神经元中表达。我们还发现T27A3.1亚型在与哺乳动物HAP1相似的亚细胞定位中表达。为了进一步了解T27A3.1的作用，我们建议1)产生针对T27A3.1的抗体，并使用这些抗体在细胞和亚细胞水平上对各种异构体进行免疫细胞化学定位；2)识别沉默少量T27A3.1异构体或突变敲除T27A3.1所导致的行为表型，并评估哺乳动物HAP1挽救这些表型的能力；3)表征T27A3.1与Huntingtin之间的相互作用，并确定控制HAP1表达水平对线虫模型行为表型的影响。这些研究将作为一个更大的校外计划的基础，该计划将研究线虫中19种不同的狩猎素相互作用。公共卫生相关性：本申请中提出的实验旨在表征T27A3.1的定位和功能，T27A3.1是一种线虫蛋白，与哺乳动物亨廷顿相关蛋白1(HAP1)有很强的相似性，HAP1与亨廷顿蛋白结合，亨廷顿蛋白携带导致亨廷顿病的突变，亨廷顿氏病是一种神经退行性疾病，在美国每10,000人中就有一人受到影响，患病风险约为亨廷顿病风险的4倍。由于线虫与人类之间的基因和代谢途径高度保守，从这些研究中获得的信息将有助于我们进一步了解HD的神经发病机制，并有助于设计新的治疗方法。例如，如果沉默或过度表达T27A3.1可以抑制HD蠕虫的表型，那么控制HAP1水平的类似策略可能对亨廷顿病患者有一些好处。