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

描述（由申请人提供）：亨廷顿相关蛋白1 （HAP1）是通过与亨廷顿病（HD）中突变的蛋白亨廷顿蛋白（huntingtin）相互作用而鉴定出来的。在HD中，亨廷顿蛋白含有扩大的聚谷氨酰胺拉伸，影响其与其他蛋白质的相互作用，包括与HAP1结合的增加。在啮齿类动物和灵长类动物中，HAP1主要存在于大脑中，并在神经元中表达。虽然HAP1具有多种功能，但其生理作用尚未确定。为了进一步了解HAP1的作用，我们开始研究它在秀丽隐杆线虫中的同系物T27A3.1。为了绘制T27A3.1a-e亚型的表达图谱，我们培育了几个转基因虫系。我们发现在T27A3.1启动子的控制下，一种荧光报告蛋白在包括头部和尾部的化学感觉神经元在内的一个神经元亚群中表达。我们还发现T27A3.1亚型的亚细胞定位表达与哺乳动物HAP1相似。为了进一步了解T27A3.1的作用，我们提出1)生成针对T27A3.1的抗体，并利用这些抗体在细胞和亚细胞水平上对各种亚型进行免疫细胞化学定位；2)鉴定T27A3.1小片段沉默或突变敲除导致的行为表型，并评估HAP1挽救这些表型的能力；3)表征T27A3.1和亨廷顿蛋白之间的相互作用，并确定操纵HAP1表达水平对HD线虫模型行为表型的影响。这些研究将为研究秀丽隐杆线虫中19种不同的亨廷顿蛋白相互作用物的更大的校外建议提供基础。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是秀丽隐杆线虫的一种蛋白质，与哺乳动物亨廷顿蛋白相关蛋白1 （HAP1）有很强的相似性，该蛋白与亨廷顿蛋白结合，该蛋白携带导致亨廷顿病的突变，亨廷顿病是一种神经退行性疾病，在美国每10000人中就有1人患病，患病风险约为4倍。由于秀丽隐杆线虫和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进一步了解HD神经发病机制和设计新的治疗方法。例如，如果沉默或过表达T27A3.1可以抑制HD蠕虫的表型，那么操纵HAP1水平的类似策略可能对亨廷顿病患者有一些好处。

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.