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（HAP 1）通过其与亨廷顿蛋白（亨廷顿病（HD）中突变的蛋白质）的相互作用进行鉴定。在HD中，亨廷顿蛋白含有扩展的多聚谷氨酰胺片段，其影响其与其他蛋白质的相互作用，包括增加其与HAP 1的结合。在啮齿动物和灵长类动物中，HAP 1主要存在于大脑中，在神经元中表达。虽然已经提出了HAP 1的几种功能，但其生理作用尚未确定。为了进一步了解HAP 1的作用，我们开始研究其C。线虫同源物T27A3.1。为了绘制T27 A3.1a-e同种型的表达图，我们已经产生了几种转基因蠕虫品系。我们已经发现，在T27A3.1的启动子的控制下，在包括头部和尾部的化学感觉神经元的神经元的子集的荧光报告蛋白的表达。我们还发现T27A3.1同种型在与哺乳动物HAP 1类似的亚细胞定位中表达。为了进一步理解T27A3.1的作用，我们提出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同种型进行免疫细胞化学定位，（二）鉴定由沉默小组T27A3.1同种型或突变敲除产生的行为表型，并评估哺乳动物HAP 1拯救这些表型的能力，以及3）以表征T27A3.1和亨廷顿蛋白之间的相互作用，并确定操纵HAP 1表达水平对HDC行为表型的影响。elegans模型这些研究将作为一个更大的校外建议的基础，以研究19种不同的亨廷顿蛋白相互作用在C。优雅公共卫生相关性：本申请中提出的实验旨在表征T27A3.1，一种C.这种蛋白质与哺乳动物亨廷顿蛋白相关蛋白1（HAP 1）具有很强的相似性，HAP 1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C.从这些研究中获得的信息将有助于我们进一步理解HD的神经发病机制和设计新的治疗方法。例如，如果T27 A3.1的沉默或过表达可以抑制HD蠕虫的表型，那么用于操纵HAP 1水平的类似策略可能对患有亨廷顿病的患者具有一些益处。

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.