The roles of Huntingtin Associated Protein 40 in Huntingtin functions and Huntingtons disease pathogenesis

亨廷顿相关蛋白 40 在亨廷顿功能和亨廷顿病发病机制中的作用

基本信息

批准号：
10608221
负责人：
Sheng Zhang
金额：
$ 7.6万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10608221
关键词：
Affect Affinity Chromatography Animal Model Animals Autophagocytosis Binding Biochemical Biological Assay Brain Cell physiology Cells Complex Corpus striatum structure Development Disease Disease model Distant Drosophila genus Drug Targeting Etiology Evaluation Evolution Experimental Models Fibroblasts Foundations Genes Genetic Genetic Models Genetic Transcription Glutamine Health Heterogeneity Homologous Gene Human Huntington Disease Huntington gene Huntington protein Knock-out Lead Mammalian Cell Mammals Molecular Molecular Conformation Mus Mutate Mutation Neurons Pathogenesis Pathogenicity Patients Phenotype Physiological Prevention Proteins Proteomics Rattus Regulation Reporting Role Sampling Scaffolding Protein Sequence Alignment Structure Testing Tissues Toxic effect fly gain of function genetic testing in vivo knockout animal loss of function multidisciplinary mutant neuronal survival neurotoxicity novel novel therapeutics overexpression polyglutamine vesicle transport

项目摘要

Huntington's disease (HD) is caused by an abnormal expansion of the glutamine tract (polyQ) in Huntingtin (HTT). A clear understanding on how endogenous HTT is regulated in vivo is critical both for elucidating HD etiology and for identifying effective drug targets. HTT has numerous reported HTT associated partners (HAPs) and is functionally implicated in a growing list of cellular processes. However, little is known how HTT itself is regulated and whether such regulation is altered in HD. We previously characterized the HTT homolog (dHtt) in model organism Drosophila. Given the significant functional conservation of HTT from the fly to mammals, we hypothesized that the core regulators of HTT likely are among the numerous known HAPs and should also be conserved in Drosophila. In a proteomic study for such conserved central regulators of HTT in Drosophila, we isolated dHap40, the fly homolog of HAP40, as the strongest dHtt interactor. Importantly, converging evidence from studies in multiple species all support that in vivo HTT protein normally exists in a complex with HAP40, and HAP40 binding stabilizes the conformation of HTT. Further, in samples from HD patients, a ~10- fold increase of the levels of endogenous HAP40 were observed as compared to controls. However, despite these findings, by now there is no reported functional study of HAP40 in any physiological settings, and its effect on HTT's normal functions and mutant HTT toxicity remains unclear. Our preliminary studies support the significantly conserved physical and functional interactions between HTT and HAP40, implying a highly important regulatory relationship that constrains their co-evolution from flies to humans. Our findings not only establish Drosophila as a relevant genetic model to study the physiological roles of HAP40, but also lead to our hypothesis that HAP40 is a conserved central regulator of HTT and potentially a critical modulator of mutant HTT toxicity. Using established assays and HD models in Drosophila and cultured mammalian cells, we will systematically test this hypothesis. In Aim 1, we will carry out a comprehensive phenotypic analyses of dhap40 gene and test its genetic interactions with dhtt, so as to obtain a first systematic evaluation of HAP40 in a physiological setting and clarify its relationship with HTT at whole-animal level. In Aim 2, we will systematically test whether HAP40 is a central regulator of HTT's subcellular dynamics and its diverse cellular functions, so as to elucidate its relationship with HTT at molecular and cellular levels. In Aim 3, taking advantage of the well- established HD models in Drosophila and mammalian neurons, we will rigorously interrogate the role of HAP40 on mutant HTT toxicity. From these multidisciplinary studies, we will obtain a first comprehensive evaluation on the physiological functions of HAP40, its effect on endogenous HTT functions and on HD pathogenesis. The results potentially lay foundation on novel therapeutic avenues against HD via HAP40.

亨廷顿氏病（HD）是由亨廷顿蛋白中谷氨酰胺道（Polyq）异常扩张引起的（htt）。对内源性HTT在体内如何调节的清晰了解对于阐明HD至关重要病因和确定有效的药物靶标。 HTT有许多报告的HTT相关伙伴（HAP）并在功能上与越来越多的细胞过程列表有关。但是，鲜为人知的htt本身是多么受监管以及该调节是否在HD中改变。我们以前表征了HTT同源物（DHTT）在模型生物果蝇中。考虑到HTT从果蝇到哺乳动物的显着功能保护，我们假设HTT的核心调节剂可能是众多已知的HAP，也应该在果蝇中保守。在果蝇中HTT的这种保守的中央调节剂的蛋白质组学研究中，我们将HAP40的苍蝇同源物分离为最强的DHTT相互作用器。重要的是，融合来自多种物种研究的证据都支持体内HTT蛋白通常存在于一个复合体中 HAP40和HAP40结合稳定了HTT的构象。此外，在高清患者的样品中，约10- 与对照组相比，观察到内源性HAP40水平的倍数增加。但是，尽管如此这些发现，到目前对HTT正常功能和突变HTT毒性的影响尚不清楚。我们的初步研究支持 HTT和HAP40之间显着保守的物理和功能相互作用，这意味着高度重要的监管关系，将其共同发展从苍蝇限制为人类。我们的发现不仅建立果蝇作为研究HAP40生理作用的相关遗传模型，但也导致我们假设HAP40是HTT的保守中央调节剂，并且可能是突变体的关键调节剂 HTT毒性。在果蝇和培养的哺乳动物细胞中使用既定的测定法和高清模型，我们将系统地检验这一假设。在AIM 1中，我们将对DHAP40进行全面的表型分析基因并测试其与DHTT的遗传相互作用，以便获得对A中HAP40的首次系统评估生理环境并阐明其与HTT的关系在整个动物层面。在AIM 2中，我们将系统地测试HAP40是否是HTT亚细胞动力学及其多种细胞功能的中心调节剂，因此为了阐明其在分子和细胞水平上与HTT的关系。在AIM 3中，利用福祉在果蝇和哺乳动物神经元中建立的高清模型，我们将严格询问HAP40的作用关于突变的HTT毒性。从这些多学科研究中，我们将对 HAP40的生理功能，其对内源性HTT功能和HD发病机理的影响。这结果可能是通过HAP40针对HD的新型治疗途径的基础。