Function of the DRPLA Gene Product Atrophin

DRPLA基因产物萎缩蛋白的功能

• 批准号: 6369946
• 负责人: CRAIG D BLACKSTONE
• 金额: $ 13万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2001-09-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6369946
• 关键词: atrophy; complementary DNA; deafness; dynamin; dystonia; gene mutation; guanosinetriphosphatases; immunocytochemistry; immunoprecipitation; laboratory rat; membrane transport proteins; mitochondrial membrane; molecular pathology; neural degeneration; pathologic process; protein protein interaction; protein purification; protein structure function; tissue /cell culture; yeast two hybrid system

The goal of this project is to elucidate the function of the atrophin protein, mutations of which underlie the neurodegenerative disease dentatorubral-palliudoluysin atrophy (DRPLA). These mutations are expansions of a polyglutamine repeat motif (coded by CAG trinucleotide repeats in the mRNA) within the protein, making DRPLA one of several known "glutamine repeat disorders." In this group of neurodegenerative diseases, an expansion of polyglutamine repeat in a number of different proteins confers the various disease phenotypes. However, in spite of the relatively restricted patterns of differential cell death in DRPLA and the other disorders, the gene products are widely distributed in both the central nervous system and peripheral tissues, and not their normal functions and mechanisms of disease are largely unknown. The DRPLA gene product atrophin has a number of advantages as a model protein for study; it is hydrophilic, of moderate size, and contains a number of intriguing peptide motifs which may be important in its function. This study will examine the function of atrophin through four specific aims. First, antibodies will be generated against purified atrophin, bacterial fusion proteins, and small synthetic peptides. These regents will be used to probe the regional, cellular, and subcellular localizations of atrophin in brain. The focus of the project, however, will be the identification of proteins that interact with atrophin, using yeast two hybrid screening and affinity purification techniques. The effect of expansion of polyglutamine repeat on any identified interactions will then be assessed. A focus on protein-protein interactions is critical while evaluating the function of atrophin, as alterations in which interactions due to the polyglutamine repeat expansion may be involved in the pathogenesis of DRPLA. Finally, the effects of differential alternative splicing of the atrophin gene on atrophin protein-protein interactions will be examined. Hereditary disorders such as DRPLA and the other glutamine repeat diseases are of particular interest of neurologists, as identification of the mechanism of neurodegeneration in these hereditary disorders will likely shed light on similar mechanisms in the more common acquired neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. During the course of this research, sponsored by Morgan Sheng MBBS, PhD, the candidate expects to be trained in the cutting edge techniques of molecular genetics and protein biochemistry that will empower him to pursue a productive career in molecular neurology.

本项目的目标是阐明萎缩蛋白的功能，其突变是神经退行性疾病齿状红核-苍白球蛋白质萎缩（DRPLA）的基础。这些突变是蛋白质内多聚谷氨酰胺重复基序（由mRNA中的CAG三核苷酸重复编码）的扩展，使DRPLA成为几种已知的“谷氨酰胺重复障碍”之一。“在这组神经退行性疾病中，多聚谷氨酰胺重复序列在许多不同蛋白质中的扩增赋予了各种疾病表型。然而，尽管DRPLA和其他疾病中的差异性细胞死亡模式相对有限，但基因产物广泛分布于中枢神经系统和外周组织中，并且其正常功能和疾病机制在很大程度上是未知的。DRPLA基因产物atrophin作为研究的模型蛋白具有许多优点;它是亲水性的，大小适中，并且含有许多有趣的肽基序，这些肽基序可能在其功能中很重要。本研究将通过四个具体目标来研究萎缩蛋白的功能。首先，将产生针对纯化的萎缩蛋白、细菌融合蛋白和小的合成肽的抗体。这些试剂将被用来探测脑内萎缩蛋白的区域、细胞和亚细胞定位。然而，该项目的重点将是使用酵母双杂交筛选和亲和纯化技术鉴定与萎缩蛋白相互作用的蛋白质。然后将评估多聚谷氨酰胺重复序列扩增对任何已鉴定相互作用的影响。在评价萎缩蛋白的功能时，关注蛋白质-蛋白质相互作用是至关重要的，因为多聚谷氨酰胺重复扩增引起的相互作用可能参与DRPLA的发病机制。最后，将研究萎缩蛋白基因的差异选择性剪接对萎缩蛋白-蛋白相互作用的影响。遗传性疾病如DRPLA和其他谷氨酰胺重复疾病是神经学家特别感兴趣的，因为这些遗传性疾病中神经变性机制的鉴定可能会揭示更常见的获得性神经变性疾病如阿尔茨海默病，肌萎缩性侧索硬化症和帕金森病中的类似机制。在这项由Morgan Sheng MBBS博士赞助的研究过程中，候选人希望接受分子遗传学和蛋白质生物化学前沿技术的培训，这将使他能够在分子神经学领域从事富有成效的职业生涯。