PROTEIN PROTEIN INTERACTION TO AMYLOID PRECURSOR PROTEIN PROCESSING

蛋白质与淀粉样前体蛋白加工的相互作用

• 批准号: 6315627
• 负责人: RAYMOND SCOTT TURNER
• 金额: $ 22.35万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6315627
• 关键词: Alzheimer's disease; Caenorhabditis elegans; Semliki Forest virus; amyloid proteins; brain metabolism; cell line; cell type; gene expression; human tissue; immunocytochemistry; in situ hybridization; intracellular transport; neuritic plaques; neuropathology; plasmids; postmortem; presenilin; protein localization; protein metabolism; protein protein interaction; protein transport; transfection /expression vector; virus infection mechanism

The major components of amyloid in Alzheimer's disease (AD) brain are Abeta peptides that are derived by proteolytic cleavage of amyloid precursor protein (APP). Our preliminary data demonstrates that the X11alpha protein strongly influences APP metabolism in transfected HEK 293 cells. Specifically, X11alpha prolongs the half-life of cellular APP and retards recovery of its metabolic fragments, including the secreted amino-terminal fragments APPs, as well as Abeta40 and Abeta42 in conditioned medium. These effects are mediated by direct binding of the PTB-PI domain of Xllalpha to the YENPTY motif in the intracellular carboxy-terminus of APP. In addition, to a PTB-PI domain, X11alpha also contains two PDZ domains as well as an extended amino-terminus that may modulate Xllalpha effects on APP processing. We have recently found that Xllalpha exists as a heterotrimeric complex in mouse brain complexed with the mammalian homologues of the C. elegans Lin-2 and Lin-7 proteins. We propose to determine the potential modulatory influence of mammalian Lin-2 and Lin-7 on the inhibitory effects of X11alpha on cellular APP metabolism. We will study these effects by over-expression of wild type and dominant negative constructs of X11alpha, mLin-2 and mLin-7. Initial studies will be performed with transfected Hek 293 cells. Since APP metabolism is to some degree cell-type specific and Xllalpha is primarily a neuronal protein, we will also analyze the effects of Xllalpha, mLin-2 and mLin-7 on APP processing in neurons. We will perform these experiments in NT2 neurons infected with Semliki Forest Virus expressing the wild type and dominant negative constructs. We hypothesize that Xllalpha influences PP metabolism by altering its cellular trafficking. Thus, we will examine the cellular localization of PP, Xllalpha, mLin-2, and mLin-7 in wild type and infected cells. Finally, we hypothesize that the regional expression of APP, X11alpha, mLin-2, and mLin-7 in brain may inversely correlate with the regional neuropathology of AD, particular amyloid plaques. We will determine the regional expression and localization of these genes/proteins in normal and AD brain sections by immunohistochemistry and in situ hybridization. Collectively, this data will provide insights into the regulation of cellular APP trafficking and metabolism via these specific protein-protein interactions, and their potential roles in the development of AD. This information may lead to novel therapeutic strategies to delay the onset or slow the progression of amyloid deposition, dementia, and AD.

阿尔茨海默氏病（AD）大脑中淀粉样蛋白的主要成分是通过淀粉样蛋白前体蛋白（APP）蛋白水解裂解得出的Abeta肽。我们的初步数据表明，X11Alpha蛋白强烈影响转染的HEK 293细胞中的APP代谢。具体而言，X11Alpha延长了细胞应用程序的半衰期，并延迟其代谢片段的回收，包括分泌的氨基末端片段应用程序，以及在条件培养基中的Abeta40和Abeta42。这些效应是通过Xllalpha的PTB-PI结构域与APP的细胞内羧基末端中的Yenpty基序的直接结合来介导的。此外，对于PTB-PI域，X11Alpha还包含两个PDZ域以及一个扩展的氨基末端，可能会调节Xllalpha对应用程序处理的影响。我们最近发现，Xllalpha作为与秀丽隐杆线虫Lin-2和Lin-7蛋白的哺乳动物同源物复合的小鼠大脑中的异三聚体复合物存在。我们建议确定哺乳动物LIN-2和LIN-7对X11alpha对细胞APP代谢的抑制作用的潜在调节作用。我们将通过过度表达X11Alpha，Mlin-2和Mlin-7的野生型和显性负构建体的过表达来研究这些效果。最初的研究将使用转染的HEK 293细胞进行。由于APP代谢在某种程度上是细胞类型的特异性，而Xllalpha主要是一种神经元蛋白，因此我们还将分析Xllalpha，Mlin-2和Mlin-7对神经元应用程序处理的影响。我们将在感染的Semliki森林病毒感染的NT2神经元中执行这些实验，这些病毒表达了野生类型和显性负构建体。我们假设Xllalpha通过改变其细胞运输来影响PP代谢。因此，我们将研究野生型和感染细胞中PP，Xllalpha，Mlin-2和Mlin-7的细胞定位。最后，我们假设大脑中APP，X11Alpha，Mlin-2和Mlin-7的区域表达可能与AD的区域神经病理（特定的淀粉样蛋白斑块）成反比。我们将通过免疫组织化学和原位杂交确定这些基因/蛋白在正常和AD脑切片中的区域表达和定位。总的来说，这些数据将通过这些特定的蛋白质 - 蛋白质相互作用及其在AD的发展中的潜在作用，从而为细胞APP运输和代谢的调节提供见解。这些信息可能会导致新的治疗策略，以延迟或减慢淀粉样蛋白沉积，痴呆和AD的进展。