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: 6216980
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6216980
• 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.

阿尔茨海默病 (AD) 大脑中淀粉样蛋白的主要成分是 Abeta 肽，它是通过淀粉样前体蛋白 (APP) 的蛋白水解裂解而衍生的。我们的初步数据表明，X11alpha 蛋白强烈影响转染 HEK 293 细胞中的 APP 代谢。具体来说，X11alpha 延长了细胞 APP 的半衰期，并延迟了其代谢片段的恢复，包括分泌的氨基末端片段 APP，以及条件培养基中的 Abeta40 和 Abeta42。这些效应是通过 Xllalpha 的 PTB-PI 结构域与 APP 胞内羧基末端的 YENPTY 基序直接结合来介导的。此外，除了 PTB-PI 结构域外，X11alpha 还包含两个 PDZ 结构域以及一个扩展的氨基末端，可调节 Xllalpha 对 APP 加工的影响。我们最近发现 Xllalpha 在小鼠大脑中以异三聚体复合物的形式存在，与秀丽隐杆线虫 Lin-2 和 Lin-7 蛋白的哺乳动物同源物复合。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影响。我们将通过 X11alpha、mLin-2 和 mLin-7 野生型和显性失活构建体的过度表达来研究这些影响。初步研究将使用转染的 Hek 293 细胞进行。由于 APP 代谢在某种程度上具有细胞类型特异性，并且 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 对神经元中 APP 处理的影响。我们将在感染了表达野生型和显性失活结构的塞姆利基森林病毒的 NT2 神经元中进行这些实验。我们假设 Xllalpha 通过改变 PP 的细胞运输来影响 PP 代谢。因此，我们将检查 PP、Xllalpha、mLin-2 和 mLin-7 在野生型和感染细胞中的细胞定位。最后，我们假设大脑中 APP、X11α、mLin-2 和 mLin-7 的区域表达可能与 AD 的区域神经病理学（特别是淀粉样斑块）呈负相关。我们将通过免疫组织化学和原位杂交确定这些基因/蛋白质在正常和 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-蛋白质相互作用来深入了解细胞 APP 运输和代谢的调节，以及它们在 AD 发展中的潜在作用。这些信息可能会带来新的治疗策略，以延缓淀粉样蛋白沉积、痴呆和 AD 的发生或减缓其进展。