PROTEIN PROTEIN INTERACTION TO AMYLOID PRECURSOR PROTEIN PROCESSING

阿尔茨海默病（AD）脑中淀粉样蛋白的主要组分是通过淀粉样前体蛋白（APP）的蛋白水解裂解而衍生的Abeta肽。我们的初步数据表明，X11 α蛋白强烈影响APP代谢转染HEK 293细胞。具体而言，X11 alpha延长细胞APP的半衰期，并延缓其代谢片段的恢复，包括分泌的氨基末端片段APP，以及条件培养基中的Abeta 40和Abeta 42。这些作用是通过X11 α的PTB-PI结构域与APP细胞内羧基末端的YENPTY基序直接结合介导的。此外，除了PTB-PI结构域之外，X11 α还含有两个PDZ结构域以及一个延伸的氨基末端，其可以调节X11 α对APP加工的作用。我们最近发现X11 α在小鼠脑中以异源三聚体复合物的形式存在，与哺乳动物中的C.线虫Lin-2和Lin-7蛋白。我们建议确定哺乳动物Lin-2和Lin-7对X11 alpha对细胞APP代谢的抑制作用的潜在调节作用。我们将通过X11 α、mLin-2和mLin-7的野生型和显性负性构建体的过表达来研究这些效应。将使用转染的Hek 293细胞进行初始研究。由于APP代谢在某种程度上是细胞类型特异性的，并且X11 α主要是神经元蛋白，因此我们还将分析X11 α、mLin-2和mLin-7对神经元中APP加工的影响。我们将在用表达野生型和显性阴性构建体的塞姆利基森林病毒感染的NT 2神经元中进行这些实验。我们推测Xllalpha通过改变其细胞运输来影响PP代谢。因此，我们将检查PP、X11 α、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APP，X11 α，mLin-2和mLin-7在脑中的区域表达可能与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来确定这些基因/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，以及它们在AD发展中的潜在作用，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，以延迟淀粉样蛋白沉积，痴呆和AD的发作或减缓其进展。

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.