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成分是由淀粉样前体蛋白(APP)蛋白分解而来的Aβ多肽。我们的初步数据表明，X11pha蛋白强烈地影响了转基因HEK 293细胞中APP的代谢。具体地说，X11α延长了细胞APP的半衰期，并延缓了其代谢片段的恢复，包括分泌的氨基末端片段APP，以及条件培养液中的Abeta40和Abeta42。这些作用是通过XllAlpha的PTB-PI结构域与APP细胞内羧基末端的YENPTY基序直接结合而介导的。此外，除了PTB-PI结构域，X11pha还包含两个PDZ结构域以及一个延伸的氨基末端，可能调节XllAlpha对APP加工的影响。我们最近发现，XllAlpha在小鼠脑中以异三聚体的形式存在，与线虫LIN-2和LIN-7蛋白的哺乳动物同源物形成复合体。我们建议确定哺乳动物LIN-2和LIN-7对X11α对细胞APP代谢的抑制作用的潜在调制影响。我们将通过过度表达野生型和显性负结构的X11pha、Mlin-2和Mlin-7来研究这些影响。初步研究将在转基因的Hek293细胞上进行。由于APP的代谢在某种程度上是细胞类型特异性的，而XllAlpha主要是一种神经元蛋白质，我们还将分析XllAlpha、Mlin-2和Mlin-7对神经元中APP处理的影响。我们将在感染塞姆利基森林病毒的NT2神经元上进行这些实验，表达野生型和显性负结构。我们假设Xllpha通过改变PP的细胞运输来影响PP的新陈代谢。因此，我们将研究PP、Xllpha、Mlin-2和Mlin-7在野生型和感染细胞中的细胞定位。最后，我们推测APP、X11Alpha、Mlin-2和Mlin-7在脑内的区域表达可能与AD的局部神经病理，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，这些数据将为通过这些特定的蛋白质-蛋白质相互作用调节细胞APP运输和新陈代谢，以及它们在AD发展中的潜在作用提供见解。这些信息可能导致新的治疗策略，以延迟或减缓淀粉样蛋白沉积、痴呆和AD的发病或进展。

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.