Functional analysis of proteins, which interact with Alzheimer's disease amyloid precursor protein and presenilins

与阿尔茨海默病淀粉样前体蛋白和早老素相互作用的蛋白质的功能分析

• 批准号: 17590242
• 负责人: TANAHASHI Hiroshi
• 金额: $ 2.24万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590242/
• 关键词: Alzheimer's disease; transcriptional regulation; neurodegenerative disease; Fe65L2; BACE; proteasome-dependent degradation; nonsense-mediated mRNA decay; nonsense-mediated mRNA decay; プロテオゾーム; NMD

1.Using the yeast two-hybrid system, we screened for proteins interacting with APP-binding protein Fe65L2 and cloned the cDNA large subunit of RNA polymerase 2 (RPB1). The cDNA encoded the RPB1 C-terminal domain containing the repeat domain consisting of 52 repeats of seven amino acids. Fe65L2 preferentially interacted with hypo-phosphorylated form of RPB1. Reporter gene assay using GAL4-dependent reporter gene and Fe65L2-GAL4 DNA-binding domain fusion showed transcriptional suppression. These results suggest that Fe65L2 forms complexes with RNA polymerase 2 in nuclei and regulates transcription.2.Gel shift assay using DNA probe containing c954C>T polymorphism sequence indicated the existence of nuclear protein interacting with the probe sequence.3.Overexpression of PS2-binding protein in HEK293 cells that stably overe pressed APP, DRAL increased the secretion of soluble APP and decreased the secretion of Aβ, suggesting that DRAL affect the metabolism of APP.4.We found a novel splicing variant of BACE, 1-127 which is regulated by nonsense-mediated mRNA decay (NMD). This transcript was slightly detected in the human brain. When expressed in cells, 1-127 is retained in ER without its propeptide removal, unstable and degraded by proteasome-dependent manner. Thus 1-127 is regulated by both NMD and proteasome-dependent degradation.

1.利用酵母双杂交系统筛选与APP结合蛋白Fe 65 L2相互作用的蛋白质，并克隆了RNA聚合酶2大亚基（RPB 1）的cDNA。该cDNA编码RPB 1的C-末端结构域，该结构域包含由52个重复的7个氨基酸组成的重复结构域。Fe 65 L2优先与低磷酸化形式的RPB 1相互作用。利用GAL 4依赖性报告基因和Fe 65 L2-GAL 4 DNA结合域融合的报告基因分析显示转录抑制。结果表明，Fe 65 L2与RNA聚合酶2在细胞核内形成复合物，调节转录; 2.凝胶迁移实验表明，含有c954 C>T多态性序列的DNA探针与核蛋白相互作用; 3.在稳定过表达APP的HEK 293细胞中，过表达PS 2结合蛋白，DRAL可增加可溶性APP的分泌，减少Aβ的分泌，4.我们发现了一种新的BACE剪接变异体1-127，它受无义介导的mRNA衰变（NMD）的调控。这种转录本在人类大脑中被轻微检测到。当在细胞中表达时，1-127保留在ER中而不去除其前肽，不稳定并通过蛋白酶体依赖性方式降解。因此，1-127受NMD和蛋白酶体依赖性降解的调节。