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Molecular Biological Studies on Trafficking and Processing of Amyloid Precursor Protein

淀粉样前体蛋白运输和加工的分子生物学研究

基本信息

批准号：
04454304
负责人：
FUKATSU Ryo
金额：
$ 3.2万
依托单位：
SAPPORO MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

Intracellular trafficking and processing of amyloid precursor protein (APP) to generate amyloid beta protein (Abeta) was studied.1.Antibodies against specific protein in intracellular compartments : We were successful in raising specific antibodies against intracellular compartment specific protein, ERGIC53, galactosyltransferase, cathepsin D.2.APP trafficking in cultured cells : APP and Abeta immunoreactivities were observed in cell surface, and vesicular structures in cytoplasm. Western blot analyzes showed 50,20 kDa amyloidogenic fragments, and 4 kDa, apparently Abeta, in microsomal fraction.3.Effect of brefeldin A and monensin on APP processing : Brefeldin A and monensin are able to inhibit protein trafficking from endoplasmic reticulum (ER) to Golgi apparatus, and from Golgi apparatus to another cellular compartments. Under brefeldin A treatment, numerous vacuoles appeared in the cytoplasm of the cultured cells, and the vacuoles were immunolabeled with anti-ERGIC53. With monensin treatment, vacuoles appeared in perinuclear cytoplasm, and were stained with anti-APP and anti-Golgi antibodies. Western blot analysis showed reduced intensity of Abeta 4 kDa band.4.APP trafficking in Hela cells transfected with wild type APP770, and Swedish mutant cDNA : Wild type APP770 and Swedish mutant APP were overexpressed in cultured HeLa cells. APP immunoreactivities appeared to increase in ER,cell surface, and vesicles in the cytoplasm of these transfected cells. APP mRNA and Swedish type mRNA were determined with Northern ELISA,and turned out to increase about 3 to 6 fold compared to plasmid controls.Our data provide new evidence indicating that APP may process to generate Abeta either in ER,or lysosomes, or both compartments may be involved.

研究了淀粉样蛋白前体蛋白（APP）在细胞内运输和加工生成β淀粉样蛋白（Abeta）的过程。针对细胞内区室特异性蛋白的抗体：我们成功培养了针对细胞内区室特异性蛋白ERGIC53、半乳糖转移酶、组织蛋白酶D.2的特异性抗体。APP在培养细胞中的转运：在细胞表面观察到APP和Abeta的免疫反应，在细胞质中观察到囊泡结构。Western blot分析显示微粒体片段中有50、20 kDa淀粉样蛋白片段和4 kDa，明显为β。brefeldin A和莫能菌素对APP加工的影响：brefeldin A和莫能菌素能够抑制蛋白质从内质网（ER）向高尔基体的运输，以及从高尔基体向其他细胞室的运输。在brefeldin A的作用下，培养细胞的细胞质中出现了大量的空泡，这些空泡用抗ergic53免疫标记。莫能菌素处理后，核周细胞质出现空泡，抗app和抗高尔基抗体染色。Western blot分析显示Abeta 4kda条带强度降低。转染野生型APP770和瑞典突变体cDNA的Hela细胞中APP转运：野生型APP770和瑞典突变体APP在培养的Hela细胞中过表达。这些转染细胞的内质网、细胞表面和细胞质中的囊泡的APP免疫反应性似乎增加。应用Northern ELISA法检测APP mRNA和瑞典型mRNA，结果显示与质粒对照相比，APP mRNA和瑞典型mRNA增加了约3 ~ 6倍。我们的数据提供了新的证据，表明APP可能在内质网或溶酶体中产生β，或者两个区室都可能参与。