Molecular Biological Studies on Processing of Amyloid Precursor Protein in Lysosomal Pathway

淀粉样前体蛋白在溶酶体途径中加工的分子生物学研究

• 批准号: 05670817
• 负责人: TAKAHATA Naohiko
• 金额: $ 1.28万
• 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 (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670817/
• 关键词: amyloid precursor protein; amyloid beta protein; proteolytic processing; lysosome pathway; chloroquine; transferrin receptor; rab 7; ライソゾーム系輸送経路; モノクローナル抗体; 細胞内輸送システム; ライソゾーム経路; 細胞内処理過程; 熱ショック; beta蛋白

Proteolytic proccessing of amyloid precursor protein (APP) to generate amyloid beta protein (Abeta) in lysosomal pathway, and an effect of chloroquine, a potent lysosomotropic agent, on the processing were studied using variety of cultured cells.1.Antibodies raised against specific protein to intracellular compartments under lysosomal pathway : Lysosomal pathway consists of endosome, primary, secondary lysosomes, and transport vesicles. There are specific proteins to these intracellular compartments, transferrin receptor for endosome, rab 7 for primary lysosome, and cathepsin D for secondary lysosome. Antibodies against synthetic peptides homologous to amino acid sequence of these proteins were successfully established.2.Proteolytic processing of APP to A beta in cultured human cells under Chloroquine treatment : HUT78, derived from human T cell, U937, derived from human monocyte, and HeLa cell were cultured.1) Proteolytic processing of APP to A beta in cultured human cells without Chl … More oroquine treatment(1) Immunohistochemical studies : Antibodies against intracellular compartment specific protein stained unique intracellular structures respectively. Transferrin receptor antibody labeled relatively large vesicles, and rab 7 antibody immunostained numerous small vesicles. Cathepsin D antibody labeled intracellular granular materials. APP and Abeta (Abeta17-28) antibodies demonstrated cell surface dot-like structures and cytoplasmic fine granular materials.(2) Biochemical studies of APP processing : Many amyloidogenic and non-amyloidogenic APP fragments were detectable in cell fractionate. Western blot analyzes showed that 4 kDa Abeta fragment, was observed in mitochondrial and cytosolic fractions.2) Proteolytic processing of APP to Abeta in cultured human cells under Chloroquine treatment : Chloroquine induced vacuoles in cytoplasm of the cultured cells after 12 hours. Vacuoles increased in number until up to 24 hours.(1) Immunohistochemical studies : A number of these vacuoles were stained with transferrin receptor antibody, rab 7 antibody, and were also labeled with both APP and Abeta antibodies.(2) Biochemical studies of APP processing : 4 kDa Abeta fragment became visualized more intensely after chloroquine treatment in mitochondrial and cytosolic fractions.Our data provides further evidence for participation of acidic compartments, for example, lysosomal pathway, in the generation of amyloidogenic processing proteolytic cleaved APP products and Abeta. Less

本实验研究了淀粉样前体蛋白（APP）在溶酶体途径中水解生成淀粉样β蛋白（Abeta）的过程，以及氯喹对该过程的影响。1.溶酶体途径：溶酶体途径由内体、初级溶酶体、次级溶酶体和转运囊泡组成。这些细胞内区室有特定的蛋白质，内体有转铁蛋白受体，初级溶酶体有rab 7，次级溶酶体有组织蛋白酶D。2.氯喹处理下人细胞APP蛋白水解为A β的过程：培养人T细胞HUT 78、人单核细胞U937和HeLa细胞; 1）不加Chl处理的人细胞APP蛋白水解为A β; 2）细胞培养液中加入Chl处理的人细胞APP蛋白水解为A β; 3）细胞培养液中加入Chl处理的人细胞APP蛋白水解为A β ...更多信息 （1）免疫组化研究：抗细胞内区室特异性蛋白抗体分别染色独特的细胞内结构。转铁蛋白受体抗体标记相对较大的囊泡，rab 7抗体免疫染色许多小囊泡。组织蛋白酶D抗体标记的细胞内颗粒物质。APP和Abeta（Abeta 17 -28）抗体显示细胞表面点状结构和细胞质细颗粒物质。(2)APP加工的生化研究：在细胞碎片中可检测到许多淀粉样蛋白和非淀粉样蛋白APP片段。Western blot分析显示，在线粒体和胞浆中均观察到4kDa的A β片段。2）氯喹处理培养的人细胞中APP蛋白水解为A β：氯喹处理12 h后，培养的细胞质中出现空泡。直至24小时，微球数量增加。(1)免疫组织化学研究：用转铁蛋白受体抗体、rab 7抗体对这些空泡中的一些进行染色，并且还用APP和Abeta抗体进行标记。(2)APP加工的生化研究：氯喹处理后，线粒体和胞浆中的4 kDa Abeta片段变得更强烈，我们的数据进一步证明了酸性区室，例如溶酶体途径，参与了淀粉样蛋白形成过程中蛋白水解裂解的APP产物和Abeta的产生。少

项目成果

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