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

利用多种培养细胞，研究了溶酶体途径中淀粉样前体蛋白（APP）的蛋白水解过程生成淀粉样β蛋白（Abeta），以及强效溶酶体增厚剂氯喹对该过程的影响。针对溶酶体途径下细胞内区室的特定蛋白产生抗体：溶酶体途径由内核体、初级溶酶体、次级溶酶体和运输囊泡组成。这些细胞内区室有特定的蛋白质，内核体有转铁蛋白受体，初级溶酶体有rab7，次级溶酶体有组织蛋白酶D。成功地建立了针对这些蛋白氨基酸序列同源的合成肽的抗体。在氯喹处理下，APP在培养的人细胞中蛋白水解为A β：培养人T细胞来源的HUT78、人单核细胞来源的U937和HeLa细胞。1)在培养的人细胞中，APP在不含Chl的情况下水解为A β。更多的奥罗奎处理(1)免疫组织化学研究：针对细胞内室特异性蛋白的抗体分别染色出独特的细胞内结构。转铁蛋白受体抗体标记相对较大的囊泡，rab7抗体免疫染色大量小囊泡。组织蛋白酶D抗体标记细胞内颗粒物质。APP和Abeta （Abeta17-28）抗体显示细胞表面呈点状结构，胞质呈细颗粒状物质。(2) APP加工的生化研究：在细胞分馏酸中检测到许多淀粉样和非淀粉样的APP片段。Western blot分析显示，线粒体和细胞质中均有4kda片段。2)氯喹作用下培养的人细胞中APP蛋白水解为Abeta的过程：氯喹作用12小时后，培养细胞的细胞质中产生空泡。液泡数量增加，直至24小时。(1)免疫组化研究：许多液泡用转铁蛋白受体抗体、rab 7抗体染色，同时用APP和Abeta抗体标记。(2) APP加工的生化研究：氯喹处理后线粒体和细胞质组分中4kda Abeta片段的可视化更加强烈。我们的数据进一步证明了酸性区室（例如溶酶体途径）参与淀粉样蛋白水解裂解的APP产物和β的生成。少

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