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Control mechanisms of autophagy and apoptosis by a new group H^+/Cl^- symporting antibiotics

新型H^/Cl^-同向抗生素控制自噬和凋亡的机制

基本信息

批准号：
11470483
负责人：
OHKUMA Shoji
金额：
$ 9.22万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

In this project, we have investigated the role of autophagy in the induction of neurite outgrowth (NOG), growth inhibition and apoptosis by a new type of H^+/CL^- symporting antibiotics like prodigiosins.We found the followings: (1) Prodigiosins, like bafilomycins, inhibited pancreatic cancer transplanted into nude mice. (2) Prodigiosins also induced apoptosis and NOG in RNA_-, protein-syntheses, and serine/threonine kinase-dependent manners, and K_-252_<a-> or A_-kinase-independent manners, but induced apoptosis differently from NOG in its in-sensitivity to tyrosine phosphates inhibitors. (3) E-58591, a prodigiosin-like tambjamine antibiotics, also showed H^+/CL^- symport activity and strongly inhibited spleen cell immune responses, gastric acid secretion and inhibited osteoclastosis and induced NOG at higher concentrations. (4) We have succeeded in the synthesis of affinity probe for concanamycins (analogs of bafilomycins) and showed concanamycin-induced NOG from outside of plasma membranes. (5) 3-Methyladenine, and inhibitor against autophagy, did not induce apoptosis in the presence of serum, suggesting little participation of autophapy on the bafilomycin-induced apoptosis. (6) We isolated human-homologs of autophagy-related genes from HeLa cells. They showed cell death of CHO in the absence of amino acid- or serum-free medium. (7) We established a system to look into degradation of GFP-labeled organelles (mitochondria and peroxisomes). (8) We showed that apoptosis and NOG was not induced by pH-differences between inside and outside of cells, because they were not induced by NH_4Cl. (9) Prodigiosins also strongly and reversibly uncoupled (H^+/K^+) ATPase-dependent proton-translocation from rabbit gastric mucosa. (10) We have succeeded in the cloning, proton pump-dependent ATP synthesis, and determation of operon structure of V-ATPase from a eubact rium (Thermus termophilus).

本课题研究了自噬在新型H^+/CL^-共转运抗生素灵菌红素（prodigiosins）诱导神经突生长（NOG）、生长抑制和凋亡中的作用，发现：（1）灵菌红素与巴非霉素一样，对胰腺癌裸鼠移植瘤有抑制作用。(2)灵菌红素也以RNA、蛋白质合成和丝氨酸/苏氨酸激酶依赖的方式诱导细胞凋亡和NOG，但不依赖于K-252<a->或A-激酶，其诱导细胞凋亡的方式与NOG不同，对酪氨酸磷酸酶抑制剂不敏感。(3)E-58591是一种类似灵菌红素的坦比明类抗生素，在较高浓度下也表现出H^+/CL^-协同转运活性，并强烈抑制脾细胞免疫反应和胃酸分泌，抑制破骨细胞增生和诱导NOG。(4)我们已经成功地合成了亲和探针的concanamycins（类似物的bafilomycins），并显示concanamycin诱导的NOG从质膜外。(5)自噬抑制剂3-甲基腺嘌呤在血清存在下不诱导细胞凋亡，表明自噬对巴非霉素诱导的细胞凋亡几乎没有参与。(6)我们从HeLa细胞中分离出自噬相关基因的人类同源物。它们显示在不存在无氨基酸或无血清培养基的情况下CHO的细胞死亡。(7)我们建立了一个系统来研究GFP标记的细胞器（线粒体和过氧化物酶体）的降解。(8)结果表明，细胞内外pH值的差异不诱导细胞凋亡和NOG的产生，因为NH_4Cl不诱导细胞凋亡和NOG的产生。(9)灵菌红素还能强烈地可逆地解偶联（H^+/K^+）ATP酶依赖的兔胃粘膜质子转位。(10)我们成功地克隆了嗜热栖热菌（Thermus termophilus）的V-ATP酶基因，并对其质子泵依赖的ATP合成和操纵子结构进行了研究。