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

在本项目中，我们研究了一种新型的H^+/CL^-同源抗生素如prodigiosins在自噬诱导神经突生长（neurite outgrowth， NOG）、生长抑制和凋亡中的作用。结果表明：(1)Prodigiosins与巴非霉素一样，对裸鼠胰腺癌移植有抑制作用。(2) Prodigiosins在RNA_-、蛋白质合成、丝氨酸/苏氨酸激酶依赖以及K_-252_<a->或a-激酶独立的方式诱导细胞凋亡和NOG，但其对酪氨酸磷酸酯抑制剂的敏感性不同于NOG。(3)类灵鼓胺抗生素E-58591在较高浓度下也表现出H^+/CL^-同调活性，并能强烈抑制脾细胞免疫反应和胃酸分泌，抑制破骨和诱导NOG。(4)我们成功合成了conncanamycin（巴非霉素类似物）的亲和探针，并在质膜外显示了conncanamycin诱导的NOG。(5) 3-甲基腺嘌呤和抗自噬抑制剂在血清存在的情况下不诱导细胞凋亡，提示自噬在巴非霉素诱导的细胞凋亡中作用不大。(6)我们从HeLa细胞中分离到人类同源的自噬相关基因。结果显示，在没有氨基酸或无血清培养基的情况下，CHO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)我们发现细胞内外ph值差异不诱导细胞凋亡和NOG，因为它们不是由NH_4Cl诱导的。(9) Prodigiosins还能从兔胃粘膜强烈可逆地解耦（H^+/K^+） atp酶依赖的质子移位。（10）我们成功地克隆了一种细菌，合成了依赖质子泵的ATP，并测定了v -ATP酶的操纵子结构。

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).