MECHANISM FOR NUTRITIONAL AND MOLECULAR REGULATION OF MAMMALIAN AUTOPHAGY

本研究项目旨在阐明哺乳动物自噬的营养、生理和分子调控机制，自噬主要参与细胞内蛋白质的大量水解，两年来取得的成果如下：A.自噬的营养作用-大米蛋白、麸质和大豆蛋白对肝脏体内蛋白水解的影响，通过从每融合大鼠肝脏中初始缬氨酸释放来测量，进行了比较。大米蛋白的蛋白水解速率比大豆蛋白快一倍，谷蛋白饮食相当于大豆蛋白饮食。结果发现，膳食蛋白质的质量会影响肝脏中蛋白质的周转率。氨基酸自噬形成的调节机制- a）寻找肝质膜上氨基酸的“受体”。亮氨酸类似物Leu-MAP用作推定的“受体”的配体。加入阿萨并生物素化，使阿萨与靶蛋白结合。 ...更多信息 n通过光亲和标记并通过抗生物素蛋白树脂纯化靶标。B）使用α-毒素透化的肝细胞寻找用于氨基酸调节的未知信使分子。制备了具有稳定药效的重组α毒素。利用该毒素，我们可以反复得到最初的发现，即肝脏提取物对调节性氨基酸的刺激表现出抑制作用！ve对a-毒素细胞中蛋白质水解的影响。通过单独用亮氨酸刺激显示了有效性。此外，乙酸乙酯的可提取部分也是活性的。c）蛋白磷酸化可能参与氨基酸信号传导-结果表明mTOR信号传导途径不参与对肝自噬的氨基酸有效性。然而，膜蛋白LC 3，参与自噬形成，是由氨基酸信号。LC 3 I向II型的转化被氨基酸抑制。这强烈表明氨基酸信号传导的靶分子之一是LC 3.C。自噬体膜融合体外检测方法的建立--以胞浆酶甜菜碱同型半胱氨酸甲基转移酶（BHMT）的p32片段为探针，建立自噬体与溶酶体膜融合的体外检测方法，以鉴定参与该过程的胞浆因子。少

This research project deals with clarifying the mechanism for nutritional, physiological and molecular regulation of mammalian autophagy, which mainly contributes to bulk proteolysis in the cell.Results obtained through the project for these 2 years are as follows : A. Nutritional role of AUTOPHAGY - The effect of rice protein, gluten and soy protein on hepatic in vivo proteolysis, measured by initial valine release from per fused rat liver, was compared. Feeding rice protein showed twice a faster proteolytic rate than that of soy protein, and a gluten diet e qualed a soy protein diet. The results led to the discovery the quality of dietary protein could af feet protein turnover rates in the liver.B. Regulatory mechanism of autophagic formation by amino acids - a) The "receptor" of amino acids on the hepatic plasma membrane was searched. The leucine analog, Leu-MAP, was employed as a ligand for putative "receptor". It was added with ASA and biotinylated to bind ASA with a target protei … More n by photoaffinity labeling and to purify the target by avidin resin, b) The unknown messenger molecule(s) for amino acid regulation was searched using α-toxin permeabilized hepatocytes. The recombinant α-toxin was prepared for steady effectiveness. By using this toxin,we could get repeatedly the original findings that the liver extract stimulated by regulatory amino acids showed a suppress!ve effect on proteolysis in a-toxin cell. The effectiveness was shown by that stimulated by leucine alone. More over, The extractable fraction by ethylacetate was also active, c) Possible involvement of protein phosphorylation in amino acid signaling - It turned out that the mTOR signaling pathway was not involved in the amino acid effectiveness on hepatic autophagy. However, a membrane protein LC3, involved in autophagic formation, was responded by amino acid signaling. The conversion of LC3 I to II form was suppressed by amino acids. It strongly suggested one of the target molecules of amino acid signaling is LC3.C. Development of in vitro assay method of autophagic membrane fusion - Using a p32 fragment of cytosolic enzyme, betaine homocysteine methyltransferase (BHMT) as a probe, an in vitro assay of membrane fusion between autophagosome and lysosome is to be established in order to identify cytosolic factors involved in this process. Less