MECHANISM FOR NUTRITIONAL AND MOLECULAR REGULATION OF MAMMALIAN AUTOPHAGY

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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