Autophagic Response Triggered By Chloroquine And Decomposition Of Atg12-140-Atg5Conjugate In Ubiquitin-Like Conjugation Systems

Autophagy is an extremely important cellular activity, it is commonly found inmost eukaryotic cells.It is a complex process for the lysosome degradating thestructures in cells. In normal state, cells can eliminate injured or aging organelles,intracellular protein aggregation and other biological macromolecules that are nolonger needed by autophagy in order to maintain the stability of internal environment.In addition, autophagy is also involved in many other cellular activities, such as celldifferentiation, tissue repair, growth regulation, adaptation to the unfavorableenvironment, cellular immunity and so on. This thesis focuses on two majordiscoveries in the study of autophagy, one is that we discovered and confirmed thatchloroquine can effectively induce strong autophagic response in mammalian cells;theother is that we discovered and confirmed the Atg12-140-Atg5protein complexwhich plays very important roles in autoghagy can decompose to generate free formsof Atg12-140and Atg5proteins.To clarify the autophagic response induced by chloroquine in mammalian cells,we took the human lung adenocarcinoma A549cell line,the mouse embryonicfibroblast cell line and the mouse primary hepatocytes as objects for studying.Indirectimmunofluorescence and Western blot techniques were used and a kind of autophagy-related protein—LC3-Ⅱ was taken as the criteria for judgement. We proved that theautophagic response was significantly enhanced in the three kinds of cells under thetreatment of chloroquine. The studies showed that the autophagic response in cellswas significantly enhanced when the treatment duration of chloroquine was prolongedproperly.In addition,the higher concentration of chloroquine could induce strongerautophagic response when treatment duration was the same.We also found thescavenger of the reactive oxygen species—N-acetylcysteine could block theautophagic response induced by chloroquine.This indicated that the reason why chloroquine can induce autophagic response in mammalian cells is probably becausethe reactive oxygen species can be induced in cells by chloroquine.We found that Atg12-140-GFP and Atg12-140-3×Flag can decompose togenerate free forms of Atg12-140/GFP and Atg12-140/3×Flag proteins in cells.According to that,we thought Atg12-140-Atg5protein complex which is an importantcomponent in the ubiquitin-like conjugation systems can also decompose to generatefree forms of Atg12-140and Atg5proteins.By validating that exogenous GFP-Atg12-140, GFP-Atg12-187and Atg12-187proteins were all able to be conjugated withAtg5to form protein complexes in cells and there is no other free form of Atg5incells,we confirmed that the Atg5s which were conjugated with the three exogenousproteins were derived from the Atg12-140-Atg5protein complexes in cells.So thededuction that the Atg12-140-Atg5protein complex can decompose wasdemonstrated to be correct. Therefrom,the traditional view that the conjugationbetween Atg12and Atg5is irreversible will be probably overturned.The two studies are both groundbreaking discoveries in the area of autophagy. Thechloroquine-induced autophagic cell model was also used in the second study. Thetwo studies can definitely provide theoretical supports and new ideas for exploring themolecular mechanism of autophagy deeply in the future.