The Location Of ATG4B In Mitochondria And Its Role In Cell Growth Of HCC Cells

ATG4B(Autophagy related 4B), a member of C54 peptidases, plays a key role in the regulation of autophagy in mammalian cells. Autophagy(here refereed to macroautophagy) is a highly conserved process of metabolic degradation. During the process, the cytoplasmic constituents including damaged organelles and long-lived proteins are packaged by a double-membrane compartment called autophagosome. Then the autophagosome fuses with lysosome and the contents were degraded by lysosomal hydrolases. In mammalian cells, MAP1LC3/LC3 plays an essential role in the formation of autophagosome. The pro-LC3 can be cleaved and further transformed to the lipidated LC3(LC3-II), which is the necessary component of autophagosome. Previous studies have confirmed that ATG4 B regulate autophagy through either cleaving pro-LC3 B for its further lipidation or delipidating LC3B-II from autophagosome, which could lead to the change of autophagy level. More and more researches have demonstrated that ATG4 B participate in kinds of physiological and pathological processes including cell metabolism, infection and inflammation, nervous system disease and tumor through affecting autophagy flux. As to tumors, targeting ATG4B-mediated autophagy significantly affected the growth of osteosarcoma cells in vitro and in vivo. Moreover, recent study in colon cancer cells indicated that ATG4 B could promote cell proliferation independent of its role on autophagy, which opens a new sight for the diverse regulation mechanisms of ATG4 B in tumors. Our previous studies have indicated that ATG4 B may locate in mitochondria, but the the related function is still unclear. As the power house of the cell, mitochondria produces most of energy-rich molecule adenosine triphosphate(ATP) as the energy supply. The dysfunction of mitochondria can result in the pathological energy metabolism. In addition, The Reactive Oxygen Species(ROS) that mainly in mitochondria can server as an important messenger of signaling transduction in cells. Multiple evidence have demonstrated that the dysfunction of mitochondria is tightly related to the tumor malignant progression, but it has still not reported that ATG4 B could affect the phenotype of tumor cells. Therefore, in this study we will confirm the location of ATG4 B in mitochondria and its role in HCC cell growth in order to be beneficial for clinical treatments.Purpose:to verify the location of ATG4 B in mitochondria and investigate the related roles and mechanisms in the growth of HCC cells.Methods: 1. Isolate mitochondria from different cell lines and patient-derived tumor and the corresponding adjacent tissues, then detect the level of ATG4 B in mitochondria using Western blot assay.2. Construct GFP-labbled recombinant plasmid and transfect into cells. Then the mitochondria of cells were labeled with fluorescent dye and the cells were obsereved under a fluorescence microscope.3. Analyze the sub-mitochondrial localization of ATG4 B using immunogold-labeling electron microscopy, mitochondrial sub-fractionations isolation and protease K protection assay.4. Screen out the binding partners of ATG4 B in mitochondria using the combined methods of immunoprecipitation and mass spectrum(IP/MS).5. Investigate that ATG4 B could interact with thesubunites of mitochondrial ATP synthase(ATP5A1,ATP5 B and ATP5C1).6. Establish ATG4 B hemizygous knockout cells(ATG4B+/- cells) using CRISPR/Cas9-mediated genome editing system. Then compare the diffecrence of F0F1-ATP synthase activity between ATG4B+/- and ATG4B+/+ cells and evaluate the alternation of F0F1-ATP synthase activity with exogenous ATG4 B overexpression.7. Estimate the change of ATG4B-mediated signaling pathways and the roles in HCC cell growth.8. Detect the role of activated AKT on ATG4 B mitochondrial translocation using Western blot, CO-IP and fluorescence imaging methods.9. Explore the mechanism of AKT-enhanced ATG4 B mitochondrial translocation using different methods including bioinformatics and CO-IP.10. Cells were transfected with different recombinant plasmids and the significance of AKT-mediated phosphorylation of ATG4 B at Ser34 in HCC cell growth was detected using several methods including CCK-8 assay.11. Cells were transfected with different recombinant plasmids and the role of AKT-mediated phosphorylation of ATG4 B at Ser34 in autophagy was assessed using several methods including Western blot assay.12. Develop the method for detection of ATG4 B activity in living cells. Then measure the activity of ATG4 in ATG4BWT-overexpressed cells and ATG4BS34A-overexpressed cells.Results:1. ATG4 B can locate in the mitochondria of cells, especially in the inside of mitochondria.2. Mitochondrial ATG4 B interacts with F1 complex of ATP synthase and inhibits the activity of ATP synthase.3. The activated AKT enhances the mitochondrial translocation of ATG4 B.4. The Ser34 of ATG4 B is a novel target of AKT.5. AKT-enhanced ATG4 B mitochondrial translocation is highly dependent on the phosphorylation of ATG4 B at Ser34.6. AKT-mediated phosphorylation of ATG4 B at Ser34 is related to HCC cell growth, which could be independent of autophagy;Conclusion:In HCC cells, AKT-mediated phosphorylation of ATG4 B at Ser34 enhanced ATG4 B mitochondrial translocation and subsequent inhibition of F0F1-ATP synthase, leading to the alternation of cell metabolism, and ultimately promotes HCC cell growth.