Atg7 Modulates AKT/pPDCD4 Axis To Regulate Cell Cycle During Metabolic Stress

Objective:Cellular homeostasis is the basis of cellular physiological activities,and imbalance in cellular homeostasis is likely to induce tumorigenesis or apoptosis.Therefore,investigating the mechanisms of cellular homeostasis is an important prerequisite for studying tumor development.In order to maintain cellular homeostasis,cells survive in a series of mechanisms,such as cell cycle checkpoints and autophagy.The presence of cell cycle checkpoints allows cells to stop temporarily at various periods in order to repair cellular damage,wait for exogenous stimulus signals to disappear or wait for the supply of growth factors,hormones and nutrients.Autophagy,on the other hand,provides nutrients to cells by degrading and recycling a variety of cellular components,such as long-lived proteins and damaged organelles,through the fusion of autophagic vesicles with lysosomes.Therefore,probing the mechanisms by which cells maintain homeostasis under metabolic stress can provide new insights into the process of tumor development.Autophagy Related Protein 7(Atg7),an E1-like ubiquitin-activating enzyme,is involved in the covalent binding of Atg12 to Atg5 and Atg8 to phosphatidylethanolamine lipids(PE),which are important proteins in the autophagy chain.By mass spectrometry we found that Atg7 is able to interact with factors that inhibit protein synthesis,i.e.negative regulatory proteins,reducing the production of non-essential proteins and further increasing the nutrients available in the cell.Programmed cell death protein 4(PDCD4)is one of the representatives of this class of proteins.PDCD4 is a negative regulatory protein that competitively binds to eIF4(eukaryotic initiation factor 4A)through its MA3 domain,which is homologous to MA1 in its structure,thereby inhibiting RNA unwinding and its associated protein translation.Therefore,we speculate that in response to metabolic stress,Atg7 is able to "produce"nutrients through autophagy,inhibit non-essential protein synthesis through PDCD4,and"cut the flow" while blocking the cell cycle to wait for exogenous nutrients to be supplied so that the cell can safely The cell cycle is also blocked while waiting for exogenous nutrients to be supplied,so that the cells can pass through the stress period and avoid death.Our study was based on the ability of Atg7 to degrade proteins and found that Atg7 increased the ubiquitinated degradation of PDCD4 by upregulating its phosphorylation level.The interaction of Atg7 with PDCD4 was weakened under metabolic stress,resulting in a weakened level of PDCD4 Ser67 phosphorylation,reduced ubiquitination degradation,and increased PDCD4 protein levels.It completes G1 phase cell cycle block and reduces apoptosis by affecting p21,Cdc2,Cdc25C and other cycle-associated eggs.Methods:1.To verify the interaction between Atg7 and PDCD4 using immunoprecipitation assay,GST-Pulldown,and immunofluorescence assay.2.Explore the effect of Atg7 on PDCD4 protein modification,i.e.phosphorylation modification and ubiquitination modification,using Western blot assay.3.To verify the effect of Atg7 on PDCD4 protein half-life using immunofluorescence assay and Western blot assay.4.To investigate the mechanism of Atg7 and PDCD4 in cell cycle and apoptosis under metabolic stress using flow cytometry and Western blot experiments..Results:5.Atg7 has a direct interaction with PDCD4.6.Overexpression of Atg7 promotes PDCD4 Ser67 phosphorylation,and knockdown of Atg7 attenuates PDCD4 Ser67 phosphorylation.7.Overexpression of Atg7 promoted AKT binding to PDCD4,and knockdown of Atg7 significantly weakened AKT binding to PDCD4.8.Atg7 degrades PDCD4 by promoting the level of PDCD4 ubiquitination and changes its half-life.9.Overexpression of PDCD4 promotes increased p21 protein expression and decreases Cdc2 and Cdc25C protein levels.10.Knockdown of PDCD4 rescues cell cycle disorder caused by Atg7 deletion(p<0.001).11.Knockdown of PDCD4 rescued apoptosis caused by Atg7 deletion(p<0.01)Conclusions:1.AKT,Atg7 and PDCD4 interact with each other.2.Atg7 promotes AKT-dependent phosphorylation of PDCD4 Ser67 and degrades PDCD4.3.Metabolic stress inhibits Atg7-PDCD4 pathway blocking cells to reduce apoptosis.