BAG3 Reduces Cellular Adaptive Response To Metabolic Stress Via P53-dependent And-independent Manners

Objective: Glucose is the most important energy substrate for energy metabolism during cell proliferation and differentiation,and is the main carbon source in cells.During the occurrence and development of solid malignant tumors,continuous or intermittent glucose deficiency is a feature of solid tumors due to excessive glucose consumption caused by metabolic reprogramming and insufficient blood supply inside the tumors caused by uncontrolled proliferation of solid tumor cells.Therefore,how solid tumors adapt to nutrient deficiency,especially glucose deficiency,so as to maintain their normal survival and proliferation under the environment of metabolic stress is still an unresolved problem.Human BAG(Bcl-2-associated athanogene)proteins form an evolutionarily conserved family of antiapoptotic proteins.BAG3 is a member of BAG family.Participate in regulation of many life activities,including cell proliferation apoptosis adhesion transfer of oxidative stress autophagy etc recently discovered BAG3 can directly with Hexokinase HK2(2,Hexokinase HK2),pancreatic cancer cells glycolysis,BAG3 may participate in the process of tumor cell metabolic regulation but BAG3 under the condition of stress because of the lack of glucose metabolism play a role of what is not clear.As a tumor suppressor,p53 hasreports confirmed that it participates in the metabolism and metabolic regulation to maintain steady,cells in inhibition of cancer occurrence and development play a key role in the regulation of p53 gene mutation or missing change the glycolysis of tumor cells and the balance of aerobic respiration,promote glycolysis and reduce oxidative phosphorylation,so as to promote the development of tumorigenesis and metastasis when organism or cell in different stress states,activation of p53 by participating in fatty acid metabolism in sugar metabolism of ROS levels related to regulate signaling pathways that affect various metabolic pathways,and then by inducing cell cycle arrest repairSenescence or apoptosis,and ultimately to regulate the cellular metabolism or stress when cell DNA damage or deficiency,higher degree of p53 protein phosphorylation,slow degradation,p53 protein expression in cell volume rapidly rising,stagnation of cells will thus in G1 phase,inhibiting anabolic promoting catabolism,which benefits the survival of cells while inadequacy of p53 transcriptional activity in glucose metabolic stress protect cells play a key role,but the specific mechanism of action and the downstream target molecules did not clarify.The preliminary results of this study showed that: glucose deficiency significantly reduced the expression of BAG3 in p53 wild-type HCT116 cells,and had no significant effect on the expression of p53-deficient HCT116 cells.However,glucose deficiency did not affect the expression level of other members of the BAG family,so we took BAG3 as the next major research objectAs a molecular chaperone,BAG3 can bind to many proteins.Experiments have further confirmed that BAG3 can bind to p53 to promote the degradation of p53 and regulate the expression of p53 downstream genes.This project aims to explore the role and specific mechanism of BAG3 in metabolic stress caused by glucose deficiency based on previous resultsMethods: I.Colon cancer cell lines HCT116(p53+/+)and HCT116(p53-/-)were cultured to construct p53+/+ and p53-/-mouse MEF cells.Western blot was used to detect BAG3 protein expression under conditions of adequate glucose and glucose deficiency,and qRT-PCR was used to detect the expression of BAG3 total mRNA.II.HCT116(p53+/+)and HCT116(p53-/-)cell lines that stably overexpressing BAG3 were constructed by lentivirus infection.BAG3 konckin mice were constructed and MEF cells were extracted.1.Cell counting assay was used to detect the cell survival of HCT116 and MEF cells under the conditions of glucose sufficiency and glucose deficiency.2.Flow cytometry was used to detect the cell cycle changes of HCT116 and MEF cells under the conditions of glucose sufficiency and glucose deficiency.3.qRT-PCR was used to detect the expression of total p53 mRNA under the conditions of glucose sufficiency and glucose deficiency.CHX was used to inhibit intracellular protein synthesis,and Western blot was used to detect the expression of BAG3 and p53 at each dosing time point.4.The degradation pathway of p53 in the absence of glucose culture was analyzed after the addition of proteasome hydrolysis inhibitor MG132 and autophagic lysosomal inhibitor E64D/PepstatinA.5.Detection of direct interaction between BAG3 and p53 using Doulink technique;BAG3 and p53 protein domain deletion expression vectors were constructed,and the binding regions of BAG3 and p53 were determined by CO-IP technology.6.BAG3 luciferase reporter gene expression vector was constructed and transfected into cells,and luciferase activity was detected.7.HPG was used to detect the synthesis of cell new protein in the condition of adequate glucose and lack of glucose.8.PCR Array was used to detect the RNA expression of p53 downstream target gene under the conditions of glucose sufficiency and glucose deficiency,and its RNA and Western blot were verified to detect the protein expression in HCT116 and MEF cells.9.P21,GADD4 a and SESN2 expression vectors were constructed and transfected into cells,and crystal violet staining was used to detect the viability of cells under conditions of sufficient glucose and insufficient glucose culture.10.RIP assay detects the recruitment of SESN2 mRNA by RNA-binding protein BAG3 itself.The SESN2 5 ’UTR,CR and 3’ UTR luciferase reporter gene expression vectors were constructed and transfected into the cells.Luciferase activity was detected 48 h later.11.RNA expression of SESN2 was detected by qRT-PCR and SESN2 expression was detected by Western blot in BAG3 cells with locus deletion of 67-76 aa,261-294 aa and 473-485 aa.12.Biotin pull-down assay with biotin markers was performed to detect the binding sites of BAG3 to SESN2 transcript and whether the binding sites were direct or not.Results: 1.Compared with normal culture,BAG3 mRNA and protein levels were down-regulated in a p53-dependent manner under the condition of glucose deficiency.2.BAG3 reduces the survival rate of low-glucose cultured tumor cells through cell cycle arrest caused by metabolic stress and activation of p53.3.Duolink experiment confirmed the direct interaction between BAG3 and p53.4.BAG3 promotes calpain-dependent degradation of p53 and weakens its protective effect on cells in metabolic stress.5.HPG intragination confirmed that BAG3 promoted the synthesis of new proteins in response to metabolic stress,leading to greater cell anabolism than catabolism,which was not conducive to cell survival.6.PCR Array was used to screen out the significantly increased expression of GADD45 a p21 SESN2 under low glucose stress.The induced expression of GADD45 a p21 was dependent on p53,and the induced expression of GADD45 a p21 was independent of p53.7.The expression of GADD45 a p21 SESN2 was inhibited by BAG3 at the RNA and protein levels under low glucose culture conditions.8.BAG3 inhibits SESN2 mRNA stability during metabolic stress.9.Luciferase reporter results confirm that BAG3 acted on the 3’UTR region of SESN2 mRNA and reduces the stability of the transcript.10.RIP assay showed that BAG3 recruitment of SESN2 mRNA could be detected in HCT116 cells under normal culture and glucose deficiency.11.In vitro pull down experiments using BAG3 domain deletion mutants and biotin markers demonstrate that BAG3 binds directly to SESN2 3 ’UTR via its 67-76 aa and 261-294 aa sites,thereby reducing SESN2 stability.Conclusion: During the metabolic stress caused by glucose deficiency,on the one hand,BAG3 directly interacts with p53 protein to promote the degradation of p53 and inhibit the activation of p53-dependent p21 and GADD45 a,thus damaging the protective effect of p53 on cells in the metabolic stress.On the other hand,the direct binding of BAG3 to SESN2 mRNA promotes the degradation of its transcripts and reduces the adaptability of tumor cells to metabolic stress,suggesting that BAG3 can selectively kill tumor cells lacking glucose tolerance,thus providing new ideas for reducing the recurrence rate of tumor cells.