BAG3 Promotes Autophagy And Glutaminolysis Via Stabilizing Glutaminase

Objective: Autophagy is an evolutionarily conserved catabolic process that is important to maintain cellular homeostasis.Although autophagy was considered to be a random process for many years.With the development of scientific research,accumulating data now support that autophagy is a selective process and receives tight regulation.Metabolic reprogramming is one of the hallmarks of malignant tumors.Changes in cellular metabolism enable tumors to sustain their increased energetic needs.As malignant proliferating tumor cells usually acquired energy by glycolysis,most studies focus on glycometabolism.Glutaminolysis initiates with deamination of glutamine by glutaminase(GLS),by which yields glutamate and ammonia in mitochondria.Glutamine is avidly consumed in rapidly proliferating tumor cells,and Warburg effect was replaced by glutaminolysis to produce ATP,and which acts as a source of energy for tumor cells.Researchers recently have paid more attention to glutaminolysis.Glutaminolysis takes place in all proliferating cells and plays a critical role in maintaining bioenergetics and providing nitrogen,sulfur and carbon skeletons for macromolecular biosynthesis.Glutaminolysis also plays an important role in regulating redox balance,m TOR signaling.In addition,glutaminolysis is an important source of cellular ammonia,which induces autophagy in tumor cells.BAG3(Bcl-2 associated athanogene)is an important molecule that maintains oncogenic features of cancer cells via diverse mechanisms.One of the important functions assigned to BAG3 is to degrade misfolded proteins or aggregates by regulating selective macroautophagy/autophagy under stressful conditions,and maintain protein homeostasis.As a regulator of autophagy,BAG3 plays an important role in maintaining cell homeostasis,which has attracted much attention in recent years.However,the mechanism underlying regulation of autophagy by BAG3 has not been well defined.We have reported that BAG3 plays a critical role in Beclin1-and Ptd Ins3K-independent noncanonical autophagy under proteasome inhibition previously.Downregulation of BAG3 expression by sh RNA can significantly inhibit the autophagy induced by proteasome inhibitors,but the molecular mechanism of BAG3 promoting autophagy remains unclear.It has been reported that SIRT5 is responsible for desuccinylation of GLS1.Silencing SIRT5 can promote glutaminolysis and autophagy,but the succinylation sites of GLS is still unclear.In previous study,we found that BAG3 increased glutamine consumption,ammonia production and GLS expression,and BAG3 increased succinylation levels at Lys 158 and Lys 164 sites.Therefore,we speculate that BAG3 may promote glutaminolysis and autophagy by increasing the expression of GLS succinylation.By analyzing the mechanism of BAG3 regulating glutaminolysis and autophagy in cells,the molecular mechanism of BAG3 upregulating GLS and its succinylation was revealed,and the important role of GLS and its succinylation in cell energy metabolism and autophagy was clarified,which could provide more theoretical and experimental basis for finding new drug targets for cancer treatment.Methods: 1.Hep G2 or MCF7 cells were infected with lentivirus containing empty,BAG3,GLS and their mutants GLS(K158/164A),GLS(K158/164E)construct.Hep G2-BAG3 cells and MCF7-BAG3 cells were infected with scramble sh RNA or sh RNA specific against GLS(sh GLS).2.Western blot was used to detect the expression of BAG3,GLS,GLS(K158/164A),GLS(K158/164E)and autophagy-related proteins.3.Hep G2 or MCF7 cells stably overexpressing pc DNA3.1-EGFP-LC3 B were infected with lentivirus containing empty or BAG3 construct.4.The punctate distribution of EGFP-LC3 B was visualized under the florescence microscopy.5.The ultrastructure was analyzed using transmission electron microscopy.6.Assay kit was used to detect the consumption of glutamine in culture medium.7.The production of glutamate and ?-ketoglutarate in cells was detected by kit.8.Ammonia production in culture medium was detected by kit.9.The total RNA was extracted and purified,and the reverse transcription was used to synthesize the c DNA.RT-q PCR was used to detect the expression level of the target gene GLS.10.Cycloheximide(CHX)was used to inhibit intracellular protein synthesis.Western blot was used to detect the expression of GLS and its mutants GLS(K158/164A)and GLS(K158/164E)at different time points.11.Mass spectrometry and IP were used to detect the succinylation sites and succinylation of GLS.12.Co-immunoprecipitation was used to detect protein interaction.13.In vivo ubiquitination assay was used to detect the ubiquitination of GLS and its mutants GLS(K158/164A)and GLS(K158/164E).Results: 1.Ectopic BAG3 expression induces autophagy.2.BAG3 overexpression induces autophagy independent of Beclin1 and Ptd Ins3 K.3.BAG3 promotes autophagy via upregulation of glutaminase(GLS).4.BAG3 stabilizes GLS by suppression its proteasomal degradation.5.Ectopic BAG3 expression increases GLS succinylation at Lys158 and Lys164 sites.6.Succinylation at Lys158 and Lys164 sites stabilizes GLS via suppression its Lys48-linked ubiquitination.Conclusion: BAG3 enhances succinylation of GLS at Lys158 and Lys164 sites via both SIRT5 reduction and interaction with GLS,inhibits its proteasomal degradation of GLS and stabilizes GLS,thereby promoting glutaminolysis and activating autophagy.