SUCLA2-coupled Regulation Of GLS Succinylation And Activity To Counteract Oxidative Stress Of Pancreatic Cancer Cells

[OBJECTIVE] Glutamine metabolism plays an important role in the development and progression of pancreatic cancer.GLS,the first key enzyme of glutaminolysis,is associated with tumor "glutamine addiction" and is considered as an important target for cancer therapy.However,the mechanism underlying glutaminase activity regulation is largely unknown.In this study,the effect of succinylation on the enzyme activity of glutaminase was investigated,and the specific mechanism of the regulation of GLS succinylation and its effect on pancreatic cancer was further studied.[METHODS] Firstly,the expression levels of GLS in normal human pancreatic duct epithelial cells and pancreatic cancer cells were detected by Western blot.The expression levels of GLS in pancreatic cancer tissues and adjacent normal human pancreatic tissues in TCGA database were analyzed.The dependence of pancreatic cancer cells on glutamine and GLS was determined.The GLS succinylation sites were identified by mass spectrometry and the main sites of GLS succinylation were clarified by point mutation and in vitro succinylation assay.Next,the effect of GLS succinylation on its enzymatic activity was studied by in vitro glutaminase activity assay.Molecular dynamics simulation and other methods were used to explore the specific mechanism of GLS enzyme activity alteration induced by succinylation modification.Then,the molecular mechanisms involved in the regulation of GLS succinylation were explored.Finally,the effect of GLS succinylation on the redox state of pancreatic cancer cells and its effect on the growth of pancreatic cancer were examined.[RESULTS] We demonstrated that pancreatic cancer cells showed a stronger dependence on glutamine,and knockdown of GLS significantly reduced the growth of pancreatic cancer cells.Importantly,GLS K311 succinylation promotes GLS oligomerization,which is likely due to a hydrogen bond formation between succinylated K311 and H475 of an adjacent monomer,leading to subsequently enhanced GLS activity.Importantly,GLS K311 succinylation is regulated by GLS-associated succinyl-Co A synthetase ADP-forming ? subunit(SUCLA2).Upon oxidative stress,SUCLA2 dissociates from GLS and is unable to catalyze local succinyl-Co A,resulting in enhanced K311 succinylation and activity of GLS.GLS K311 succinylation plays an important role in the maintenance of redox balance in pancreatic cancer cells.Activated GLS promotes tumor cell proliferation and tumor development in mice.[CONCLUSION] GLS is succinylated at K311 in pancreatic cancer cells by succinyl-Co A in a succinyl-Co A concentration-dependent manner,leading to enhanced GLS activity.SUCLA2 associates with GLS,and this association is disrupted by oxidative stress,resulting in increased available succinyl-Co A to enhance K311 succinylation and activity of GLS.Activated GLS helps maintain the redox balance of pancreatic cancer cells and promote tumor cell proliferation.