The Study On The Function And The Mechanism Of ATP-Citrate Lyase O-GLCNAcylation In Tumor Cell Proliferation

In contrast to differentiated cells,tumor cells need to undergo significant metabolic reprogramming to meet the increasing demands for biosynthetic material,energy,and reducing power required for their proliferation as well as oxidative stress mitigation.One of the best characterized metabolic alterations observed in tumor cells is the vigorous de novo synthesis of fatty acids,which can produce sufficient membrane lipids for dividing tumor cells.It is known that acetyl-CoA in mitochondria produced by glucose catabolism is the main raw material for de novo synthesis of fatty acids.However,de novo synthesis of fatty acids occurs in the cytoplasm,and acetyl-CoA cannot pass directly through the mitochondrial membrane.It is necessary to condense with mitochondrial oxaloacetate to form citrate,and to be transported to the cytoplasm in the form of citrate.Citrate can then be catalyzed by ATP-citrate lyase（ACLY）to lysis citrate to form acetyl-CoA,acting as the raw material for de novo synthesis of fatty acids.Although the reaction catalyzed by ACLY does not involve the elongation of two-carbon units during de novo fatty acid synthesis,the availability of acetyl-CoA in the cytoplasm is the key for the de novo fatty acid synthesis initiation.Thus,ACLY is widely defined as the first rate-limiting enzyme in the de novo fatty acid synthesis pathway and is regarded as a key regulator connecting glucose and lipid metabolism.It has been well documented that the activity of ACLY can be regulated by a variety of covalent modifications,such as phosphorylation,acetylation,ubiquitination,etc.Recently,ACLY was also found to be O-GlcNAcylated.O-GlcNAcylation is a mode of protein post-translational modification widely occurring on serine or threonine residues,and its donor UDP-GlcNAc is closely related to the metabolism of four biological macromolecules;thus,O-GlcNAcylation is recognized as the"nutrient sensor"of cells.However,the functional consequence of O-GlcNAcylation of ACLY as well as its implication in tumor cell proliferation are still unknown.In the present study,firstly,we analyzed the O-GlcNAcylation levels of ACLY in tumor cells and tumor tissues and identified the modification sites.1)We confirmed the existence of ACLY O-GlcNAcylation in tumor cells and tumor tissues and compared the O-GlcNAcylation levels of ACLY between normal and tumor cell lines,and between human lung cancer tissues and adjacent tissues.The results showed that the O-GlcNAcylation level of ACLY was significantly up-regulated in tumor cells and human lung cancer tissues;Meanwhile,we evaluated the ratio of O-GlcNAcylated ACLY in rapidly proliferating tumor cells and found that its abundance was about10%.2)Subsequently,by using mass spectrometry techniques and point mutation experiments,we detected the O-GlcNAcylation site of ACLY and identified that Ser⁹⁷⁹was the major site of ACLY undergoing O-GlcNAcylation.3)The effects of overexpression of O-GlcNAc transferase（OGT）or treatment with O-GlcNAcase（OGA）inhibitor revealed that the O-GlcNAcylation of Ser⁹⁷⁹is regulated by OGT and OGA.Secondly,we explore the role and mechanism of O-GlcNAcylation of Ser⁹⁷⁹in the regulation of ACLY enzymatic activity and fatty acid anabolism in tumor cells.1)By overexpression of OGT or use of OGA inhibitor,we found the Ser⁹⁷⁹O-GlcNAcylation level was increased,and ACLY activity was significantly enhanced detected by using an enzyme activity assay kit.The results showed that increasing the level of ACLY O-GlcNAcylation at Ser⁹⁷⁹could significantly enhance its activity.2)Furthermore,we explored the mechanism of Ser⁹⁷⁹O-GlcNAcylation on ACLY enzymatic activity.Proved by enzymatic kinetic experiments,Ser⁹⁷⁹O-GlcNAcylation could up regulate ACLY activity by promoting the association of ACLY to its substrate CoA.The consistent results were verified by using biofilm interferometric and isothermal titration calorimetry assays.3)The effect of Ser⁹⁷⁹O-GlcNAcylation of ACLY on fatty acid anabolism in tumor cells was analyzed by HPLC-tandem mass spectrometry.The results showed that the level of ACLY O-GlcNAcylation at Ser⁹⁷⁹was highly positively correlated with the abundance of fatty acids and their products.Next,the influencing factors on ACLY O-GlcNAcylation were investigated.1)The results showed that the level of ACLY O-GlcNAcylation at Ser⁹⁷⁹could be directly regulated by glucose concentration,and the increase of Ser⁹⁷⁹O-GlcNAcylation level mediated by high-level glucose could significantly up regulate the enzyme activity of ACLY,so as to promote the de novo synthesis of fatty acids.2)The activated-type modification of ACLY,Ser⁴⁵⁵phosphorylation,is also known to be regulated by the glucose concentration.This suggests a possible crosstalk between ACLY Ser⁴⁵⁵phosphorylation and Ser⁹⁷⁹O-GlcNAcylation.Various mutants(ACLY^S455A,ACLY^S455D,and ACLY^S979A)were used to test for an interaction between the two modifications.The results showed that both Ser⁴⁵⁵phosphorylation and Ser⁹⁷⁹O-GlcNAcylation level and ACLY activity could respond to glucose concentration changes,but the two modifications did not affect each other.Additionally,the effect of Ser⁹⁷⁹O-GlcNAcylation on ACLY activity was independent of Ser⁴⁵⁵phosphorylation and played a regulatory role comparable to Ser⁴⁵⁵phosphorylation.3)The role of the O-GlcNAcylation of ACLY at Ser⁹⁷⁹in fatty acid anabolism regulated by glucose was analyzed using the[U-¹³C]glucose tracer metabolic flow technique.The result showed that glucose level can regulate ACLY O-GlcNAcylation at Ser⁹⁷⁹,and thereby promoting the entry of glucose-derived carbon units into de novo fatty acid synthesis pathways.Finally,the effects of ACLY O-GlcNAcylation on tumor cell proliferation and tumor growth were evaluated at the cellular and animal levels.1)Cell counting and crystal violet staining showed that ACLY Ser⁹⁷⁹O-GlcNAcylation was positively correlated with tumor cell proliferation rate;proliferation of ACLY^WTcells increased with glucose concentration,but ACLY^S979Acells did not respond to the alteration of glucose concentration.2)Mouse tumor-bearing experiments using ACLY^WTand ACLY^S979Astable transgenic cell lines showed that removal of ACLY O-GlcNAcylation at Ser⁹⁷⁹(ACLY^S979A)significantly inhibited tumor growth.Compared to that of ACLY^WTcells,tumor composed of the ACLY^S979Astable transgenic cells was significantly reduced both in volume and weight in mice and exhibited less expression of the proliferation marker Ki67.In conclusion,we proposed a new regulation mechanism of ACLY activity.In tumor cells,Ser⁹⁷⁹O-GlcNAcylation enhances ACLY activity by promoting ACLY binding to its substrate CoA,and subsequently promotes de novo fatty acid synthesis,favoring tumor cell proliferation.More importantly,the O-GlcNAcylation of ACLY at Ser⁹⁷⁹can"sense"the changes in glucose concentration,thus acting as a key regulator linking glucose metabolism to fatty acid synthesis.The present study not only reveals a new mechanism of glucose and lipid metabolism turnover in tumor cells,but also provides new ideas and new targets for tumor therapy based on the regulation of glucose-lipid metabolism in future.