| Objective:Crotonylation,as a new type of protein post-translational modified(PTM),exists widely in eukaryotic cells and is evolutionarily conserved.Crotonylation can be reversibly regulated by crotonyltransferase and decrotonylase.Proteomic analysis shows that many key cellular proteins that regulate physiological function and participate in the pathogenesis of the disease can be modified by crotonylation.However,the role of crotonylation in the pathogenesis of colorectal cancer(CRC)is still unclear.PRKACA is the main catalytic subunit of protein kinase A(PKA),and its mutation can lead to adrenocortical tumors.As a key enzyme in glycolysis pathway,overexpression of ENO1(α-enolase)in CRC is a carcinogenic factor.Our purpose is to study the relationship between crotonylation of PRKACA and ENO1 and CRC,and to explore its effect on the biological function of CRC and its downstream pathway,in order to identify the key links in the pathogenesis of CRC and provide potential targets for the diagnosis and treatment of CRC.Methods:We first detected the whole crotonylation level in orthotopic colorectal cancer and adjacent normal tissues by immunoblotting,and quantitatively identified crotonylated proteins and sites by 4D-LFQ proteomics combined with crotonylomics and bioinformatics analysis.Then,the crotonyltransferase and decrotonylase of PRKACA and ENO1 were screened by immunoprecipitation.Then,the expression of SIRT3 in CRC tissue was detected by immunoblotting and immunohistochemistry,and the effect of SIRT3 on the proliferation,invasion,and migration of HCT116 was detected by CCK8,colony formation test,Transwell method and wound healing assay.Next,the main crotonylation sites of PRKACA and ENO1 were identified by Tims TOF Pro MS/MS.PRKACA-K84 Q,PRKACA-K84 R,ENO1-K420 Q or ENO1-K420 R plasmids were constructed and transfected into colon cancer cells,and the following studies were carried out: to detect the effects of PRKACA-K84 Q,K84R,ENO1-K420 Q,420R on the growth,invasion,migration,PKA enzyme activity and ENO1 activity of colon cancer cells;to construct the crystal structure of PKA and immunoprecipitation to explore the interaction between PRKACA K84 and regulatory subunits.The intracellular and extracellular lactic acid content of ENO1 K420 Q and K420 R and the cell survival rate under glucose-free culture were measured.At the same time,si PRKACA+ K84Q/ WT and si ENO1+K420Q/ WT HCT116 cells were sequenced by RNAseq,the differentially expressed genes were analyzed,and the downstream pathways were verified by fluorescence quantitative PCR and Western blotting.Finally,the expression levels of PRKACA and ENO1 crotonylation in CRC carcinoma and paracancerous tissues were analyzed by immunoprecipitation.Results:Crotonylated proteins in colon cancer are mainly located in the cytoplasm,followed by mitochondria and nuclei.The motif of crotonylation is characterized by negatively charged amino acids.Many biological processes,such as metabolic process,cell junction,enzymatic reaction and so on,are involved in many biological processes,including PRKACA and ENO1.CBP upregulated the crotonylation levels of PRKACA and ENO1,while SIRT3 and SIRT2 down-regulated the crotonylation levels of PRKACA and ENO1,respectively.The expression of SIRT3 in CRC was significantly decreased,and overexpression in vitro could inhibit the progression of CRC.K84 and K420 are the main crotonylation sites of PRKACA and ENO1,respectively.PRKACA K84 Q and ENO1 K420 Q enhanced the proliferation,invasion,and migration of colon cancer cells,while K84 R and K420 R showed the opposite effect.With or without Forskolin stimulation,PRKACA K84 Q increased PKA activity and phosphorylated CREB Ser133,while K84 R had the opposite effect.The crystal structure of PKA shows that K84 is located at the junction of PRKACA and PRKAR1 A.Immunoprecipitation assay showed that the binding ability of PRKACA K84 Q to endogenous and exogenous PRKAR1 A decreased,and RP-c AMPS could not inhibit the increased PKA activity of K84 Q.The differentially expressed genes between PRKACA WT and K84 Q focus on tumor-related pathways and functions.PRKACA K84 Q phosphorylated FAK Y397,Akt S473,Paxillin Y118 and increased the level of CCNE2 protein.This effect was inhibited by H89,but the effect of K84 R was opposite.ENO1 K420 Q significantly increased the activity of ENO1,increased the content of lactate inside and outside the cell,and decreased the apoptosis rate of HCT116 cells in the absence of glucose,while ENO1K420 R had the opposite effect.The differentially expressed genes of ENO1 K420 Q and WT were also enriched in tumor-related pathways and crossed with genes affected by PRKACA K84 Q.PRKACA and ENO1 crotonylation increased in CRC tumor tissues,and had nothing to do with protein expression,different TNM stages and clinicopathological features of patients.Conclusion:The level of crotonylation in CRC tissue is up-regulated and participates in a variety of tumor-related functional pathways;CBP is the crotonyltransferase of PRKACA and ENO1;SIRT3 and SIRT2 are its decrotonylase respectively;SIRT3 is lacking in CRC tissue and is the tumor suppressor of CRC.Our study revealed that PRKACA-K84 crotonylation promotes the growth and metastasis of CRC by reducing the binding of PRKACA to regulatory subunits and structurally activating the activity of PKA,resulting in phosphorylation of downstream FAK/AKT pathway and further up-regulation of Paxillin phosphorylation and CCNE2 protein expression.ENO1-K420 crotonylation can increase the content of lactic acid inside and outside the tumor cells by activating ENO1 enzyme activity,thus maintaining the vitality of cancer cells when glucose is deficient.Crotonylation of PRKACA and ENO1 is related to the occurrence and development of colon cancer,but has nothing to do with the progression of colon cancer,and ENO1-K420 crotonylation and PRKACA-K84 crotonylation affect common tumor-related genes,suggesting that crotonylation is a potential carcinogenic factor in CRC,which provides a new insight for the study of the pathogenesis of CRC. |
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