矩阵热转移技术鉴定有机酸类代谢物的结合蛋白质OA北大核心CSTPCDMEDLINE

Organic acid metabolites exhibit acidic properties.These metabolites serve as inter-mediates in major carbon metabolic pathways and are involved in several biochemical path-ways,including the tricarboxylic acid(TCA)cycle and glycolysis.They also regulate cellular activity and play crucial roles in epigenetics,tumorigenesis,and cellular signal transduction.Knowledge of the binding proteins of organic acid metabolites is crucial for understanding their biological functions.However,identifying the binding proteins of these metabolites has long been a challenging task owing to the transient and weak nature of their interactions.Moreover,traditional methods are unsuitable for the structural modification of the ligands of organic acid metabolites because these metabolites have simple and similar structures.Even minor structural modifications can significantly affect protein interactions.Thermal proteome profiling(TPP)provides a promising avenue for identifying binding proteins without the need for structural modifications.This approach has been successfully applied to the identification of the binding proteins of several metabolites.In this study,we investigated the binding proteins of two TCA cycle intermediates,i.e.,succinate and fumarate,and lactate,an end-product of glycolysis,using the matrix thermal shift assay(mTSA)technique.This technique involves combining single-temperature(52 ℃)TPP and dose-response curve analysis to identify ligand-binding pro-teins with high levels of confidence and determine the binding affinity between ligands and pro-teins.To this end,HeLa cells were lysed,followed by protein desalting to remove endogenous metabolites from the cell lysates.The desalted cell lysates were treated with fumarate or succi-nate at final concentrations of 0.004,0.04,0.4,and 2 mmol/L in the experimental groups or 2 mmol/L sodium chloride in the control group.Considering that the cellular concentration of lac-tate can be as high as 2-30 mmol/L,we then applied lactate at final concentrations of 0.2,1,5,10,and 25 mmol/L in the experimental groups or 25 mmol/L sodium chloride in the control group.Using high-sensitivity mass spectrometry coupled with data-independent acquisition(DIA)quantification,we quantified 5 870,5 744,and 5 816 proteins in succinate,fumarate,and lactate mTSA experiments,respectively.By setting stringent cut-off values(i.e.,signifi-cance of changes in protein thermal stability(p-value)＜0.001 and quality of the dose-response curve fitting(square of Pearson's correlation coefficient,R2)＞0.95),multiple binding pro-teins for these organic acid metabolites from background proteins were confidently determined.Several known binding proteins were identified,notably fumarate hydratase(FH)as a binding protein for fumarate,and a-ketoglutarate-dependent dioxygenase(FTO)as a binding protein for both fumarate and succinate.Additionally,the affinity data for the interactions between these metabolites and their binding proteins were obtained,which closely matched those repor-ted in the literature.Interestingly,ornithine aminotransferase(OAT),which is involved in ami-no acid biosynthesis,and 3-mercaptopyruvate sulfurtransferase(MPST),which acts as an an-tioxidant in cells,were identified as lactate-binding proteins.Subsequently,an orthogonal assay technique developed in our laboratory,the solvent-induced precipitation(SIP)technique,was used to validate the mTSA results.SIP identified OAT as the top target candidate,validating the mTSA-based finding that OAT is a novel lactate-binding protein.Although MPST was not identi-fied as a lactate-binding protein by SIP,statistical analysis of MPST in the mTSA experiments with 10 or 25 mmol/L lactate revealed that MPST is a lactate-binding protein with a high level of confidence.Peptide-level empirical Bayes t-tests combined with Fisher's exact test also sup-ported the conclusion that MPST is a lactate-binding protein.Lactate is structurally similar to pyruvate,the known binding protein of MPST.Therefore,assuming that lactate could potentially occupy the binding site of pyruvate on MPST.Overall,the novel binding proteins identified for lactate suggest their potential involvement in amino acid synthesis and redox balance regulation.