有机酸代谢物表现出酸性性质。这些代谢物充当主要碳代谢途径的中间体,并参与几种生化途径。包括三羧酸(TCA)循环和糖酵解。它们还调节细胞活动并在表观遗传学中起关键作用,肿瘤发生,和细胞信号转导。了解有机酸代谢物的结合蛋白对于理解其生物学功能至关重要。然而,鉴定这些代谢物的结合蛋白长期以来一直是一项具有挑战性的任务,因为它们相互作用的短暂和弱性质。此外,传统的方法不适用于有机酸代谢物配体的结构修饰,因为这些代谢物具有简单和相似的结构。即使是微小的结构修饰也会显著影响蛋白质相互作用。热蛋白质组谱分析(TPP)为鉴定结合蛋白提供了一种有希望的途径,而无需进行结构修饰。该方法已成功应用于几种代谢物的结合蛋白的鉴定。在这项研究中,我们研究了两个TCA循环中间体的结合蛋白,即,琥珀酸盐和富马酸盐,和乳酸,糖酵解的最终产物,使用基质热移位测定(mTSA)技术。该技术涉及结合单温度(52℃)TPP和剂量反应曲线分析,以确定具有高置信度的配体结合蛋白,并确定配体与蛋白之间的结合亲和力。为此,HeLa细胞被裂解,然后进行蛋白质脱盐以从细胞裂解物中去除内源性代谢物。将脱盐的细胞裂解物用富马酸盐或琥珀酸盐处理,实验组的终浓度为0.004、0.04、0.4和2mmol/L,对照组的终浓度为2mmol/L的氯化钠。考虑到乳酸的细胞浓度可高达2-30mmol/L,然后我们在实验组中施用终浓度为0.2,1,5,10和25mmol/L的乳酸,在对照组中施用25mmol/L的氯化钠.使用高灵敏度质谱与数据无关采集(DIA)定量相结合,我们量化了琥珀酸中的5870、5744和5816种蛋白质,富马酸盐,和乳酸mTSA实验,分别。通过设置严格的截止值(即,蛋白质热稳定性(p值)<0.001和剂量反应曲线拟合质量变化的显著性(皮尔逊相关系数的平方,R2)>0.95),从背景蛋白中确定了这些有机酸代谢物的多种结合蛋白。鉴定了几种已知的结合蛋白,特别是富马酸水合酶(FH)作为富马酸盐的结合蛋白,和α-酮戊二酸依赖性双加氧酶(FTO)作为富马酸盐和琥珀酸盐的结合蛋白。此外,获得了这些代谢物与其结合蛋白之间相互作用的亲和力数据,这与文献中报道的非常吻合。有趣的是,鸟氨酸转氨酶(OAT),参与氨基酸的生物合成,和3-巯基丙酮酸硫转移酶(MPST),作为细胞中的抗氧化剂,被鉴定为乳酸结合蛋白。随后,我们实验室开发的正交测定技术,溶剂诱导沉淀(SIP)技术,用于验证mTSA结果。SIP将OAT确定为最高目标候选,验证了基于mTSA的发现,即OAT是一种新型的乳酸结合蛋白。尽管MPST未被SIP鉴定为乳酸结合蛋白,在使用10或25mmol/L乳酸的mTSA实验中,对MPST的统计学分析显示,MPST是一种具有高置信度的乳酸结合蛋白。肽水平经验贝叶斯t检验结合Fisher精确检验也支持MPST是乳酸结合蛋白的结论。乳酸在结构上与丙酮酸相似,MPST的已知结合蛋白。因此,假设乳酸可能潜在地占据MPST上丙酮酸的结合位点。总的来说,发现的新型乳酸结合蛋白提示其可能参与氨基酸合成和氧化还原平衡调节。
Organic acid metabolites exhibit acidic properties. These metabolites serve as intermediates in major carbon metabolic pathways and are involved in several biochemical pathways, 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 proteins with high levels of confidence and determine the binding affinity between ligands and proteins. 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
succinate 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 lactate 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 5870, 5744, and 5816 proteins in
succinate, fumarate, and lactate mTSA experiments, respectively. By setting stringent cut-off values (i.e., significance 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 proteins 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 α-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 reported in the literature. Interestingly, ornithine aminotransferase (OAT), which is involved in amino acid biosynthesis, and 3-mercaptopyruvate sulfurtransferase (MPST), which acts as an antioxidant 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 identified 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 supported 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.