Studies on the clinical proteomics with bile acid tagged proteins as the targeted molecules

以胆汁酸标记蛋白为靶分子的临床蛋白质组学研究

• 批准号: 17590046
• 负责人: IKEGAWA Shigeo
• 金额: $ 2.24万
• 依托单位: Kinki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590046/
• 关键词: bile acid; LC; MS; acyl-adenylate; glutathione; CoA-thioester; lithocholic acid; rat; protein; 抗体; 胆汁酸CoAリガーゼ; 二次元電気泳動

Formation of covalently bound protein adducts with lithocholic acid (LCA) has been proposed as a possible explanation for hypersensitivity and toxic responses of this bile acid. We therefore carried out to capture of LCA-modified cellular proteins in the bile duct-ligated rat liver by immunoprecipitation using an antibody with high affinity to the 3α-hydroxy-5β-steroid moiety of LCA. Results of the structural analysis of the captured proteins by the basic proteomic technique of 2-DE combined with the use of MALDI-TOFMS and computer assisted programs indicated the presence of LCA bound Rab proteins, which may be the course of LCA-induced liver toxicity.Further studies on the formation of the glutathione (GSH) conjugates via trans-acylation reactions between GSH and reactive acyl-linked metabolites of bile acids have been carried out to demonstrate the presence of detoxification mechanism. For this purpose, the authentic GSH conjugates of five major bile acids were chemically synthesized … More and their structures were characterized by ESI-tandem mass spectrometry in negative and positive ion modes. Incubation of cholyl-adenylate and cholyl-CoA, reactive metabolites of cholic acid (CA), with GSH in a phosphate buffer (pH 7.5) solution demonstrated that both compounds are able to acylate GSH in vitro. In addition, we cloned and expressed rat and human liver bile acid CoA-ligase to better understand the mechanism for the formation of acyl-adenylate and bile acid bound proteins.Finally, our efforts were directed to explore the proteins having affinity with LCA in rat liver by using affinity adsorbents, in which LCA was covalently bound to an activated agarose through the bridges introduced at the C-3 and C-24 positions of LCA. Results of LC/ESI-MS^n analysis of the captured proteins after separation by SDS-PAGE revealed the presence of functional proteins, i.e., carbamoyl phosphate synthase I, glutamate dehydrogenase, acyl-CoA acyltransferase, glutathione-S-transferase, and catalase, indicating the hypothesis involved in the appearance of the liver toxicity by inhibition of these enzymes by LCA. Less

与石胆酸（LCA）形成共价结合的蛋白加合物被认为是这种胆汁酸过敏和毒性反应的可能解释。因此，我们使用一种对LCA的3α-羟基-5β-类固醇部分具有高亲和力的抗体，通过免疫沉淀法捕获胆管结连大鼠肝脏中LCA修饰的细胞蛋白。利用2-DE基本蛋白质组学技术结合MALDI-TOFMS和计算机辅助程序对捕获的蛋白质进行结构分析，结果表明存在LCA结合的Rab蛋白，这可能是LCA诱导的肝毒性过程。通过谷胱甘肽（GSH）与胆汁酸的活性酰基连接代谢物之间的反式酰化反应形成谷胱甘肽（GSH）偶联物的进一步研究已经证实了解毒机制的存在。为此，化学合成了5种主要胆汁酸的GSH偶联物，并通过esi -串联质谱法在正离子和负离子模式下对其结构进行了表征。胆酸（CA）的活性代谢物胆酰腺苷酸和胆酰辅酶a与谷胱甘肽在磷酸盐缓冲液（pH 7.5）中孵育表明，这两种化合物都能体外酰基化谷胱甘肽。此外，我们克隆并表达了大鼠和人肝脏胆汁酸辅酶a连接酶，以更好地了解酰基腺苷酸和胆汁酸结合蛋白的形成机制。最后，我们利用亲和吸附剂探索大鼠肝脏中与LCA有亲和关系的蛋白，其中LCA通过在LCA的C-3和C-24位置上引入的桥与活化的琼脂糖共价结合。SDS-PAGE分离后，对捕获的蛋白质进行LC/ESI-MS^n分析，发现存在功能蛋白，即氨甲酰磷酸合酶I、谷氨酸脱氢酶、酰基辅酶a酰基转移酶、谷胱甘肽- s转移酶和过氧化氢酶，表明LCA抑制这些酶导致肝毒性出现的假设。少