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

石胆酸(LCA)与共价结合蛋白加合物的形成可能解释了胆酸的超敏反应和毒性反应。因此，我们采用免疫沉淀法从胆管结扎的大鼠肝脏中捕获经低密度脂蛋白修饰的细胞蛋白，用一种对低密度脂蛋白的3-α-羟基-5-β类固醇部分具有高亲和力的抗体。利用2-DE基础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的结果表明，存在LCA结合的Rab蛋白，这可能是LCA诱导肝毒性的过程。进一步研究了GSH与胆汁酸的反应性酰化代谢产物通过反酰化反应形成谷胱甘肽(GSH)偶联物的解毒机制。为此，化学合成了5种主要胆汁酸的正品谷胱甘肽结合物…并用电喷雾级联质谱对其结构进行了表征。在磷酸盐缓冲液(pH 7.5)中，胆酸(CA)的活性代谢物胆酰腺苷和胆酰辅酶A与GSH孵育，表明两种化合物在体外都能酰化GSH。此外，我们还克隆并表达了大鼠和人肝胆汁酸辅酶A连接酶，以更好地了解乙酰腺苷和胆汁酸结合蛋白的形成机制。最后，我们利用亲和吸附剂，通过在LCA的C-3和C-24位引入桥，将LCA与活化的琼脂糖共价结合，探索与LCA有亲和力的蛋白质。对分离后的蛋白质进行LC/ESI-MS分析，结果表明存在氨基甲酰磷酸合成酶I、谷氨酸脱氢酶、酰辅酶A酰基转移酶、谷胱甘肽-S转移酶和过氧化氢酶等功能蛋白，这一假说可能是由于低分子肝素对这些酶的抑制而引起的。较少