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

已经提出，与岩性酸（LCA）共价结合的蛋白质成立是一种可能解释该胆汁酸的过敏性和毒性反应的解释。因此，我们使用对LCA的3α-羟基-5β-类固醇的高亲和力，通过免疫沉淀来捕获LCA修饰的细胞蛋白通过免疫沉淀在胆管绑扎大鼠肝脏中的LCA。 Results of the structural analysis of the captured proteins by the basic proteinomic 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已经进行了胆汁酸的代谢物，以证明存在解毒机制。为此，在化学合成的五个主要胆汁酸的真实GSH缀合物中……更多，其结构的特征是在负和正离子模式下以ESI-TANDEM质谱法进行了特征。在磷酸盐缓冲液中的GSH（pH 7.5）溶液中孵育胆烯基辅酶A和胆囊-COA，反应性代谢物（Ca），证明两种化合物都能够在体外酰化GSH。 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 LCA的C-24位置。通过SDS-PAGE分离后，LC/ESI-MS^n分析的LC/ESI-MS^n分析的结果表明，功能性蛋白质的存在，即，即氨基甲酰磷酸合酶I，谷氨酸脱氢酶，酰基-COA酰基酰基酰基转移酶，谷胱甘肽 - 甲基硫酸 - 甲基转移酶的效果涉及的特殊情况涉及甲基转移酶，谷氨酸脱氢酶，酰基-COA酰基转移酶，表明了脑含量的特殊性。 LCA的酶。较少的