study of Physiological activity of Hepatotoxins, Mirocystins Produced by freshwater Cyanobacteria

淡水蓝藻产生的肝毒素、微囊藻毒素的生理活性研究

• 批准号: 14572115
• 负责人: HARADA Ken-ich
• 金额: $ 2.18万
• 依托单位: Meijo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572115/
• 关键词: Microcystin; Protein phosphatase; Okadaic acid; Oxidative stress; Mass spectrometry; Phosphorylation of proteins; Binding proteins; Hepato toxicity; 代謝; アフィニティーカラム; DNAマイクロアレイ; 網羅的解析; 親和性タンパク質

Microcystin-LR (MCLR) produced by freshwater cyanobacteria is potent hepatotoxin, and inhibits protein serine/threonine phosphatases 1 and 2A (PP1 and PP2A). A toxicological mechanism has not been elucidated, so that an appropriate treatment has not been yet established for Microcystin toxicosis. In order to clarify this acute toxicity mechanism by MC we have searched a targeted compound and related compounds for MCLR in the liver during the toxicological process. In this study, we always compared the behavior with MC and that of okadaic acid (OA), which does not show such toxicity but the similar PP inhibitory activities.1.Mt-binding proteins were comprehensively searched in mouse livers using MC affinity column, two-dimensional gel electrophoresis and MALD1-TOFMS. The results obtained from the competitive inhibition experiments using the affinity chromatography with OA indicated that the complex of PP1 with the inhibitory subunit NIPP1 was only detected as the MC-binding proteins.2.In order to investigate proteins increased in phosphorylation in mouse livers, resulting phosphoproteins were chemically modified with cleavable biotin reagent on a phosphate moiety, purified by an avidin column, and then analyzed by ESI-LC/MS/MS. Consequently, a phosphoprotein, formyltetrahydrofolate dehydrogenase was only detected in both the MCLR-and OA-treated mice. These results suggested that the phosphorylation was caused by PP2A inhibition, which was not related to the liver injury.2.In order to investigate proteins increased in phosphorylation in mouse livers, resulting phosphoproteins were chemically modified with cleavable biotin reagent on a phosphate moiety, purified by an avidin column, and then analyzed by ESI-LC/MS/MS. Consequently, a phosphoprotein, formyltetrahydrofolate dehydrogenase was only detected in both the MCLR-and OA-treated mice. These results suggested that the phosphorylation was caused by PP2A inhibition, which was not related to the liver injury.

淡水蓝藻产生的微囊藻毒素（microcytin - lr， MCLR）是一种强效肝毒素，可抑制蛋白丝氨酸/苏氨酸磷酸酶1和2A （PP1和PP2A）。微囊藻毒素中毒的毒理学机制尚未阐明，因此尚未建立适当的治疗方法。为了阐明MC的急性毒性机制，我们在肝脏毒理学过程中寻找了MCLR的靶化合物及相关化合物。在本研究中，我们总是将其与MC和冈田酸（OA）的行为进行比较，后者没有这种毒性，但具有类似的PP抑制活性。利用MC亲和柱、二维凝胶电泳和MALD1-TOFMS对小鼠肝脏中mt结合蛋白进行全面搜索。OA亲和层析的竞争抑制实验结果表明，PP1与抑制亚基NIPP1的复合物仅作为mc结合蛋白被检测到。为了研究小鼠肝脏中磷酸化增加的蛋白，用可切割生物素试剂在磷酸段上对所得磷酸化蛋白进行化学修饰，用亲和素柱纯化，然后用ESI-LC/MS/MS分析。因此，磷蛋白甲酰基四氢叶酸脱氢酶仅在mclr和oa处理的小鼠中检测到。这些结果表明，磷酸化是由PP2A抑制引起的，与肝损伤无关。为了研究小鼠肝脏中磷酸化增加的蛋白，用可切割生物素试剂在磷酸段上对所得磷酸化蛋白进行化学修饰，用亲和素柱纯化，然后用ESI-LC/MS/MS分析。因此，磷蛋白甲酰基四氢叶酸脱氢酶仅在mclr和oa处理的小鼠中检测到。这些结果表明，磷酸化是由PP2A抑制引起的，与肝损伤无关。

项目成果

Susumu Imanishi, Ken-ichi Harada: "Proteomics approach on microcystin binding proteins in mouse liver for investigation of microcystin toxicity"Toxicon. (in press).

Susumu Imanishi、Ken-ichi Harada：“小鼠肝脏中微囊藻毒素结合蛋白的蛋白质组学方法，用于研究微囊藻毒素毒性”Toxicon。

Susumu Imanishi et al.: "Proteomics approach on microcystin binding proteins in mouse liver for investigation of microcystin toxicity"Toxicon. 43(6). 651-659 (2004)

Susumu Imanishi 等人：“用于研究微囊藻毒素毒性的小鼠肝脏中微囊藻毒素结合蛋白的蛋白质组学方法”Toxicon。

Susumu Imanishi et al.: "Proteomics approach on microcystin binding proteins in mouse liver for investigation of microcystin toxicity"Toxicon.

Susumu Imanishi 等人：“用于研究微囊藻毒素毒性的小鼠肝脏中微囊藻毒素结合蛋白的蛋白质组学方法”Toxicon。