Studies on mechanism of toxicity of methylmercury

甲基汞毒性机制研究

基本信息

批准号：
15209004
负责人：
NAGANUMA Akira
金额：
$ 32.45万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15209004/
关键词：
methylmercury toxicity resistance ubiquitin F-box proteins Cdc34 Rad23 酵母

项目摘要

We have identified a gene, CDC34 that confers resistance to methylmercury in Saccharomyces cerevisiae by screening a yeast genomic DNA library. CDC34 encodes one of the ubiquitin-conjugating enzymes (Cdc34) that catalyze ubiquitin transfer onto substrate proteins, an important step for protein degradation via ubiquitin-proteasome pathway. In yeast cells, the ubiquitin-conjugating activity of Cdc34 is essential for the Cdc34-mediated resistance to methylmercury. It is possible to conclude that proteins involved in expression of methylmercury toxicity might exist in the cells and the proteins are degraded after its ubiquitination in proteasomes. In ubiquitin system, substrate proteins are recognized by F-box proteins and ubiquitinated by ubiquitin-conjugating enzymes. Thus, we searched F-box proteins which confer resistance to methylmercury, and found that Hrt3 and Ylr224w have role in protection against methylmercury toxicity. Proteins that bind to these F-box proteins might have activity to intensify the toxicity of methylmercury. We identified three proteins that bind to the F-box proteins, Hrt3 and Ylr224w. Overexpression of the genes for these proteins conferred a hyper susceptibility to methylmercury. Moreover, these proteins are found to be ubiquitinated in the cells. These results suggest that the F-box protein-bounding proteins identified by the present study have significant roles in expression of methylmercury toxicity and are decomposed by the ubiquitin-proteasome pathway. Two of these proteins are enzymes involved in synthesis of pyruvate, and overexpression of these proteins might increase cellular concentration of pyruvate. Addition of excess pyruvate into the culture medium significantly prevents toxicity of methylmercury. Increase in cellular production of pyruvate might have an important role in enhancement of toxicity of methylmercury.

我们通过筛选酵母基因组 DNA 文库，鉴定出一种基因 CDC34，该基因赋予酿酒酵母对甲基汞的抗性。 CDC34 编码一种泛素结合酶 (Cdc34)，可催化泛素转移到底物蛋白上，这是通过泛素-蛋白酶体途径进行蛋白质降解的重要步骤。在酵母细胞中，Cdc34 的泛素结合活性对于 Cdc34 介导的甲基汞抗性至关重要。可以得出结论，细胞中可能存在参与甲基汞毒性表达的蛋白质，并且这些蛋白质在蛋白酶体中泛素化后被降解。在泛素系统中，底物蛋白被 F-box 蛋白识别，并被泛素结合酶泛素化。因此，我们搜索了赋予甲基汞抗性的F-box蛋白，发现Hrt3和Ylr224w具有防止甲基汞毒性的作用。与这些 F-box 蛋白结合的蛋白可能具有增强甲基汞毒性的活性。我们鉴定了三种与 F-box 蛋白结合的蛋白：Hrt3 和 Ylr224w。这些蛋白质基因的过度表达赋予了对甲基汞的高度敏感性。此外，发现这些蛋白质在细胞中被泛素化。这些结果表明，本研究鉴定的F-box蛋白结合蛋白在甲基汞毒性的表达中具有重要作用，并被泛素-蛋白酶体途径分解。其中两种蛋白质是参与丙酮酸合成的酶，这些蛋白质的过度表达可能会增加丙酮酸的细胞浓度。在培养基中添加过量的丙酮酸可显着防止甲基汞的毒性。丙酮酸细胞生成的增加可能在增强甲基汞的毒性方面发挥重要作用。