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Physiological significance of taurine chloramine induced inhibition of NF-_KB activation

牛磺酸氯胺诱导抑制 NF-_KB 激活的生理意义

基本信息

批准号：
12660112
负责人：
MIYAMOTO Yusei
金额：
$ 2.62万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

When this grant application was submitted, preliminary data showed modification of α-isoform of inhibitory protein (I_KB_α) by cell treatment with taurine chloramine (TauCl) that was judged by band shift observed in Western blots, suppression of I_KB_α degradation in response to stimulation and subsequent inhibition of nuclear factor _KB (NF_KB) activation. This suppression of I_KB_α degradation seemed to be due to its chlorination by TauCl. Therefore, this research was started with seeking amino acid residues of I_KB_α that are chlorinated by TauCl. Because the amino acid residue that is most easily chlorinated is tyrosine, tyrosine residues (eight tyrosine residues in I_KB_α) were mutated to alanine one by one. However, a mutant whose tyrosine residues were all converted to alanine showed band shift by TauCl treatment, indicated that tyrosine residue is not the chlorination site. Similar experiments were performed in regard to tryptophan. However, tryptophan was not a chlorination site, either. Then, amino acid residues modified by TauCl were further searched with deletion mutants of I_KB_α. As a result, the 45th methionine was found to be a site susceptible to TauCl modification. This methionine modification was estimated to be oxidation to form methionine sulfoxide by analysis of an oligo-peptide containing the 45th methionine with high performance liquid chromatography and mass spectrometry. These results lead to a unique hypothesis that protein oxidation at methionine residues may play a part in the cellular signal transduction.

在提交该基金申请时，初步数据显示，牛磺酸氯胺（TauCl）处理细胞后，抑制蛋白（I_KB_α）的α-异构体（I_KB_α）发生了修饰，这是通过蛋白质印迹中观察到的条带移动、抑制I_KB_α对刺激的响应降解以及随后抑制核因子_KB（NF_KB）活化来判断的。TauCl对I_KB_α降解的抑制作用可能与其氯化作用有关。因此，本研究从寻找I_KB_α经TauCl氯化后的氨基酸残基开始。由于最容易被氯化的氨基酸残基是酪氨酸，因此酪氨酸残基（IKB α中的8个酪氨酸残基）被逐一突变为丙氨酸。然而，突变体的酪氨酸残基全部转化为丙氨酸显示带移动由TauCl处理，表明酪氨酸残基不是氯化位点。对色氨酸进行了类似的实验。然而，色氨酸也不是氯化位点。然后，用IKB α缺失突变体进一步寻找TauCl修饰的氨基酸残基。结果，发现第45位甲硫氨酸是对TauCl修饰敏感的位点。通过高效液相色谱和质谱分析含有第45位甲硫氨酸的寡肽，估计这种甲硫氨酸修饰是氧化形成甲硫氨酸亚砜。这些结果导致了一个独特的假设，蛋白质氧化在蛋氨酸残基可能在细胞信号转导的一部分。