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Comprehensive analysis of redox regulations of plant by the disulfide proteome.

二硫键蛋白质组综合分析植物氧化还原调控。

基本信息

批准号：
16510151
负责人：
YANO Hiroyuki
金额：
$ 2.37万
依托单位：
National Agriculture and Food Research Organization
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16510151/
关键词：
Redox Thioredoxin Disulfide Rice Germination Proteome ジスルフィドプロテオームアレルゲン

项目摘要

Redox regulation in biology is as important as regulation by phosphorylation / de-phosphorylation. The head investigator of this project recently developed the disulfide proteome technique that allows comprehensive analysis of redox regulation of proteins. This study was aimed to apply the technique to clarify new redox regulations in rice seed germination.First, proteins were extracted from aleurone layer of rice seeds. When NADPH and NADPH-dependent thioredoxin reductase (NTR) were added to the extract, embryo-specific protein (ESP)-2 disappeared. On the other hand, when the experiment was done in the presence of leupeptine, a cysteine-protease inhibitor, degradation of ESP2 did not occur. These results suggested that the endogenous thioredoxin in the aleurone layer activated a cystein protease that digested ESP2. The ESP2 has a high disulfide content (l6S-S/392aa). So thioredoxin seemed to reduce the disulfide bonds to unfold the molecular structure of ESP2. Next, when isolated aleurone layer was incubated in the presence of gibberellic acid and CaCl_2, similar results were obtained as the previous in vitro studies. So, in rice seed germination, endogenous thioredoxin likely activates cysteine protease and concurrently unfolds its substrate, ESP2, to facilitate prompt degradation. It has been reported that a serine-protease, thiocalsin, that digests globulins is synthesized de novo and activated thioredoxhvdependently in the presence of Ca^<2+> in the later stage of seed germination. In our in vivo study, globulins remained intact when ESP2 was digested by the cystein protease. These observations suggest that in seed germination, thioredoxin activates different proteases step by step, and concurrently unfolds the substrate of the respective protease to facilitate degradation. Thus new redox regulation was clarified in the present study.

生物中的氧化还原调节与磷酸化/去磷酸化的调节一样重要。该项目的首席研究员最近开发了二硫键蛋白质组技术，可以全面分析蛋白质的氧化还原调节。本研究旨在应用该技术阐明水稻种子萌发过程中新的氧化还原调节机制。当加入NADPH和依赖NADPH的硫氧还蛋白还原酶(NTR)时，胚胎特异性蛋白(ESP)-2消失。另一方面，当实验在半胱氨酸蛋白酶抑制剂亮氨酸的存在下进行时，ESP2没有发生降解。这些结果表明糊粉层中的内源硫氧还蛋白激活了半胱氨酸蛋白酶，从而消化了ESP2。ESP2的二硫键含量较高(16S-S/392aa)。因此，硫氧还蛋白似乎减少了二硫键，从而揭示了ESP2的分子结构。其次，将分离的糊粉层在赤霉酸和CaCl2的存在下孵育，得到了与以前的体外研究类似的结果。因此，在水稻种子萌发中，内源硫氧还蛋白可能激活半胱氨酸蛋白酶并同时展开其底物ESP2，以促进迅速降解。据报道，在种子萌发的后期，一种消化球蛋白的丝氨酸蛋白酶--硫代钙素被重新合成，并在钙离子存在下被硫氧还原酶激活。在我们的活体研究中，当ESP2被半胱氨酸蛋白酶消化时，球蛋白保持完整。这些观察表明，在种子萌发过程中，硫氧还蛋白一步一步地激活不同的蛋白水解酶，并同时展开相应的蛋白水解酶的底物以促进降解。因此，在本研究中阐明了新的氧化还原调节。