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

生物学中的氧化还原调控与磷酸化/去磷酸化调控同样重要。该项目的首席研究员最近开发了二硫蛋白质组技术，可以对蛋白质的氧化还原调节进行全面分析。本研究旨在应用该技术阐明水稻种子萌发过程中氧化还原的新调控。首先，从水稻种子糊粉层中提取蛋白质。在提取液中加入NADPH和NADPH依赖性硫氧还蛋白还原酶（NTR）后，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胰肽的存在下进行时，ESP2的降解没有发生。这些结果表明，糊粉层内源性硫氧还蛋白激活了消化ESP2的半胱氨酸蛋白酶。ESP2具有高二硫化物含量（l6S-S/392aa）。因此，硫氧还蛋白似乎通过减少二硫键来展开ESP2的分子结构。然后，将分离的糊粉层在赤霉素酸和CaCl_2的作用下孵育，得到与之前体外实验相似的结果。因此，在水稻种子萌发过程中，内源性硫氧还蛋白可能激活半胱氨酸蛋白酶，同时展开其底物ESP2，以促进迅速降解。据报道，一种消化球蛋白的丝氨酸蛋白酶硫钙蛋白（thiocalsin）是在种子萌发后期在Ca^<2+>存在的情况下重新合成并激活硫氧自由基的。在我们的体内研究中，当ESP2被半胱氨酸蛋白酶消化时，球蛋白保持完整。这些观察结果表明，在种子萌发过程中，硫氧还蛋白会逐步激活不同的蛋白酶，并同时展开相应蛋白酶的底物以促进降解。因此，本研究阐明了新的氧化还原调控。

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.