Abstract：
Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins-PsbO, PsbP, and PsbQ-integral components of photosystem II\'s oxygen-evolving complex. Subsequent biochemical assays and mass spectrometry experiments revealed the involvement of CrCEP1, a previously uncharacterized papain-like cysteine protease, as the catalyst of this labelling reaction. Further experiments with recombinant CrCEP1 and PsbO proteins replicated the reaction in vitro. Our data unveiled that endopeptidase CrCEP1 also has transpeptidase activity, ligating probes and peptides to the N-termini of Psb proteins, thereby expanding the repertoire of its enzymatic activities. The hitherto unknown transpeptidase activity of CrCEP1, working in conjunction with its proteolytic activity, unveils putative complex and versatile roles for proteases in cellular processes during stress responses.
摘要：
蛋白酶,植物应激反应的基本调节剂，在其精确的功能角色中仍然神秘。通过使用基于活动的探针进行实时监控，本研究旨在探讨暴露于过氧化氢诱导的氧化应激下莱茵衣藻的蛋白酶活性。然而,我们的工作表明，基于活性的探针强烈标记三个非蛋白水解蛋白-PsbO，PsbP,和PsbQ-光系统II的析氧复合物的积分分量。随后的生化测定和质谱实验揭示了CrCEP1的参与，CrCEP1是一种以前未表征的木瓜蛋白酶样半胱氨酸蛋白酶，作为这种标记反应的催化剂。用重组CrCEP1和PsbO蛋白进行的进一步实验在体外复制了该反应。我们的数据揭示了内肽酶CrCEP1也具有转肽酶活性，将探针和肽连接到Psb蛋白的N末端，从而扩大其酶活性的库。CrCEP1迄今未知的转肽酶活性与其蛋白水解活性一起工作，揭示了蛋白酶在应激反应过程中在细胞过程中的复杂和多功能作用。
