Max-Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany.
Biochemistry. 2010 Feb 9;49(5):1042-52. doi: 10.1021/bi901813e.
In a process contributing to the innate immunity of higher plants, Arabidopsis thaliana cyclophilin ROC1 induces the self-cleavage of Pseudomonas syringae putative cysteine protease AvrRpt2, triggering limited cleavage of A. thaliana RIN4, a negative regulator of plant immunity. We report an increase in AvRpt2 activity in hydrolysis of decapeptide substrates at -GG- sites of more than 5 orders of magnitude, in the presence of cyclophilin-like peptidyl prolyl cis/trans isomerases including ROC1 or hCyp18. Both full-length AvrRpt2 and its 21 kDa self-cleavage product (AvrRpt2(72-255)) were found to be equally active under these conditions. In contrast to classical isomer-specific proteolysis, inertness toward cleavage of a cis/trans prolyl bond isomer at the substrate P4 subsite is not the cause of cyclophilin-mediated activation of the proteolytic reaction. Monitoring single- and double-jump kinetics of proteolytic reactions in the presence of the PPIase inhibitor cyclosporin A revealed that the cis/trans ratio of potentially relevant prolyl bonds of AvrRpt2(72-255) remained the same in the functionally inactive state of AvrRpt2(72-255) and the productive AvrRpt2(72-255)-cyclophilin-substrate complex.
在高等植物固有免疫的过程中，拟南芥环孢素 ROC1 诱导丁香假单胞菌假定半胱氨酸蛋白酶 AvrRpt2 的自我切割，触发植物免疫负调节剂 A. thaliana RIN4 的有限切割。我们报告了在环孢素样肽基脯氨酰顺/反式异构酶包括 ROC1 或 hCyp18 存在下，AvrRpt2 在水解 GG-位点的十肽底物中的活性增加超过 5 个数量级。全长 AvrRpt2 及其 21 kDa 的自我切割产物（AvrRpt2(72-255)）在这些条件下均具有同等活性。与经典的异构特异性蛋白水解相反，对底物 P4 亚位点的顺/反脯氨酰键异构化的无切割性不是环孢素介导的蛋白水解反应激活的原因。在 PPIase 抑制剂环孢菌素 A 的存在下监测蛋白水解反应的单跳和双跳动力学，发现 AvrRpt2(72-255)的潜在相关脯氨酰键的顺/反比值在无活性状态的 AvrRpt2(72-255)和产生活性的 AvrRpt2(72-255)-环孢素-底物复合物中保持不变。
