Design and characterization of a hybrid miniprotein that specifically inhibits porcine pancreatic elastase
Design and characterization of a hybrid miniprotein that specifically inhibits porcine pancreatic elastase
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DOI：
10.1074/jbc.m212152200
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发表时间：
2003-07-04
影响因子：
4.8
通讯作者：
Höhne, W
中科院分区：
文献类型：
--
作者：
Hilpert, K;Wessner, H;Höhne, W
研究蛋白酶/肽抑制剂相互作用是理解分子识别的有用工具，并且与具有治疗潜力的抑制剂的合理设计尤其重要。将抑制性肽（PMTLEYR）衍生自火鸡卵球形抑制剂的第三结构域，并针对特异性猪胰腺弹性酶抑制作用进行了优化，以增加抑制剂支架，以提高肽的蛋白水解稳定性。从Ecballium Elaterium中选择了胰蛋白酶特异性的南瓜抑制剂EETI II作为脚手架。由此产生的杂化抑制剂Hei-toE I（来自大肠球大肠杆菌的杂化抑制剂和土耳其卵球形抑制剂的第三个结构域的优化结合环）显示出对猪胰腺弹性酶的特异性和亲和力，类似于自由抑制肽。等温滴定量热法表明，Hei-toe I的弹性酶结合具有小的不利正焓贡献，较大的有利的正熵变化以及较大的负热容量变化。此外，用猪胰腺弹性酶治疗后，抑制性肽和杂化抑制剂HEI-TOE保护内皮细胞免受降解。
Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I ( hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase.