Fluorescent Probes for Studying Thioamide Positional Effects on Proteolysis Reveal Insight into Resistance to Cysteine Proteases
Fluorescent Probes for Studying Thioamide Positional Effects on Proteolysis Reveal Insight into Resistance to Cysteine Proteases
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DOI：
10.1002/cbic.201900115
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发表时间：
2019-08-16
期刊：
影响因子：
3.2
通讯作者：
Petersson, E. James
中科院分区：
文献类型：
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作者：
Liu, Chunxiao;Barrett, Taylor M.;Petersson, E. James
肽主链的硫代酰胺取代已被证明可以减少蛋白水解降解，并且该特性可用于产生竞争性蛋白酶抑制剂并稳定肽以防止体内降解。在这里，我们提出了一种简单的传感器设计，可以通过使用实时荧光系统地研究硫代酰胺取代的位置效应。对五种木瓜蛋白酶家族半胱氨酸蛋白酶的肽底物进行的硫代酰胺扫描表明，在易断键处或附近的硫代酰胺在所有情况下都可以减缓蛋白水解，但影响的程度随位置和蛋白酶的不同而变化，尽管序列同源性很高。木瓜蛋白酶水解的机制研究表明，硫代酰胺效应源自亲和力 (K-M) 和周转数 (k(cat)) 的降低。计算模型可以根据关键酶-底物氢键的破坏来理解这些效应，为未来合理使用硫代酰胺以赋予半胱氨酸蛋白酶抗性提供模型。
Thioamide substitutions of the peptide backbone have been shown to reduce proteolytic degradation, and this property can be used to generate competitive protease inhibitors and to stabilize peptides toward degradation in vivo. Here, we present a straightforward sensor design that allows a systematic study of the positional effects of thioamide substitution by using real-time fluorescence. Thioamide scanning in peptide substrates of five papain family cysteine proteases demonstrates that a thioamide at or near the scissile bond can slow proteolysis in all cases, but that the magnitude of the effects varies with position and protease in spite of high sequence homology. Mechanistic investigation of papain proteolysis reveals that the thioamide effects derive from reductions in both affinity (K-M) and turnover number (k(cat)). Computational modeling allows these effects to be understood based on disruption of key enzyme-substrate hydrogen bonds, providing a model for future rational use of thioamides to confer cysteine protease resistance.