A Chemical Strategy for Protease Substrate Profiling
A Chemical Strategy for Protease Substrate Profiling
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DOI：
10.1016/j.chembiol.2019.03.007
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发表时间：
2019-06-20
影响因子：
8.6
通讯作者：
Bachovchin, Daniel A.
中科院分区：
文献类型：
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作者：
Griswold, Andrew R.;Cifani, Paolo;Bachovchin, Daniel A.
二肽基肽酶（DPPs）通过从氨基酸末端切割脯氨酸后的二肽来调节激素、细胞因子和神经肽。由于技术上的挑战，许多DPP基材仍然未知。在这里，我们介绍了一种简单的方法，称为chop（蛋白酶底物的化学富集），用于发现蛋白酶底物。CHOPS利用2-吡啶羧醛(2PCA)-生物素探针，选择性地对蛋白质n -末端进行生物素化，除了脯氨酸在第二位置的那些。理论上，chop可以发现任何蛋白酶的底物，但特别适合发现典型的DPP底物，因为劈裂的而不是完整的DPP底物可以通过凝胶电泳或质谱识别。通过chop，我们发现通过未知机制控制Nlrp1炎性体的酶DPP8和DPP9不能直接切割Nlrp1。我们进一步表明，DPP9强有力地切割短肽，而不是全长蛋白。更一般地说，这项工作描述了一种鉴定蛋白酶底物的实用技术，我们预计这将补充现有的“n -末端”方法。
The dipeptidyl peptidases (DPPs) regulate hormones, cytokines, and neuropeptides by cleaving dipeptides after proline from their amino termini. Due to technical challenges, many DPP substrates remain unknown. Here, we introduce a simple method, termed CHOPS (chemical enrichment of protease substrates), for the discovery of protease substrates. CHOPS exploits a 2-pyridinecarboxaldehyde (2PCA)-biotin probe, which selectively biotinylates protein N-termini except those with proline in the second position. CHOPS can, in theory, discover substrates for any protease, but is particularly well suited to discover canonical DPP substrates, as cleaved but not intact DPP substrates can be identified by gel electrophoresis or mass spectrometry. Using CHOPS, we show that DPP8 and DPP9, enzymes that control the Nlrp1 inflammasome through an unknown mechanism, do not directly cleave Nlrp1. We further show that DPP9 robustly cleaves short peptides but not full-length proteins. More generally, this work delineates a practical technology for identifying protease substrates, which we anticipate will complement available "N-terminomic" approaches.