A proteomic approach for the discovery of protease substrates
A proteomic approach for the discovery of protease substrates
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DOI：
10.1073/pnas.0402353101
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发表时间：
2004-08-10
影响因子：
11.1
通讯作者：
Ley, TJ
中科院分区：
文献类型：
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作者：
Bredemeyer, AJ;Lewis, RM;Ley, TJ
用于发现蛋白酶底物的标准化、全面的平台一直非常难以创建。蛋白酶特异性的筛选现在经常基于肽底物的切割模式，肽底物含有活性位点内易裂键切割所需的小识别基序。然而，这些研究没有确定体内底物，也不能导致解释蛋白酶生物特异性的大分子特征的定义。为了在蛋白酶底物的蛋白质组学筛选中使用正确折叠的蛋白质，我们使用2D差异凝胶电泳和串联MS来鉴定诱导细胞凋亡的蛋白酶颗粒酶B的底物。我们证实了这种酶的关键底物之一的半胱天冬酶原-3的裂解，并确定了几种以前未知的底物，以及这些底物之一的裂解位点。我们还能够观察到的底物裂解和裂解产物积累的动力学，通过使用2D差异凝胶电泳方法。因此，“蛋白酶蛋白质组学”可能是发现各种蛋白酶的天然底物的重要工具。
Standardized, comprehensive platforms for the discovery of protease substrates have been extremely difficult to create. Screens for protease specificity are now frequently based on the cleavage patterns of peptide substrates, which contain small recognition motifs that are required for the cleavage of the scissile bond within an active site. However, these studies do not identify in vivo substrates, nor can they lead to the definition of the macromolecular features that account for the biological specificity of proteases. To use properly folded proteins in a proteomic screen for protease substrates, we used 2D difference gel electrophoresis and tandem MS to identify substrates of an apoptosis-inducing protease, granzyme B. We confirmed the cleavage of procaspase-3, one of the key substrates of this enzyme, and identified several substrates that were previously unknown, as well as the cleavage site for one of these substrates. We were also able to observe the kinetics of substrate cleavage and cleavage product accumulation by using the 2D difference gel electrophoresis methodology. "Protease proteomics" may therefore represent an important tool for the discovery of the native substrates of a variety of proteases.