Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay
Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay
复制标题
DOI：
10.1073/pnas.1215076110
复制
发表时间：
2013-02-12
影响因子：
11.1
通讯作者：
Verhelst, Steven H. L.
中科院分区：
文献类型：
--
作者：
Vosyka, Oliver;Vinothkumar, Kutti R.;Verhelst, Steven H. L.
菱形蛋白酶是进化保守的膜内丝氨酸蛋白酶。由于菱形结构在许多重要的生物途径中发挥着新兴作用，因此成为潜在的药物靶点。不幸的是，很少有化学工具可用于他们的研究。在这里，我们描述了一种基于质谱的测定法来测量菱形底物裂解和抑制。我们已经鉴定了异香豆素抑制剂并开发了基于活性的菱形蛋白酶探针。由于活性位点丝氨酸的共价、可逆结合和组氨酸残基的稳定修饰，探针可以区分活性和非活性菱形。最后，基于异香豆素的抑制剂与大肠杆菌菱形 GlpG 的结构揭示了活性位点的不寻常结合模式，并表明异香豆素抑制剂上的 3-取代基与蛋白酶上的疏水残基之间的相互作用反映了 S' 亚位点结合。总体而言，这些探针代表了菱形研究的宝贵工具，其结构见解可能有助于未来的抑制剂设计。
Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S' subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design.