Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2
Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2
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DOI：
10.1016/j.ab.2005.04.023
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发表时间：
2005-08-15
影响因子：
2.9
通讯作者：
Filipuzzi, I
中科院分区：
文献类型：
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作者：
Tirat, A;Schilb, A;Filipuzzi, I
去泛素化酶（DUBs）催化从靶蛋白的氨基上去除附着的泛素分子。DUB大家族在调节不同蛋白质的细胞内稳态中起重要作用，并因此影响关键事件，如细胞分裂、凋亡等。DUB家族成员UCH-L3和USP 2被认为通过去除降解的触发物来抑制各种肿瘤生长促进蛋白的降解。因此，这些酶的抑制剂应该导致癌蛋白的降解增强，从而可能阻止肿瘤生长。为了开发用于搜索UCH-L3和USP 2抑制剂的酶促测定，酶促合成C-末端标记的泛素底物。我们已经使用泛素激活酶El和泛素缀合酶E2之一来将荧光赖氨酸衍生物连接到泛素的C末端。由于赖氨酸衍生物中只有ε-NH（2）基团是游离的和反应性的，所以缀合物紧密地模拟了靶蛋白的泛素和赖氨酸侧链之间的异肽键。通过这种方法合成了各种底物，并使用两种DUB进行了酶促表征。发现由大的N-末端NusA标签和用α-NH（2）-四甲基罗丹明-赖氨酸修饰的泛素之间的融合蛋白组成的变体在荧光偏振读数中给出最高的动态范围。因此，我们选择了这种基板的小型化，荧光偏振为基础的高通量筛选测定的发展。(C)2005年爱思唯尔公司All rights reserved.
Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme El and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay. (C) 2005 Elsevier Inc. All rights reserved.