Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases.
Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases.
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DOI：
10.1016/j.bbamcr.2012.06.004
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发表时间：
2012-11
影响因子：
5.1
通讯作者：
Strickler, James E.
中科院分区：
文献类型：
--
作者：
Orcutt, Steven J.;Wu, Jian;Eddins, Michael J.;Leach, Craig A.;Strickler, James E.
随着泛素化在某些疾病状态中的重要性变得越来越明显，负责从靶蛋白去除泛素（Ub）的酶，去泛素化酶（DUB），正成为药物发现的有吸引力的靶点。为了快速鉴定改变DUB功能的化合物，体外测定必须能够在高通量筛选（HTS）期间在底物和酶浓度的宽动态范围内提供统计学上稳健的数据。用于HTS的最成熟的试剂是具有与C-末端缀合的淬灭荧光团的Ub;然而，用于检测DUB活性的基于内切酶的策略（DUB-Glo™，Promega）提供了比传统荧光试剂更宽的动态范围。不幸的是，该测定法需要高酶浓度，并且相对于其他异肽酶（例如脱糖基化酶）缺乏对DUB的特异性，因为它是基于衍生自Ub（Z-RLRGG-）的C-末端的肽的氨基荧光素（AML）衍生物。氨基荧光素与全长Ub（Ub-AML）的缀合产生具有宽动态范围的底物，但显示DUB的检测限比用DUB-Glo™观察到的低100至1000倍。Ub-AML甚至是DUB（例如JosDl和USP 14）的敏感底物，DUB（例如JosDl和USP 14）对DUB-Glo™不显示出明显的活性。hSUMO 2和NEDD 8的氨基萤光素衍生物也分别显示为脱糖基化酶和脱糖基化酶的敏感底物。Ub/Ubl-AML底物适合HTS（Z′ =0.67），产生稳健的信号，并为Ub/Ubl异肽酶提供替代药物发现平台。
As the importance of ubiquitylation in certain disease states becomes increasingly apparent, the enzymes responsible for removal of ubiquitin (Ub) from target proteins, deubiquitylases (DUBs), are becoming attractive targets for drug discovery. For rapid identification of compounds that alter DUB function, in vitro assays must be able to provide statistically robust data over a wide dynamic range of both substrate and enzyme concentrations during high throughput screening (HTS). The most established reagents for HTS are Ubs with a quenched fluorophore conjugated to the C-terminus; however, a luciferase-based strategy for detecting DUB activity (DUB-Glo™, Promega) provides a wider dynamic range than traditional fluorogenic reagents. Unfortunately, this assay requires high enzyme concentrations and lacks specificity for DUBs over other isopeptidases (e.g. desumoylases), as it is based on an aminoluciferin (AML) derivative of a peptide derived from the C-terminus of Ub (Z-RLRGG-). Conjugation of aminoluciferin to a full-length Ub (Ub-AML) yields a substrate that has a wide dynamic range, yet displays detection limits for DUBs 100- to 1000-fold lower than observed with DUB-Glo™. Ub-AML was even a sensitive substrate for DUBs (e.g. JosD1 and USP14) that do not show appreciable activity with DUB-Glo™. Aminoluciferin derivatives of hSUMO2 and NEDD8 were also shown to be sensitive substrates for desumoylases and deneddylases, respectively. Ub/Ubl-AML substrates are amenable to HTS (Z′ =0.67) yielding robust signal, and providing an alternative drug discovery platform for Ub/Ubl isopeptidases.
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影响因子：
64.8
作者：
通讯作者：
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影响因子：
2.9
作者：
Shinde, Rajesh;Perkins, Julie;Contag, Christopher H.
通讯作者：
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影响因子：
16
作者：
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通讯作者：
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DOI：
10.2217/14796694.3.2.191
发表时间：
2007-04-01
期刊：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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