Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities.
Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities.
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DOI：
10.1110/ps.083450408
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发表时间：
2008-06
期刊：
影响因子：
--
通讯作者：
Butt TR
中科院分区：
文献类型：
--
作者：
Nicholson B;Leach CA;Goldenberg SJ;Francis DM;Kodrasov MP;Tian X;Shanks J;Sterner DE;Bernal A;Mattern MR;Wilkinson KD;Butt TR
泛素（Ub）或泛素样蛋白（UBL）与细胞蛋白质的缀合或解缀合是调节细胞生理学、控制许多关键蛋白质的寿命、定位和活性的多方面且通用的手段。Ub或UBL与蛋白质的解缀合通过一类称为异肽酶的蛋白酶进行。本文描述了一种易于定量的新型异肽酶测定平台，其由与报告酶磷脂酶A2（PLA 2）融合的Ub或UBL组成。异肽酶活性释放PLA 2，PLA 2切割其底物，产生与去泛素化酶（DUB）浓度呈线性的信号，并且能够区分DUB、去SUMO酶、去NEDD酶和去ISG酶活性。UBL-PLA 2检测的功效和灵敏度通过其区分两种冠状病毒蛋白酶的对比脱ISGylase和DUB活性的能力来证明：严重急性呼吸综合征木瓜蛋白酶样蛋白酶（SARS-CoV PLpro）和NL 63 CoV木瓜蛋白酶样蛋白酶2（PLP 2）。此外，与当前Ub-7-氨基-4-甲基香豆素（Ub-AMC）测定的直接比较表明，Ub-PLA 2测定是表征异肽酶活性调节剂的有效工具。通过分析非选择性异肽酶抑制剂NSC 632839对DUB和deSUMO酰化酶的抑制活性，扩大了这一观察结果。两者合计，这些研究说明了实用的基于酶的方法来测量异肽酶活性。
Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A2 (PLA2). Isopeptidase activity releases PLA2, which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA2 assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA2 assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.
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