A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein
A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein
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DOI：
10.1016/j.ab.2006.06.031
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发表时间：
2007-01-01
影响因子：
2.9
通讯作者：
Riddle, Steven M.
中科院分区：
文献类型：
--
作者：
Horton, Robert A.;Strachan, Elizabeth A.;Riddle, Steven M.
去泛素化酶（DUB）通过蛋白水解将泛素从泛素化蛋白质中切割，并且抑制DUB使致癌蛋白免于蛋白酶体降解是新兴的治疗兴趣。最近，USP2和UCH 37已被证明去泛素化肿瘤生长促进蛋白，其他DUB已被证明在癌细胞中过表达。因此，DUB的抑制作为治疗癌症的潜在治疗策略是令人感兴趣的。DUB需要在底物中存在适当折叠的泛素蛋白以进行有效的蛋白水解，这排除了在DUB活性测定中使用合成肽底物。由于需要全长泛素，因此难以制备适用于荧光测定以鉴定或研究DUB抑制剂的底物。我们描述了一种基于时间分辨荧光共振能量转移（FRET）的DUB底物的开发，该底物包含使用遗传编码的黄色荧光蛋白（YFP）和化学连接的铽供体进行位点特异性标记的全长泛素。完整的基板显示出高度的FRET铽和YFP之间，而DUB依赖性切割导致FRET的减少。(c)2006年爱思唯尔公司All rights reserved.
Deubiquitinating enzymes (DUBs) proteolytically cleave ubiquitin from ubiquitinated proteins, and inhibition of DUBs that rescue oncogenic proteins from proteasomal degradation is of emerging therapeutic interest. Recently, USP2 and UCH37 have been shown to deubiquitinate tumor-growth-promoting proteins, and other DUBs have been shown to be overexpressed in cancer cells. Therefore inhibition of DUBs is of interest as a potential therapeutic strategy for treating cancer. DUBs require the presence of properly folded ubiquitin protein in the substrate for efficient proteolysis, which precludes the use of synthetic peptide substrates in DUB activity assays. Because of the requirement for full-length ubiquitin, substrates suitable for use in fluorescent assays to identify or study DUB inhibitors have been difficult to prepare. We describe the development of a time-resolved fluorescence resonance energy transfer (FRET)-based DUB substrate that incorporates full-length ubiquitin that is site-specifically labeled using genetically encoded yellow fluorescent protein (YFP) and a chemically attached terbium donor. The intact substrate shows a high degree of FRET between terbium and YFP, whereas DUB-dependant cleavage leads to a decrease in FRET. (c) 2006 Elsevier Inc. All rights reserved.