Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes.
Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes.
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DOI：
10.1002/cbic.201200497
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发表时间：
2012-10-15
期刊：
影响因子：
3.2
通讯作者：
Ovaa, Huib
中科院分区：
文献类型：
--
作者：
de Jong, Annemieke;Merkx, Remco;Berlin, Ilana;Rodenko, Boris;Wijdeven, Ruud H. M.;El Atmioui, Dris;Yalcin, Zeliha;Robson, Craig N.;Neefjes, Jacques J.;Ovaa, Huib
关键词：
表位标记的活性位点定向探针被广泛用于观察细胞提取物中去泛素化酶（DUB）的活性，研究小分子DUB抑制剂的特异性和效力，以及通过质谱法分离和鉴定DUB。随着DUB作为新的潜在药物靶点的出现，需要能够以足够的量生产并满足给定实验的特定需求的探针。已建立的DUB探针生成方法使用基于内含肽的劳动密集型方法，该方法在非天然氨基酸的掺入和可获得的材料量方面具有固有的局限性。在这里，我们描述了活性位点定向探针的全化学合成及其在基于活性的功能DUBs分析和鉴定中的应用。这种合成方法允许容易地掺入用于特定应用的所需标签，例如，荧光报告分子、用于免疫沉淀或亲和下拉的手柄和可切割的接头。另外，合成方法可以按比例放大以提供显著量的探针。荧光泛素探针允许更快的，在凝胶检测活性DUBs相比，（免疫）印迹程序。一个生物素化的探针持有的光可裂解的接头，使亲和下拉和随后的温和的，光释放的DUB。此外，DUB活性水平响应于过表达或敲低以及小分子的抑制进行监测。此外，荧光探针揭示了差异DUB活性分布在一组肺癌和前列腺癌细胞。
Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.
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