Photocaged Cell-Permeable Ubiquitin Probe for Temporal Profiling of Deubiquitinating Enzymes,Journal of the American Chemical Society

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Photocaged Cell-Permeable Ubiquitin Probe for Temporal Profiling of Deubiquitinating Enzymes
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2020-11-03 , DOI: 10.1021/jacs.9b12426
Weijun Gui ₁ , Siqi Shen ₁ , Zhihao Zhuang ₁

Affiliation

Photocaged cell-permeable ubiquitin probe holds promise in profiling the activity of cellular deubiquitinating enzymes (DUBs) with the much needed temporal control. Here we report a new photocaged cell-permeable ubiquitin probe that undergoes photoactivation upon 365 nm UV treatment and enables intracellular deubiquitinating enzyme profiling. We used a semisynthetic approach to generate modular ubiquitin-based probe containing a tetrazole-derived warhead at the C-terminus of ubiquitin and employed a cyclic polyarginine cell-penetrating peptide (cR10) conjugated to the N-terminus of ubiquitin via a disulfide linkage to deliver the probe into live cells. Upon 365 nm UV irradiation, the tetrazole group is converted to a nitrilimine intermediate in situ, which reacts with nearby nucleophilic cysteine residue from the DUB active site. The new photocaged cell-permeable probe showed good reactivity toward purified DUBs, including USP2, UCHL1, and UCHL3, upon photoirradiation. The Ub-tetrazole probe was also assessed in HeLa cell lysate and showed robust labeling only upon photoactivation. We further carried out protein profiling in intact HeLa cells using the new photocaged cell-permeable ubiquitin probe and identified DUBs captured by the probe using label-free quantitative (LFQ) mass spectrometry. Importantly, the photocaged cell-permeable ubiquitin probe captured DUBs specifically in respective G1/S and G2/M phases in synchronized HeLa cells. Moreover, using this probe DUBs were profiled at different time points following the release of HeLa cells from G1/S phase. Our results showed that photocaged cell-permeable probe represents a valuable new tool for achieving a better understanding of the cellular functions of DUBs.

中文翻译：

用于去泛素化酶时间分析的光笼细胞渗透性泛素探针

光笼式细胞渗透性泛素探针有望通过急需的时间控制来分析细胞去泛素化酶 (DUB) 的活性。在这里，我们报告了一种新的光笼细胞可渗透泛素探针，该探针在 365 nm 紫外线处理后进行光活化，并能够进行细胞内去泛素化酶分析。我们使用半合成方法在泛素的 C 端生成包含四唑衍生弹头的模块化泛素探针，并采用环状聚精氨酸细胞穿透肽 (cR10) 通过二硫键与泛素 N 端偶联将探针输送到活细胞中。在 365 nm 紫外线照射下，四唑基团被原位转化为腈亚胺中间体，它与附近来自 DUB 活性位点的亲核半胱氨酸残基反应。新的光笼细胞渗透探针在光照射下对纯化的 DUB（包括 USP2、UCHL1 和 UCHL3）显示出良好的反应性。Ub-四唑探针也在 HeLa 细胞裂解物中进行了评估，并且仅在光活化时显示出强大的标记。我们使用新的光笼细胞渗透泛素探针在完整的 HeLa 细胞中进一步进行了蛋白质分析，并使用无标记定量 (LFQ) 质谱法鉴定了探针捕获的 DUB。重要的是，光笼罩的细胞渗透性泛素探针在同步的 HeLa 细胞中分别在 G1/S 和 G2/M 期特异性捕获 DUB。此外，使用该探针在 HeLa 细胞从 G1/S 期释放后的不同时间点对 DUB 进行分析。

更新日期：2020-11-03

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