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Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells
JACS Au ( IF 8.5 ) Pub Date : 2023-10-03 , DOI: 10.1021/jacsau.3c00421 Lu-Jun Liang 1, 2 , Yu Wang 3 , Xiao Hua 4 , Rujing Yuan 3 , Qiong Xia 3 , Rongtian Wang 3 , Chuntong Li 1, 2 , Guo-Chao Chu 1, 3 , Lei Liu 1, 2 , Yi-Ming Li 3
JACS Au ( IF 8.5 ) Pub Date : 2023-10-03 , DOI: 10.1021/jacsau.3c00421 Lu-Jun Liang 1, 2 , Yu Wang 3 , Xiao Hua 4 , Rujing Yuan 3 , Qiong Xia 3 , Rongtian Wang 3 , Chuntong Li 1, 2 , Guo-Chao Chu 1, 3 , Lei Liu 1, 2 , Yi-Ming Li 3
Affiliation
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Dynamic monitoring of intracellular ubiquitin (Ub) conjugates is instrumental to understanding the Ub regulatory machinery. Although many biochemical approaches have been developed to characterize protein ubiquitination, chemical tools capable of temporal resolution probing of ubiquitination events remain to be developed. Here, we report the development of the first cell-permeable and stimuli-responsive Ub probe and its application for the temporal resolution profiling of ubiquitinated substrates in live cells. The probe carrying the photolabile group N-(2-nitrobenzyl)-Gly (Nbg) on the amide bond between Ub Gly75 and Gly76 is readily prepared through chemical synthesis and can be delivered to live cells by conjugation via a disulfide bond with the cyclic cell-penetrating peptide cR10D (i.e., 4-((4-(dimethylamino)phenyl)-azo)-benzoic acid-modified cyclic deca-arginine). Both in vitro and in vivo experiments showed that Ub-modifying enzymes (E1, E2s, and E3s) could not install the Ub probe onto substrate proteins prior to removal of the nitrobenzyl group, which was easily accomplished via photoirradiation. The utility and practicality of this probe were exemplified by the time-resolved biochemical and proteomic investigation of ubiquitination events in live cells during a H2O2-mediated oxidative stress response. This work shows a conceptually new family of chemical Ub tools for the time-resolved studies on dynamic protein ubiquitination in different biological processes and highlights the utility of modern chemical protein synthesis in obtaining custom-designed tools for biological studies.
中文翻译：
用于活细胞中底物泛素化时间分辨监测的细胞渗透性刺激响应泛素探针
细胞内泛素 (Ub) 缀合物的动态监测有助于了解 Ub 调节机制。尽管已经开发了许多生化方法来表征蛋白质泛素化，但能够对泛素化事件进行时间分辨率探测的化学工具仍有待开发。在这里，我们报告了第一个细胞渗透性和刺激响应性 Ub 探针的开发及其在活细胞中泛素化底物的时间分辨率分析中的应用。Ub Gly75 和 Gly76 之间的酰胺键上带有光不稳定基团 N-(2-硝基苄基)-Gly (Nbg) 的探针可通过化学合成轻松制备，并可通过二硫键与循环细胞缀合而递送至活细胞-穿透肽cR10D（即4-((4-(二甲氨基)苯基)-偶氮)-苯甲酸修饰的环十-精氨酸）。体外和体内实验均表明，Ub 修饰酶（E1、E2s 和 E3s）在去除硝基苄基之前无法将 Ub 探针安装到底物蛋白上，而这可以通过光照射轻松实现。该探针的实用性和实用性通过对 H 2 O 2介导的氧化应激反应期间活细胞中泛素化事件的时间分辨生化和蛋白质组学研究得到了例证。这项工作展示了概念上新的化学 Ub 工具家族，用于不同生物过程中动态蛋白质泛素化的时间分辨研究，并强调了现代化学蛋白质合成在获得定制设计的生物学研究工具中的效用。
更新日期：2023-10-03
中文翻译：
用于活细胞中底物泛素化时间分辨监测的细胞渗透性刺激响应泛素探针
细胞内泛素 (Ub) 缀合物的动态监测有助于了解 Ub 调节机制。尽管已经开发了许多生化方法来表征蛋白质泛素化，但能够对泛素化事件进行时间分辨率探测的化学工具仍有待开发。在这里，我们报告了第一个细胞渗透性和刺激响应性 Ub 探针的开发及其在活细胞中泛素化底物的时间分辨率分析中的应用。Ub Gly75 和 Gly76 之间的酰胺键上带有光不稳定基团 N-(2-硝基苄基)-Gly (Nbg) 的探针可通过化学合成轻松制备，并可通过二硫键与循环细胞缀合而递送至活细胞-穿透肽cR10D（即4-((4-(二甲氨基)苯基)-偶氮)-苯甲酸修饰的环十-精氨酸）。体外和体内实验均表明，Ub 修饰酶（E1、E2s 和 E3s）在去除硝基苄基之前无法将 Ub 探针安装到底物蛋白上，而这可以通过光照射轻松实现。该探针的实用性和实用性通过对 H 2 O 2介导的氧化应激反应期间活细胞中泛素化事件的时间分辨生化和蛋白质组学研究得到了例证。这项工作展示了概念上新的化学 Ub 工具家族，用于不同生物过程中动态蛋白质泛素化的时间分辨研究，并强调了现代化学蛋白质合成在获得定制设计的生物学研究工具中的效用。
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