Quantitative monitoring of ubiquitination/deubiquitination reaction cycles by 18O-incorporation
Quantitative monitoring of ubiquitination/deubiquitination reaction cycles by 18O-incorporation
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通过 18O 掺入定量监测泛素化/去泛素化反应循环
DOI：
10.1016/j.bbrc.2020.06.008
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发表时间：
2020
影响因子：
3.1
通讯作者：
Shirakawa Masahiro
中科院分区：
文献类型：
--
作者：
Tanaka Yuka;Morimoto Daichi;Walinda Erik;Sugase Kenji;Shirakawa Masahiro
泛素化是主要的翻译后修饰之一，需要泛素分子与靶蛋白缀合。为了使游离的泛素分子可用于缀合，在细胞中不仅从头合成泛素，而且还通过切割现有的缀合的泛素分子来提供泛素，所谓的去泛素化反应。因此，细胞内泛素分子被认为是回收的，但回收频率仍然难以捉摸。缺乏这种机制细节的主要原因是原始和回收的泛素分子在化学和物理性质上无法区分。为了解决这个问题，在这里，我们应用18 O-标记来追踪泛素是如何在同时的泛素化/去泛素化反应（泛素循环反应）中循环的。由于去泛素化是一个水解反应，在18 O标记的水溶液中，泛素分子的C端羧基的两个16 O原子可以通过去泛素化被18 O原子交换。利用定量质谱技术，我们检测到在去泛素化反应过程中18 O原子掺入到泛素的C-末端羧基中，此外，我们还能够定量泛素循环反应中18 O-掺入。出乎意料的是，动力学分析表明，在去泛素化酶的存在下，泛素化反应加速。总的来说，我们已经建立了一个定量的方法来跟踪泛素循环反应，通过分析去泛素化相关的18 O-掺入到泛素。
Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesizedde novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied18O-labeling to trace how ubiquitin is recycled in a simultaneous ubiquitination/deubiquitination reaction (ubiquitin cycle reaction). Because deubiquitination is a hydrolysis reaction, the two16O atoms of the C-terminal carboxy group of a ubiquitin molecule can be exchanged with18O atoms by deubiquitination in18O-labeled aqueous solution. By using quantitative mass spectrometry, we detected18O atom incorporation into the C-terminal carboxy group of ubiquitin in the course of a deubiquitination reaction, in addition, we were able to quantify the18O-incorporation in a ubiquitin cycle reaction. Unexpectedly, kinetic analysis suggested that ubiquitination reactivity was accelerated in the presence of a deubiquitinating enzyme. Collectively, we have established a quantitative method to trace ubiquitin cycle reactions by analyzing deubiquitination-associated18O-incorporation into ubiquitin.