AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity.

中文翻译：

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前核苷酸探针揭示了人类和细菌核苷酸转移酶的 AMPylation 与 UMPylation 的不同特异性

AMPylation 是人类和细菌细胞用来调节特定蛋白质的活性和功能的翻译后修饰。人类 FICD 和细菌 VopS 等主要 AMPylators 的底物和体外靶标范围已得到广泛研究。最近，AMP 前核苷酸探针也促进了活细胞中 AMPylation 的原位分析。基于这项技术，我们在此推出一种新型 UMP 前核苷酸探针，并利用它来分析未感染和副溶血性弧菌感染的人类细胞。标记蛋白质靶标的质谱分析揭示了人类核苷酸转移酶与几乎相同的 AMP 和 UMP 化蛋白质靶标组的意外混杂。反之亦然，对副溶血弧菌及其效应子 VopS 感染的细胞的研究揭示了宿主酶仅发生 AMP 化，突出了该转移酶对 ATP 的迄今为止未知的特异性。总而言之，前核苷酸探针为关键核苷酸转移酶的原位活性特征提供了前所未有的洞察力，这与它们的体外活性有很大不同。