Institute of Biochemistry and Molecular Biology, RWTH Aachen University, Aachen, Germany.
Leiden Institute of Chemistry, Leiden University Department of Bioorganic Synthesis, Leiden, Netherlands.
Life Sci Alliance. 2022 Nov 11;6(1). doi: 10.26508/lsa.202201455. Print 2023 Jan.
The modification of substrates with ADP-ribose (ADPr) is important in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it is unknown whether they recognise ADPr, specific amino acid-ADPr linkages, or ADPr with the surrounding protein backbone. We first optimised methods to prepare extracts containing ADPr-proteins and observe that depending on the amino acid modified, the modification is heatlabile. We tested the reactivity of available reagents with diverse ADP-ribosylated protein and RNA substrates and observed that not all reagents are equally suited for all substrates. Next, we determined cross-reactivity with adenylylated RNA, AMPylated proteins, and metabolites, including NADH, which are detected by some reagents. Lastly, we analysed ADP-ribosylation using confocal microscopy, where depending on the fixation method, either mitochondrion, nucleus, or nucleolus is stained. This study allows future work dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates, as we optimised sample preparation methods and have defined the reagents suitable for specific methods and substrates.
ADP-核糖（ADPr）修饰的底物在抗病毒免疫和癌症等方面非常重要。最近，开发了几种检测 ADP-核糖基化的试剂；然而，尚不清楚它们是否识别 ADPr、特定氨基酸-ADPr 键或带有周围蛋白质骨架的 ADPr。我们首先优化了制备含有 ADPr-蛋白质提取物的方法，并观察到根据修饰的氨基酸，修饰是热不稳定的。我们测试了现有试剂与各种 ADP-核糖基化蛋白质和 RNA 底物的反应性，并观察到并非所有试剂都适用于所有底物。接下来，我们确定了与腺苷酰化 RNA、AMP 化蛋白质以及包括 NADH 在内的代谢物的交叉反应性，一些试剂可以检测到 NADH。最后，我们使用共聚焦显微镜分析了 ADP-核糖基化，根据固定方法的不同，线粒体、细胞核或核仁被染色。这项研究允许未来在细胞中对 ADP-核糖基化在蛋白质和 RNA 底物上的功能进行剖析，因为我们优化了样品制备方法，并确定了适合特定方法和底物的试剂。
