Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
Keck Structural Biology Laboratory, Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Mol Cell. 2019 Feb 21;73(4):845-856.e5. doi: 10.1016/j.molcel.2018.12.022. Epub 2019 Jan 31.
ADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or polymers, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates or their binding affinities to other proteins. However, progress in this area is hindered by a lack of tools to investigate this protein modification. Here, we describe a new method named ELTA (enzymatic labeling of terminal ADP-ribose) for labeling free or protein-conjugated ADP-ribose monomers and polymers at their 2'-OH termini using the enzyme OAS1 and dATP. When coupled with various dATP analogs (e.g., radioactive, fluorescent, affinity tags), ELTA can be used to explore PAR biology with techniques routinely used to investigate DNA or RNA function. We demonstrate that ELTA enables the biophysical measurements of protein binding to PAR of a defined length, detection of PAR length from proteins and cells, and enrichment of sub-femtomole amounts of ADP-ribosylated peptides from cell lysates.
ADP-核糖基化是指将一个或多个 ADP-核糖基添加到蛋白质上。连接的 ADP-核糖单体或聚合物，通常称为聚（ADP-核糖）（PAR），调节修饰底物的活性或它们与其他蛋白质的结合亲和力。然而，由于缺乏研究这种蛋白质修饰的工具，该领域的进展受到阻碍。在这里，我们描述了一种名为 ELTA（末端 ADP-核糖基的酶标记）的新方法，该方法使用酶 OAS1 和 dATP 在其 2'-OH 末端标记游离或与蛋白质结合的 ADP-核糖单体和聚合物。当与各种 dATP 类似物（例如放射性、荧光、亲和标签）结合使用时，ELTA 可用于使用常规用于研究 DNA 或 RNA 功能的技术探索 PAR 生物学。我们证明，ELTA 能够进行蛋白质与特定长度 PAR 结合的生物物理测量、从蛋白质和细胞中检测 PAR 长度以及从细胞裂解物中富集亚飞摩尔量的 ADP-核糖基化肽。
