用于单 ADP 核糖基转移酶的宏结构域连接免疫吸附测定（MLISA）。
A macrodomain-linked immunosorbent assay (MLISA) for mono-ADP-ribosyltransferases.
作者信息
Chen Jingwen, Lam Albert T, Zhang Yong
机构信息
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA.
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA; Department of Chemistry, Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA 90089, USA; Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90089, USA; Research Center for Liver Diseases, University of Southern California, Los Angeles, CA 90089, USA.
出版信息
Anal Biochem. 2018 Feb 15;543:132-139. doi: 10.1016/j.ab.2017.12.019. Epub 2017 Dec 13.
ADP-ribosyltransferases (ARTs) catalyze reversible additions of mono- and poly-ADP-ribose onto diverse types of proteins by using nicotinamide adenine dinucleotide (NAD) as a cosubstrate. In the human ART superfamily, 14 out of 20 members are shown to catalyze endogenous protein mono-ADP-ribosylation and play important roles in regulating various physiological and pathophysiological processes. Identification of new modulators of mono-ARTs can thus potentially lead to discovery of novel therapeutics. In this study, we developed a macrodomain-linked immunosorbent assay (MLISA) for characterizing mono-ARTs. Recombinant macrodomain 2 from poly-ADP-ribose polymerase 14 (PARP14) was generated with a C-terminal human influenza hemagglutinin (HA) tag for detecting mono-ADP-ribosylated proteins. Coupled with an anti-HA secondary antibody, the generated HA-tagged macrodomain 2 reveals high specificity for mono-ADP-ribosylation catalyzed by distinct mono-ARTs. Kinetic parameters of PARP15-catalyzed automodification were determined by MLISA and are in good agreement with previous studies. Eight commonly used chemical tools for PARPs were examined by MLISA with PARP15 and PARP14 in 96-well plates and exhibited moderate inhibitory activities for PARP15, consistent with published reports. These results demonstrate that MLISA provides a new and convenient method for quantitative characterization of mono-ART enzymes and may allow identification of potent mono-ART inhibitors in a high-throughput-compatible manner.
ADP-核糖基转移酶（ARTs）通过使用烟酰胺腺嘌呤二核苷酸（NAD）作为共底物，催化单-ADP-核糖和多-ADP-核糖可逆地添加到不同类型的蛋白质上。在人类ART超家族中，20个成员中有14个被证明可催化内源性蛋白质单-ADP-核糖基化，并在调节各种生理和病理生理过程中发挥重要作用。因此，鉴定新型单-ART调节剂可能会带来新型治疗方法的发现。在本研究中，我们开发了一种用于表征单-ARTs的宏结构域连接免疫吸附测定法（MLISA）。从聚-ADP-核糖聚合酶14（PARP14）产生的重组宏结构域2带有C端人流感血凝素（HA）标签，用于检测单-ADP-核糖基化蛋白质。与抗HA二抗结合，所产生的带有HA标签的宏结构域2对由不同单-ARTs催化的单-ADP-核糖基化显示出高特异性。通过MLISA测定了PARP15催化的自身修饰的动力学参数，与先前的研究结果吻合良好。在96孔板中，用PARP15和PARP14通过MLISA检测了8种常用的PARP化学工具，它们对PARP15表现出中等抑制活性，与已发表的报道一致。这些结果表明，MLISA为单-ART酶的定量表征提供了一种新的便捷方法，并可能以高通量兼容的方式鉴定有效的单-ART抑制剂。
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