ADP核糖基化是一种古老的蛋白质修饰,核酸,以及在所有生命王国以及某些病毒中发现的其他生物分子。ADP-核糖基化对基本(病理)生理过程的调节,包括细胞应激反应,炎症,以及对细菌和病毒病原体的免疫反应,对修饰建立和去除的研究产生了浓厚的兴趣,以探索新的治疗方法。除了人类的ADP-核糖基化,直接靶向改变宿主ADP-核糖基化信号传导的因子(例如,病毒巨域)或利用ADP-核糖基化来操纵宿主细胞行为(例如,细菌毒素)被证明可以降低毒力和疾病严重程度。然而,因此,这些治疗潜力的实现受到简单,高通量方法研究修饰“作家”和“橡皮擦”,筛选新型抑制剂。这里,我们描述了用于测量(ADP-核糖基)水解酶活性的可扩展方法。该测定依赖于(ADP-核糖基)水解酶从修饰的底物释放的ADP-核糖通过磷酸二酯酶NudT5研究为AMP通过市售检测测定转化为生物发光。此外,该方法可用于研究nudix或ENPP型磷酸二酯酶在ADP-核糖基化过程中的作用,也可用于研究(ADP-核糖基)转移酶的活性。总的来说,该方法既适用于基础生化鉴定,也适用于大型药物库的筛选;它高度适应不同的项目需求。
ADP-ribosylation is an ancient modification of proteins, nucleic acids, and other biomolecules found in all kingdoms of life as well as in certain viruses. The regulation of fundamental (patho)physiological processes by ADP-ribosylation, including the cellular stress response, inflammation, and immune response to bacterial and viral pathogens, has created a strong interest into the
study of modification establishment and removal to explore novel therapeutic approaches. Beyond ADP-ribosylation in humans, direct targeting of factors that alter host ADP-ribosylation signaling (e.g., viral macrodomains) or utilize ADP-ribosylation to manipulate host cell behavior (e.g., bacterial toxins) were shown to reduce virulence and disease severity. However, the realization of these therapeutic potentials is thus far hampered by the unavailability of simple, high-throughput methods to
study the modification \"writers\" and \"erasers\" and screen for novel inhibitors.Here, we describe a scalable method for the measurement of (ADP-ribosyl)hydrolase activity. The assay relies on the conversion of ADP-ribose released from a modified substrate by the (ADP-ribosyl)hydrolase under investigation into AMP by the phosphodiesterase NudT5 into bioluminescence via a commercially available detection assay. Moreover, this method can be utilized to
study the role of nudix- or ENPP-type phosphodiesterases in ADP-ribosylation processing and may also be adapted to investigate the activity of (ADP-ribosyl)transferases. Overall, this method is applicable for both basic biochemical characterization and screening of large drug libraries; hence, it is highly adaptable to diverse project needs.