A high-throughput screening compatible assay for activators and inhibitors of methionine sulfoxide reductase A.
A high-throughput screening compatible assay for activators and inhibitors of methionine sulfoxide reductase A.
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DOI：
10.1089/adt.2009.0263
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发表时间：
2010-10
影响因子：
1.8
通讯作者：
David Brunell;H. Weissbach;P. Hodder;N. Brot
中科院分区：
文献类型：
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作者：
David Brunell;H. Weissbach;P. Hodder;N. Brot
蛋氨酸亚砜还原酶（Msr）系统已被证明在保护细胞免受氧化损伤方面发挥重要作用。这个酶家族可以修复由活性氧引起的蛋氨酸残基氧化成蛋氨酸亚砜对蛋白质造成的损伤。先前对动物的遗传研究表明，蛋氨酸亚砜还原酶A （MsrA）是Msr家族的重要成员，其水平的增加可以保护细胞免受氧化损伤并延长寿命。一种高通量筛选（HTS）相容的检测方法已经开发出来，用于寻找MsrA的激活剂和抑制剂。该分析涉及一个偶联反应，其中通过分光光度法或荧光分析测量NADPH的氧化。先前的研究表明，MsrA具有广泛的底物特异性，可以还原多种甲基亚砜化合物，包括二甲基亚砜（DMSO）。由于筛选文库中的化学物质溶解在DMSO中，这将与用于测定MsrA活性的任何标准底物竞争，因此开发了一种使用DMSO作为文库中化合物的溶剂作为MsrA酶的底物的测定方法。MsrA的特异性激活剂可能对涉及氧化损伤的疾病具有重要的治疗价值，特别是与年龄相关的疾病，而MsrA的特异性抑制剂将对各种研究具有价值。
The methionine sulfoxide reductase (Msr) system has been shown to play an important role in protecting cells against oxidative damage. This family of enzymes can repair damage to proteins resulting from the oxidation of methionine residues to methionine sulfoxide, caused by reactive oxygen species. Previous genetic studies in animals have shown that increased levels of methionine sulfoxide reductase enzyme A (MsrA), an important member of the Msr family, can protect cells against oxidative damage and increase life span. A high-throughput screening (HTS) compatible assay has been developed to search for both activators and inhibitors of MsrA. The assay involves a coupled reaction in which the oxidation of NADPH is measured by either spectrophotometric or fluorometric analysis. Previous studies had shown that MsrA has a broad substrate specificity and can reduce a variety of methyl sulfoxide compounds, including dimethylsulfoxide (DMSO). Since the chemicals in the screening library are dissolved in DMSO, which would compete with any of the standard substrates used for the determination of MsrA activity, an assay has been developed that uses the DMSO that is the solvent for the compounds in the library as the substrate for the MsrA enzyme. A specific activator of MsrA could have important therapeutic value for diseases that involve oxidative damage, especially age-related diseases, whereas a specific inhibitor of MsrA would have value for a variety of research studies.