Effective Quenchers Are Required to Eliminate the Interference of Substrate: Cofactor Binding in the HAT Scintillation Proximity Assay.
Effective Quenchers Are Required to Eliminate the Interference of Substrate: Cofactor Binding in the HAT Scintillation Proximity Assay.
复制标题
需要有效的猝灭剂来消除 HAT 闪烁邻近测定中底物：辅因子结合的干扰。
DOI：
10.1089/adt.2015.636
复制
发表时间：
2015
影响因子：
1.8
通讯作者：
Zheng,YujunGeorge
中科院分区：
文献类型：
--
作者：
Ngo,Liza;Wu,Jiang;Yang,Chao;Zheng,YujunGeorge
组蛋白乙酰转移酶（HAT）介导乙酰基从辅因子乙酰辅酶A转移到不同蛋白质底物（最显著的是核组蛋白）中的特定赖氨酸的侧链氨基。HAT的失调与许多疾病状态有关。HAT的可靠和快速的生物化学测定对于理解蛋白质乙酰化的生物学功能以及筛选HAT酶的小分子抑制剂至关重要。在这份报告中，我们提出了一个闪烁接近测定（SPA）的HAT酶活性的测量。乙酰基供体为[3 H]Ac-CoA，生物素修饰的组蛋白肽作为HAT底物。在HAT反应后，加入链霉亲和素包被的珠以诱导乙酰化底物接近澄清剂分子。然而，我们观察到辅因子和组蛋白肽底物之间的强非特异性结合，这不利地使SPA性能复杂化。为了防止这个问题，评估了一组化学试剂以消除辅因子-底物相互作用，从而提供可靠的SPA读数。通过优化，SPA对于HAT活性测量和HAT抑制剂评价显示出一致和稳健的性能。总的来说，这种混合和测量测定不需要任何洗涤程序，可以以微孔板形式使用，并且非常适合于HAT化学调节剂的高通量筛选。
Histone acetyltransferases (HATs) mediate the transfer of an acetyl group from the cofactor, acetyl-CoA, to the side chain amino group of specific lysines in diverse protein substrates, most notably nuclear histones. The deregulation of HATs is connected to a number of disease states. Reliable and rapid biochemical assays for HATs are critical for understanding biological functions of protein acetylation, as well as for screening small-molecule inhibitors of HAT enzymes. In this report, we present a scintillation proximity assay (SPA) for the measurement of HAT enzymatic activities. The acetyl donor was [3H]Ac-CoA, and a biotin-modified histone peptide served as the HAT substrate. After the HAT reaction, streptavidin-coated beads were added to induce proximity of acetylated substrate to the scintillant molecules. However, we observed strong nonspecific binding between the cofactor and the histone peptide substrates, which adversely complicated the SPA performance. To prevent this problem, a set of chemical agents were evaluated to eliminate the cofactor–substrate interaction, thus providing reliable SPA readings. With optimization, the SPA showed consistent and robust performance for HAT activity measurement and HAT inhibitor evaluation. Overall, this mix-and-measure assay does not require any washing procedure, can be utilized in the microplate format, and is well suited for high-throughput screening of HAT chemical modulators.