State Key laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People's Republic of China.
Department of Medicinal Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China; Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China.
Biosens Bioelectron. 2017 May 15;91:400-407. doi: 10.1016/j.bios.2016.12.065. Epub 2016 Dec 30.
Reversible protein acetylation, one of the key types of post-translational modifications, is composed of histone acetylation and deacetylation, which is typically catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) respectively. Herein, a label-free fluorescent method has been established for the homogeneous bioassay of HAT/HDAC activity and respective inhibitors. The proposed approach is primarily based on the electrostatic interaction between G-quadruplexes (G4s) and acetylation-related peptides, which results in marked change of fluorescent intensity of G4/Thioflavin T (ThT) complexes. This HAT (p300) activity assay is exceedingly sensitive and selective, with a linear range from 0.1 to 120nM and a detection limit of 0.05nM. Moreover, this biosensor is feasible to detect the HDAC (Sirt1) activity with a linear range from 1 to 450nM and a detection limit of 1nM. The potency of this assay is further demonstrated by detecting HAT/HDAC activity in cell lysates and evaluating HAT and HDAC-targeted inhibitors, C464 and EX 527, respectively. The proposed assay is convenient, label-free and cost-efficient, which is promising for HAT/HDAC-targeted epigenetic research and pharmaceutical development.
可逆蛋白质乙酰化是翻译后修饰的关键类型之一，由组蛋白乙酰化和去乙酰化组成，分别由组蛋白乙酰转移酶（HATs）和组蛋白去乙酰化酶（HDACs）催化。在此，建立了一种用于 HAT/HDAC 活性及其抑制剂的均相生物测定的无标记荧光方法。该方法主要基于 G-四链体（G4s）与乙酰化相关肽之间的静电相互作用，导致 G4/硫黄素 T（ThT）复合物的荧光强度发生明显变化。该 HAT（p300）活性测定法具有极高的灵敏度和选择性，线性范围为 0.1 至 120nM，检测限为 0.05nM。此外，该生物传感器可用于检测 HDAC（Sirt1）活性，线性范围为 1 至 450nM，检测限为 1nM。该测定法在细胞裂解物中检测 HAT/HDAC 活性并评估 HAT 和 HDAC 靶向抑制剂 C464 和 EX 527 的效力进一步证明了其功效。该测定法简便、无标记且具有成本效益，有望用于 HAT/HDAC 靶向的表观遗传研究和药物开发。
