Laboratory of Electroanalysis and Corrosion, Instituto Pedro Nunes, 3030-199 Coimbra, Portugal.
National Institute of Material Physics, Atomistilor 405A, 077125 Magurele, Romania.
Talanta. 2019 Jul 1;199:32-39. doi: 10.1016/j.talanta.2019.02.052. Epub 2019 Feb 12.
The majority of eukaryotic regulated protein turnover is performed by the proteasome, a multi-catalytic enzyme. Due to the fact that proteasome enzyme abnormal functioning was observed in different malignant cells, the proteasome is becoming a target for medical treatment. In order to evaluate the mechanisms of action of pharmaceutical compounds on proteasome enzyme inhibition, detecting and characterizing its activity is essential. An electrochemical assay that allows the monitoring of the chymotrypsin-like activity and inhibition of the 20S proteasome enzyme, based on the electrochemical detection of an electroactive compound released upon proteolysis of an adequate chymotrypsin-substrate is described. By employing differential pulse voltammetric measurement, the activity of the 20S proteasome enzyme was investigated for different incubation times of 20S with oligopeptide substrate as well as for different concentrations of substrate. Enzyme kinetic parameters were determined by voltammetry and the electrochemical assay compared with fluorescence spectroscopy. Electrochemical quartz crystal microbalance and atomic force microscopy were also used to investigate substrate interaction with the 20S proteasome and their adsorption at the electrode surface. Finally, the new electrochemical assay allowed to investigate the mechanisms of two different proteasome inhibitor drugs, bortezomib and oprozomib, underlying the applicability of the assay for understanding proteasome inhibitor action.
大多数真核生物调节蛋白的降解是由蛋白酶体完成的，它是一种多催化酶。由于在不同的恶性细胞中观察到蛋白酶体酶的异常功能，因此蛋白酶体正在成为治疗的靶点。为了评估药物化合物对蛋白酶体酶抑制作用的作用机制，检测和表征其活性是必不可少的。本文描述了一种基于电活性化合物在适当的胰凝乳蛋白酶底物的蛋白水解作用下释放的电化学测定，用于监测糜蛋白酶样活性和 20S 蛋白酶体酶抑制的电化学测定。通过采用差分脉冲伏安法测量，研究了 20S 与寡肽底物孵育不同时间以及不同底物浓度时的酶活性。通过伏安法和电化学测定确定了酶动力学参数，并将电化学测定与荧光光谱法进行了比较。电化学石英晶体微天平和原子力显微镜也用于研究底物与 20S 蛋白酶体的相互作用及其在电极表面的吸附。最后，新的电化学测定法允许研究两种不同的蛋白酶体抑制剂药物硼替佐米和奥普佐米的作用机制，证明了该测定法在理解蛋白酶体抑制剂作用方面的适用性。
