Barsan Madalina M, Diculescu Victor C
National Institute of Material Physics, 077125, Magurele, Romania.
Analyst. 2021 May 21;146(10):3216-3224. doi: 10.1039/d0an02426k. Epub 2021 Apr 7.
The 20S proteasome enzyme complex is involved in the proteolytic degradation of misfolded and oxidatively damaged proteins and is a focus of medical research for the development of compounds with pharmaceutical properties, which are active in cancer cells and/or neurodegenerative diseases. The present study aims to develop a biosensor for investigating the 20S proteasome activity and inhibition by means of electrochemical methods. The 20S proteasome is best immobilized at the electrode surface through bio-affinity interactions with antibodies that target different subunits on the 20S proteasome, enabling the investigation of the effect of an enzyme's orientation on biosensor response. The enzymatic activity is analyzed by fixed potential amperometry with the highest sensitivity of 24 μA cm mM and a LOD of 0.4 μM. The detection principle involves the oxidation of an electroactive probe that is released from the enzyme's substrates upon proteolysis. The most sensitive biosensor is then used to study the multicatalytic activity of the 20S proteasome, i.e. the caspase-, trypsin- and chymotrypsin-like activity, by analyzing the biosensor's sensitivity towards different substrates. The behavior of the immobilized 20S proteasome is investigated as a function of substrate concentration. The kinetic parameters are derived and compared with those obtained when the enzyme was free in solution, with K values being one to two orders of magnitude lower in the present case. Two 20S inhibitors, epoxomicin and bortezomib, are investigated by analyzing their influence on the 20S biosensor response. The proposed analytical method for proteasome activity and inhibitor screening has the main advantage of being cost-effective compared to the ones typically employed.
20S蛋白酶体酶复合物参与错误折叠和氧化损伤蛋白质的蛋白水解降解，是医学研究中开发具有药物特性化合物的重点，这些化合物在癌细胞和/或神经退行性疾病中具有活性。本研究旨在开发一种生物传感器，通过电化学方法研究20S蛋白酶体的活性和抑制作用。20S蛋白酶体通过与靶向20S蛋白酶体不同亚基的抗体进行生物亲和相互作用，最佳地固定在电极表面，从而能够研究酶的取向对生物传感器响应的影响。通过固定电位安培法分析酶活性，最高灵敏度为24 μA cm mM，检测限为0.4 μM。检测原理涉及氧化还原活性探针的氧化，该探针在蛋白水解时从酶的底物中释放出来。然后使用最灵敏的生物传感器，通过分析生物传感器对不同底物的灵敏度，研究20S蛋白酶体的多催化活性，即半胱天冬酶样、胰蛋白酶样和糜蛋白酶样活性。研究固定化20S蛋白酶体的行为作为底物浓度的函数。推导动力学参数并与酶在溶液中游离时获得的参数进行比较，在本案例中K值低一到两个数量级。通过分析两种20S抑制剂环氧霉素和硼替佐米对20S生物传感器响应的影响进行研究。所提出的蛋白酶体活性分析方法和抑制剂筛选方法与通常使用的方法相比，主要优点是具有成本效益。
