人类对氧磷酯酶2(PON2)是芳基酯酶和内酰胺酶小家族中最古老的成员,代表抵抗细菌感染的第一道防线,在ROS相关疾病如癌症中发挥重要作用,心血管疾病,神经变性,和糖尿病。特定的翻译后修饰(PTM)聚集在与pon2多态性位点相对应的两个残基附近,它们对催化活性的影响尚未完全了解。因此,本研究的目的是开发一种改进的PON2纯化方案,以获得更大量的蛋白质,适合深入的生化研究和生物技术应用.为此,我们还测试了几种化合物来稳定酶的活性单体形式。将酶与30mMThrealose一起在4°C下储存对活性的影响最好,保存了至少30天。确定了针对底物3-氧代-十二烷酰基-高丝氨酸内酯(3oxoC12-HSL)的催化参数以及干扰铜绿假单胞菌(PAO1)生物膜形成的酶能力,表明所获得的酶非常适合下游应用。最后,我们用纯化的rPON2来检测,通过直接分子钓鱼(DMF)方法,来自HeLa细胞可溶性提取物的新的推定PON2相互作用物。
The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.