Production of highly soluble native human paraoxonase 2 with potential anti-biofilm property.

Paraoxonase 2 (PON2) is considered as a potential anti-biofilm agent due to the highest lactonase activity among the PON family members implicating quorum quenching in gram-negative bacteria. However, PON2 is expressed mostly in insoluble fractions in the bacterial expression host which limits its application as an anti-biofilm agent. Therefore, obtaining the native human PON2 (HuPON2) protein in soluble form, better protein yield, stability, and enzymatic activities is essential. In this study, procedures for obtaining a high yield of the native form of HuPON2 in soluble and active forms were optimized. Guanidinium hydrochloride solubilized the HuPON2 protein, however, it is lethal for several bacteria, and thus a major problem for studying the various downstream application of the protein. Therefore, another refolding process for native HuPON2 was optimized. Owing to the promiscuous nature of HuPON2, we hypothesized that it could inhibit the biofilm formation in Mycobacterium smegmatis also. Interestingly, we observed a significant inhibition of the biofilm formation by HuPON2_Rf. However, the primary target of HuPON2 and the probable mechanism behind the quorum quenching in M. smegmatis need to be further explored, which would help widen the scope of HuPON2 as a potential anti-biofilm agent beyond the gram-negative bacteria.

Chlorpyrifos and parathion regulate oxidative stress differentially through the expression of paraoxonase 2 in human neuroblastoma cell.

Organophosphate (OP) compounds are frequently linked to both chronic and acute forms of nervous system disorders. Chlorpyrifos (CPF) and parathion (PA) are two of the most widely used OP insecticides throughout the world. These compounds are acetylcholinesterase inhibitors and cause a cholinergic crisis. However, there are other non-cholinergic effects of the OP compounds as well. The role of Paraoxonase 1 (PON1) in the metabolism of OP compounds is well established owing to its significant organophosphatase activity. Since PON2 has no paraoxonase activity and the level of its expression is 20-40 fold higher in the brain, in this article the role of PON2 in response to CPF and PA exposure concerning both cholinergic and non-cholinergic effects are explored. The effect of these OPs on cell viability, reactive oxygen species (ROS), PON2 gene expression, and function was studied. Glutathione level, esterase activity, and paraoxonase activity were also measured in CPF- and PA-treated IMR-32 cells. At these levels, both CPF and PA showed different impacts on IMR-32 cells. PA at higher concentrations (50-200 µM) proved to be less toxic than CPF. Interestingly, induction of ROS was also lower in the case of PA-treated cells as compared to the CPF. However, PON2 protein expression was increased with the increasing concentration of PA and decreased with the increasing concentration of CPF. To explore the possible mechanism of the differential regulation of PON2 gene expression by CPF and PA, we investigated the possible binding and signaling through the human M2 muscarinic acetylcholine receptor (M2AChR). Since M2AChRs are similar to G-protein coupled receptors and function through cAMP signalling, we measured the cAMP level after CPF and PA treatment. CPF- and PA-treated IMR-32 cells can be used as a model to study the mechanism by which PON2 acts as a ROS scavenger in response to xenobiotics stimulation in the brain.

Insights into the role of paraoxonase 2 in human pathophysiology.

Paraoxonase 2 (PON2) is a ubiquitously expressed intracellular enzyme that is known to have a protective role from oxidative stress. Clinical studies have also demonstrated the significance of PON2 in the manifestation of cardiovascular and several other diseases, and hence, it is considered an important biomarker. Recent findings of its expression in brain tissue suggest its potential protective effect on oxidative stress and neuroinflammation. Polymorphisms of PON2 in humans are a risk factor in many pathological conditions, suggesting a possible mechanism of its anti-oxidative property probably through lactonase activity. However, exogenous factors may also modulate the expression and activity of PON2. Hence, this review aims to report the mechanism by which PON2 expression is regulated and its role in oxidative stress disorders such as neurodegeneration and tumor formation. The role of PON2 owing to its lactonase activity in bacterial infectious diseases and association of PON2 polymorphism with pathological conditions are also highlighted.

Association between human paraoxonase 2 protein and efficacy of acetylcholinesterase inhibiting drugs used against Alzheimer's disease.

Serum Paraoxonase 2 (PON2) level is a potential biomarker owing to its association with a number of pathophysiological conditions such as atherosclerosis and cardiovascular disease. Since cholinergic deficiency is closely linked with Alzheimer's disease (AD) progression, acetylcholinesterase inhibitors (AChEIs) are the treatment of choice for patients with AD. However, there is a heterogenous response to these drugs and mostly the subjects do not respond to the treatment. Gene polymorphism, the simultaneous occurrence of two or more discontinuous alleles in a population, could be one of the important factors for this. Hence, we hypothesized that PON2 and its polymorphic forms may be hydrolyzing the AChEIs differently, and thus, different patients respond differently. To investigate this, two AChEIs, donepezil hydrochloride (DHC) and pyridostigmine bromide (PB), were selected. Human PON2 wildtype gene and four mutants, two catalytic sites, and two polymorphic sites were cloned, recombinantly expressed, and purified for in vitro analysis. Enzyme activity and AChE activity were measured to quantitate the amount of DHC and PB hydrolyzed by the wildtype and the mutant proteins. Herein, PON2 esterase activity and AChE inhibitor efficiency were found to be inversely related. A significant difference in enzyme activity of the catalytic site mutants was observed as compared to the wildtype, and subsequent AChE activity showed that esterase activity of PON2 is responsible for the hydrolysis of DHC and PB. Interestingly, PON2 polymorphic site mutants showed increased esterase activity; therefore, this could be the reason for the ineffectiveness of the drugs. Thus, our data suggested that the esterase activity of PON2 was mainly responsible for the hydrolysis of AChEI, DHC, and PB, and that might be responsible for the variation in individual response to AChEI therapy.

Insights into the role of paraoxonase 2 in human pathophysiology.

Paraoxonase 2 (PON2) is a ubiquitously expressed intracellular enzyme that is known to have a protective role from oxidative stress. Clinical studies have also demonstrated the significance of PON2 in the manifestation of cardiovascular and several other diseases, and hence, it is considered an important biomarker. Recent findings of its expression in brain tissue suggest its potential protective effect on oxidative stress and neuroinflammation. Polymorphisms of PON2 in humans are a risk factor in many pathological conditions, suggesting a possible mechanism of its anti-oxidative property probably through lactonase activity. However, exogenous factors may also modulate the expression and activity of PON2. Hence, this review aims to report the mechanism by which PON2 expression is regulated and its role in oxidative stress disorders such as neurodegeneration and tumor formation. The role of PON2 owing to its lactonase activity in bacterial infectious diseases and association of PON2 polymorphism with pathological conditions are also highlighted.

Comparative analysis of the metal-dependent structural and functional properties of mouse and human SMP30.

Senescence Marker Protein (SMP30) is a metalloenzyme that shows lactonase activity in the ascorbic acid (AA) biosynthesis pathway in non-primate mammals such as a mouse. However, AA biosynthesis does not occur in the primates including humans. Several studies have shown the role of SMP30 in maintaining calcium homeostasis in mammals. In addition, it is also reported to have promiscuous enzyme activity with an organophosphate (OP) substrate. Hence, this study aims to recombinantly express and purify the SMP30 proteins from both mouse and human, and to study their structural alterations and functional deviations in the presence of different divalent metals. For this, mouse SMP30 (MoSMP30) as well as human SMP30 (HuSMP30) were cloned in the bacterial expression vector. Proteins were overexpressed and purified from soluble fractions as well as from inclusion bodies as these proteins were expressed largely in insoluble fractions. The purified proteins were used to study the folding conformations in the presence of different divalent cations (Ca2+, Co2+, Mg2+, and Zn2+) with the help of circular dichroism (CD) spectroscopy. It was observed that both MoSMP30 and HuSMP30 acquired native folding conformations. To study the metal-binding affinity, dissociation constant (Kd values) were calculated from UV-VIS titration curve, which showed the highest affinity of MoSMP30 with Zn2+. However, HuSMP30 showed the highest affinity with Ca2+, suggesting the importance of HuSMP30 in maintaining calcium homeostasis. Enzyme kinetics were performed with γ-Thiobutyrolactone and Demeton-S in the presence of different divalent cations. Interestingly, both the proteins showed lactonase activity in the presence of Ca2+. In addition, MoSMP30 and HuSMP30 also showed lactonase activity in the presence of Co2+ and Zn2+ respectively. Moreover, both the proteins showed OP hydrolase activities in the presence of Ca2+ as well as Zn2+, suggesting the metal-dependent promiscuous nature of SMP30.