One enzyme, two functions: PON2 prevents mitochondrial superoxide formation and apoptosis independent from its lactonase activity

Abstract

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O(2)(-) dismutase activities and cytochrome c expression unaltered, and it did not oxidize O(2)(-) but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His(114) and His(133) are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys(311), was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.

FIGURE 1.

PON2 specifically but indirectly diminishes O₂^˙̄. A, naive cells with or without PEG-SOD/-catalase (Cat) pretreatment were loaded with L-012 and stimulated with DMNQ. B, naive or PON2-GFP-overexpressing cells were loaded with dihydroethidium, treated with DMNQ for 2 h, and analyzed for 2-hydroxyethidium by HPLC. n = 3–14; n.s. = not significant; *, p < 0.05. Ctr, control. Error bars indicate S.E. C, named cells were loaded with DHR123; ONOO⁻ was produced by Sin-1. Neither did DMNQ produce DHR123 fluorescence, nor did PEG-catalase alter Sin-1-induced signals (not shown). D, 40 μm H₂O₂ was added to lysates from named cells and analyzed for remaining H₂O₂ levels after 15 min. PEG-catalase pretreatment amplified H₂O₂ detoxification under such conditions (not shown). E, O₂^˙̄ was generated by X/XO and detected by L-012 in the presence of recombinant human PON2 (rhPON2) or bovine serum albumin (100 ng; upper panel) or PON2-His₆ (2.5 μg; lower panel). F, named cells were DMNQ-treated (2 h) and analyzed for SOD1 +SOD2 activities by zymography. Band intensity reflects enzyme activity.

FIGURE 2.

PON2 prevents O₂^˙̄ formation. A, indicated cells were loaded with Mito-HE, treated with solvent, antimycin, or rotenone for 2 h, and analyzed by fluorescence-activated cell sorting. n = 3–6; ***, p < 0.001. B, mitochondrial membranes from indicated cells were loaded with MCLA and treated with succinate. C, similar to B but stimulating with α-ketoglutarate. D, intact mitochondria, mitochondrial membranes (mem), or matrix fractions of named cells were loaded with MCLA and exposed to X/XO-generated O₂^˙̄. E, named cells were analyzed for cytochrome c and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein expression. F, H₂O₂ secondary to X/XO-generated O₂^˙̄ was measured in the presence of native lysates from naive (±PEG-SOD) or PON2-GFP-overexpressing cells. HA, hemagglutinin.

FIGURE 3.

Mapping of the glycosylation sites and enzymatically crucial residues of PON2. A, lysates of cells expressing the indicated PON2 proteins were analyzed by Western blotting with or without prior deglycosylation. B, relative 3OC12 lactonase activities of naive or PON2-WT/mutant-overexpressing cells. n = 3–12. n.s. = not significant; ***, p < 0.001. Error bars indicate S.E.

FIGURE 4.

Abrogation of lactonase activity does not affect the anti-oxidative or anti-apoptotic function of PON2. A, named cells were loaded with L-012 and stimulated by DMNQ followed by monitoring chemiluminescence as means of ROS production. B, named cells were treated with staurosporine (0.3 μm) or tunicamycin (1 μg/ml) and assayed for caspase-3/7 activation after 16 h. C, naive, PON2-WT-GFP-, or PON2-H133Q-GFP-overexpressing cells were treated with tunicamycin (1 μg/ml; 24 h) and analyzed for intracellular ATP content. A decrease in ATP reports initiation of cell death. D, named cells were treated with staurosporine (1 μm; 16 h) and analyzed for cell death (annexin V; 7-AAD) by flow cytometry. Graphs show relative induction of apoptosis in treated versus untreated cells. n = 3–9. n.s. = not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.