Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex - PubMed (original) (raw)

. 2013 Sep;123(9):3815-28.

doi: 10.1172/JCI67478. Epub 2013 Aug 1.

Zhiping Wu, Meliana Riwanto, Shengqiang Gao, Bruce S Levison, Xiaodong Gu, Xiaoming Fu, Matthew A Wagner, Christian Besler, Gary Gerstenecker, Renliang Zhang, Xin-Min Li, Anthony J DiDonato, Valentin Gogonea, W H Wilson Tang, Jonathan D Smith, Edward F Plow, Paul L Fox, Diana M Shih, Aldons J Lusis, Edward A Fisher, Joseph A DiDonato, Ulf Landmesser, Stanley L Hazen

Affiliations

Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex

Ying Huang et al. J Clin Invest. 2013 Sep.

Abstract

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein-associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other's function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.

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Figures

Figure 1

Figure 1. MPO and PON1 inhibit each other’s activity in vitro.

(A) PON1 (100 μg/ml) was incubated with either HOCl or H2O2 and MPO (50 nM) in 50 mM Na[PO4] buffer (pH 7.0) supplemented with isolated human HDL (1 mg/ml) and 100 mM NaCl at 37°C for 60 minutes. This was followed by quantification of paraoxonase activity relative to no oxidant exposures. Also shown are the effects of varying levels of H2O2 alone (i.e., no MPO added) or with the addition of the HOCl scavenger methionine (Met) (10-fold molar excess relative to oxidant) to the MPO/H2O2 system. (B) Effect of PON1 on peroxidase activity by TMB assay of either MPO or HRP. (C) Effect by TMB assay of PON1 (650 nM), HDL (650 nM), or both on MPO (65 nM) peroxidase activity. (D and E) Human neutrophils isolated from healthy donors (PMNWT) or MPO-deficient subjects (PMNMPO–) were incubated at 37°C in 50% serum for 1 hour in the absence or presence of phorbol 12-myristrate 13-acetate (PMA) and MPO or PON1, as indicated. Endogenous serum arachidonic acid (AA), linoleic acid (LA), 9-HODE, and 9-HETE were then quantified by stable isotope dilution LC/MS/MS as described in Methods. (F) PON1, MPO/H2O2, neither, or both were added to 50% serum, incubated at 37°C for 1 hour, and then AA, LA, or the indicated oxidation products were quantified as described in Methods. Data shown represent the mean ± SD of triplicate determinations. *P < 0.05; **P < 0.001. NA, no additions; oxFA, oxidized fatty acids.

Figure 2

Figure 2. MPO and PON1 inhibit each other’s activity in vivo.

(A) PON1 KO, WT, or PON1 Tg animals were injected i.p. with normal saline (Bl) or thioglycollate broth (T). Twenty-four hours later, peritoneal lavages were performed. Where indicated, mice were injected with zymosan (Z) 24 hours after thioglycollate, and peritoneal lavage was performed 4 hours later. Chlorotyrosine (ClTyr) content (normalized to the precursor amino acid tyrosine [Tyr]) in soluble proteins recovered from peritoneal lavage was determined by stable isotope dilution LC/MS/MS analysis. (B) MPO KO, WT, or MPO Tg animals were injected i.p. with normal saline or zymosan (Z). After 72 hours of subacute peritonitis, serum was isolated and paraoxonase activity was measured.

Figure 3

Figure 3. PON1 interaction sites on APOA1 within nHDL revealed by HDX MS.

(A) Plot indicating the percentage of deuterium incorporation of exchangeable protons within individual peptic peptides of APOA1 in nHDL in the absence (black bars) versus presence (white bars) of PON1. **P < 0.05. (B) Plot showing percentage change in deuterium incorporation of individual peptic peptides of APOA1 in nHDL in the absence or presence of PON1. Two regions of APOA1 demonstrate a significant reduction in deuterium incorporation in the presence or absence of PON1/P1 (L38GKQLNLKL46) and P2 (S201TLSEKAK208). Dotted line marks the 10% reduction in deuterium incorporation. (C) Illustration of PON1 interaction domains P1 and P2 on APOA1 within the double superhelix model of nHDL. The two APOA1 molecules are aligned in a head-to-tail antiparallel arrangement using a helix 5/helix 5 registry. N-termini are shown in dark red/blue, and C-termini are shown in light red/blue. PON1 interaction sites on nHDL (P1, blue; P2, light red).

Figure 4

Figure 4. Demonstration that APOA1 P1 and P2 regions are functionally important in the HDL-PON1-MPO ternary complex.

(A) APOA1 harboring mutations within P1 or P2 region (versus WT) were incubated with PON1 at 37°C for the indicated times, and then paraoxonase activity was determined. Results were normalized (100%) to PON1 activity measured with no addition (NA) at t = 0. (B) PON1 was incubated with reconstituted HDL (rHDL; 100:10:1, OPC/cholesterol/APOA1, mol/mol/mol) using the indicated APOA1 forms (WT versus P1 versus P2 mutants) or small unilamellar vesicles (SUV) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (16:0, 18:1 phosphatidylcholine [PC]) for the indicated times, and then paraoxonase activity was measured. Results were normalized (100%) to PON1 activity measured with no addition at t = 0. (C) TMB assay showed the effect of varying levels of the binary complex composed of PON1 and the indicated rHDL (made with WT versus P1 versus the P2 APOA1 mutant) on MPO activity. All results represent the mean ± SD from at least 3 independent experiments. In A and B, the differences between WT and mutants at the 3-hour point were significant (APOA1 WT versus APOA1 P1, P < 0.05; APOA1 WT versus APOA1 P2, P < 0.05). In C, the difference between WT and mutants at a molar ratio of rHDL/PON1 to MPO of 20 or greater were significant (rHDL WT versus rHDL P1, P < 0.05; rHDL WT versus rHDL P2, P < 0.05).

Figure 5

Figure 5. PON1 Tyr71 is a functionally important site for HDL interaction and a target for MPO-catalyzed oxidation in human atherosclerotic plaque.

(A) Schematic illustration of photocholestanyl-labeled PON1 tyrosyl residue. (B) Reconstituted HDL formed using POPC/photocholesterol/APOA1 (100:10:1) was incubated with PON1 and exposed to UV light as described in Methods. Proteomics studies revealed the photocholestanyl adduct of PON1 Tyr71 [Y(ch)] as a site on PON1 that directly interacts with cholesterol as described in Methods. m/z, mass-to-charge ratio. (C) PON1 crystal structure with superimposed location of Tyr71 (red) and hypothetical HDL binding surface based on hydrophobicity analysis. (D) Recombinant human WT PON1 versus the indicated PON1 Tyr71 site–specific mutants were generated, isolated, incubated with HDL, and then paraoxonase activity was determined as described in Methods. Results represent the mean ± SD from at least 3 independent experiments.

Figure 6

Figure 6. Methionine oxidation plays a role in PON1 activity and stability.

(A) Inverse correlation between systemic PON1 activity and protein content of ClTyr-isolated HDL from ACS (black ovals; n = 26) and healthy nondiabetic control subjects (white ovals; n = 26) from case-control cohort 1 (see Supplemental Table 1). P value shown is for the Spearman’s rank correlation between PON1 activity level and HDL ClTyr content among both control and ACS subjects. (B) Quantification of site-specific methionine oxidation (methionine sulfoxide) within PON1 recovered from isolated HDL from ACS (n = 10) and healthy nondiabetic control subjects (n = 10) enrolled in case-control cohort 2 (Supplemental Table 2). Results shown are expressed as the peak ratio of peptide harboring the indicated methionine sulfoxide residue or parent (methionine-harboring) peptide relative to the reference peptide, as described in Methods. (C) Isolated human HDL from a healthy donor was incubated with the MPO/H2O2/Cl– system (oxHDL), and then the effect of exposure to either methionine sulfoxide reductase (+MSR) or vehicle control (–MSR) on PON1 activity was determined as described in Methods. PON1 activity is expressed relative to paraoxonase activity measured in HDL prior to exposure to the MPO/H2O2/Cl– system. Data are the mean ± SD of triplicate determinations.

Figure 7

Figure 7. Structural model of a hypothetical ternary complex of MPO and PON1 bound to nHDL.

Illustration of an HDL-MPO-PON1 ternary complex composed of the double superhelix model of nHDL and the crystal structures of MPO and PON1 bound to one another with identified protein-protein interaction sites. The two predominantly α helical APOA1 chains in nHDL are aligned in a head-to-tail antiparallel arrangement. N-termini are shown in dark red/blue, and C-termini are shown in light red/blue. Phospholipids in the lipid core of nHDL are depicted in semitransparent green. PON1 binding sites on APOA1 shown are P1 (L38-L46, solid blue) and P2 (S201-K208, solid light red). Adjacent MPO binding site on APOA1 (residues A190-L203, filled light blue) is also depicted. Site-specific oxidative modifications found in PON1 recovered from isolated HDL from either atherosclerotic lesions or ACS plasma include Tyr71 (red) and Met55 and Met88 (both green). Met12 of PON1 is not shown because the N terminus (16 amino acids) of PON1 was not resolved in the PON1 crystal structure reported. Location of the openings to the two heme pockets on the MPO homodimer are predicted to be in close spatial proximity to the site-specific oxidative modifications reported in APOA1 recovered from human atherosclerotic plaque, Tyr166, and Tyr192 (solid black). Also shown are the “solar flare” regions of APOA1, presumed LCAT interaction sites (solid yellow).

Figure 8

Figure 8. Role of PON1 Tyr71 in HDL binding and catalysis.

(A) Illustration indicating how Tyr71 of PON1 may interact with the cholesteryl group (orange) of nHDL. PON1 crystal structure (PDB ID: 1V04) (purple) is shown docking with the lipid phase of nHDL (POPC molecules are shown as green lines, with the nitrogen atom of the choline group represented by blue spheres), and Tyr71 (red) protrudes into the lipid phase, interacting with cholesterol. (B) Illustration comparing the conformation of PON1 Tyr71 in the “closed” and “open” conformations of PON1 recently reported by Tawfik and colleagues (48). The presumed “lid” domain (K70-K81, cyan) in PON1 that seems to control access to the PON1 active site was resolved in the PON1 crystal structure with a lactone analog bound to the active site (PDB ID: 3SRG; closed conformation) only. If Tyr71 is nitrated, the bulky NO2 group interferes sterically with some of the residues (H184, F222, F292) (purple) in the active site walls. The halide Cl has an even larger van der Waals radius and would show similar steric interference.

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