Inhibition of human liver HCTL activity.

<p>* HCTLase activity was measured at 10 mM concentration of substrate.</p><p>† Iodoacetamide inhibited rBPHL to the same extent.</p><p>Inhibition of human liver HCTL activity.</p

SDS-PAGE analysis of pooled column fractions from (A) human liver and (B) recombinant HCTLase purification.

<p>(A) Lane 1, molecular weight markers (kDa); lane 2, liver extract; lane 3, diethylaminoethyl (DEAE) pool; lane 4, CM-1 pool; lane 5, ceramic hydroxyapatite (HA) pool; lane 6, Superdex 200 (#1) pool; lane 7, CM-2 pool; lane 8, Superdex 200 (#2) pool. Gel was stained with silver stain kit (Pierce Chemical). (B) Lane 1, molecular weight markers (kDa); lane 2, <i>E. coli</i> extract; lane 3, DEAE pool; lane 4, HA pool; lane 5, Superdex 200 pool. Gel was stained with Imperial Protein Stain (Pierce Chemical).</p

IEF activity and Coomassie stain of liver BPHL and rBPHL.

<p>(A) HCTLase activity stain of rBHPL (lane 2) and human liver BPHL (lane 3). Hemoglobin shows a faint band as well (lane 1). (B) Coomassie Blue stain of hemoglobin, rBPHL and human liver BPHL (lanes 1–3, respectively).</p

Protein absorbance (A₂₈₀) and HCTLase activity of column fractions from the chromatographic purification of human liver HCTLase (A–E) and recombinant HCTLase (F, G).

<p>A₂₈₀ nm, solid lines; HCTL activity, solid lines with open circles. (A) Carboxymethyl (CM) column 1, (B) ceramic hydroxyapatite (HA) column, (C) Superdex 200 column 1, (D) CM column 2, (E) Superdex 200 column 2; (F) HA column, (G) Superdex 200 column. Ticks represent fraction changes.</p

LC-MS chromatograms showing rBPHL-mediated HCTL cleavage at (A) t = 5 min and (B) t = 0 of reaction time, and valacyclovir ester cleavage at (C) t = 5 min and (D) t = 0.

<p>Chromatograms A and B represent the sum of two MRM channels monitoring the following transitions: <i>m/z</i> 117 > 89 (for HCTL, Rt  =  10.7 min) and <i>m/z</i> 136 > 89 (for Hcy, Rt  =  5.4 min). The inset shows the daughter ion spectrum (for <i>m/z</i> 136) of the product peak at Rt  =  5.4 min, and is consistent with the spectrum obtained from commercial Hcy. Chromatograms C and D also represent the sum of two MRM channels monitoring the following transitions: <i>m/z</i> 326 > 152 (for VC, Rt  =  18.3 min) and <i>m/z</i> 226 > 152 (for acyclovir, Rt  =  6.1 min). The inset shows the daughter ion spectrum (for <i>m/z</i> 226) of the product peak at Rt  =  6.1 min, and is consistent with the spectrum obtained from commercial acyclovir. The small product peak at t  =  0 in chromatogram D reflects the extremely rapid metabolism of VC by rBPHL, which generated a detectable amount of acyclovir in even the very short period of time (∼10 sec) between initiation and quenching of the enzymatic reaction.</p

BPHL substrates.

<p>* <i>V</i>_max from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110054#pone-0110054-g004" target="_blank">Figure 4B</a>.</p><p>† from this study.</p><p>‡ from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110054#pone.0110054-Puente2" target="_blank">[27]</a>.</p><p>BPHL substrates.</p

Purification of recombinant human BPHL (HCTLase).

<p>DEAE indicates diethylaminoethyl; and HA, hydroxyapatite.</p><p>* Units of HCTLase activity are µmol NTB produced per minute measured at ≈K_m concentration of substrate.</p><p>Purification of recombinant human BPHL (HCTLase).</p

Paraoxonase‑3 Is Depleted from the High-Density Lipoproteins of Autoimmune Disease Patients with Subclinical Atherosclerosis

Patients with autoimmune diseases have a significantly increased risk of developing cardiovascular disease. In disease, high-density lipoprotein (HDL) particles lose their anti-inflammatory and antioxidant properties and become dysfunctional. The purpose of this study was to test the hypothesis that alterations in the HDL proteomic profile are associated with subclinical atherosclerosis and HDL dysfunction in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and type 1 diabetes. Targeted proteomics was used to quantify the relative abundance of 18 proteins in HDL from SLE patients with and without atherosclerotic plaque detectable by carotid ultrasound. Changes in the proteomic profile were compared against the in vitro ability of HDL to protect against lipid oxidation. The same proteins were quantified in HDL from patients with type 1 diabetes with or without coronary artery calcification as determined by computed tomography. In each population, paraoxonase-3 (PON3), a potent antioxidant protein, was depleted from the HDL of patients with subclinical atherosclerosis. PON3 expression in HDL was positively correlated with HDL antioxidant function. These results suggest that PON3 may be an important protein in preventing atherosclerosis and highlight the importance of antioxidant proteins in the prevention of atherosclerosis in vivo

Substrate dependence and pH optimum of HCTL hydrolysis by BPHL.

<p>Substrate dependence of HCTL hydrolysis by (A) purified human liver HCTLase (BPHL) and (B) rBPHL. Closed symbols, velocity (v) vs substrate concentration [S]; open symbols, [S]/v vs [S]. K_m for HCTL hydrolysis by purified liver BPHL =  3.92 mM and for rBPHL K_m  =  3.18 mM. (C) HCTLase activity of rBPHL (% maximal activity) as a function of pH. Open squares, □―□ 50 mM citrate/Na₂HPO₄; open circles ○―○, 50 mM Tris-HCl; open triangle, ▵―▵ 50 mM Na₂PO₄.</p

Paraoxonase‑1 Deficiency Is Associated with Severe Liver Steatosis in Mice Fed a High-fat High-cholesterol Diet: A Metabolomic Approach

Oxidative stress is a determinant of liver steatosis and the progression to more severe forms of disease. The present study investigated the effect of paraoxonase-1 (PON1) deficiency on histological alterations and hepatic metabolism in mice fed a high-fat high-cholesterol diet. We performed nontargeted metabolomics on liver tissues from 8 male PON1-deficient mice and 8 wild-type animals fed a high-fat, high-cholesterol diet for 22 weeks. We also measured 8-oxo-20-deoxyguanosine, reduced and oxidized glutathione, malondialdehyde, 8-isoprostanes and protein carbonyl concentrations. Results indicated lipid droplets in 14.5% of the hepatocytes of wild-type mice and in 83.3% of the PON1-deficient animals (<i>P</i> < 0.001). The metabolomic assay included 322 biochemical compounds, 169 of which were significantly decreased and 16 increased in PON1-deficient mice. There were significant increases in lipid peroxide concentrations and oxidative stress markers. We also found decreased glycolysis and the Krebs cycle. The urea cycle was decreased, and the pyrimidine cycle had a significant increase in orotate. The pathways of triglyceride and phospholipid synthesis were significantly increased. We conclude that PON1 deficiency is associated with oxidative stress and metabolic alterations leading to steatosis in the livers of mice receiving a high-fat high-cholesterol diet