Sheldon I. Feinstein

Phospholipid Hydroperoxides Are Substrates for Non-selenium Glutathione Peroxidase

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity withsn-2-linolenoyl- orsn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM−1min−1) were k 1 = 1–3 × 105 and k 2 = 4–11 × 104. In the presence of 0.36 mm GSH, apparentK m was 120–130 μm and apparentV max was 1.5–1.6 μmol/min/mg of protein. Assays with H2O2 and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7–8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K i 4 μm. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.

1-Cys peroxiredoxin overexpression protects cells against phospholipid peroxidation-mediated membrane damage

1-Cys peroxiredoxin (1-cysPrx) is a novel antioxidant enzyme able to reduce phospholipid hydroperoxides in vitro by using glutathione as a reductant. This enzyme is widely expressed and is enriched in lungs. A fusion protein of green fluorescent protein with 1-cysPrx was stably expressed in a lung-derived cell line (NCI-H441) lacking endogenous enzyme. Overexpressing cells (C17 or C48) degraded H2O2 and t-butylhydroperoxide more rapidly and showed decreased sensitivity to oxidant stress as measured by 51Cr release. On exposure to •OH generated by Cu2+-ascorbate (Asc), overexpressing cells compared with H441 showed less increase in thiobarbituric acid-reactive substance and phosphatidylcholine hydroperoxide content. This effect was reversed by depletion of cellular glutathione. Diphenyl-1-pyrenoylphosphonium fluorescence, used as a real-time probe of membrane phospholipid peroxidation, increased immediately on exposure to Cu2+-Asc and was abolished by preincubation of cells with Trolox (a soluble vitamin E) or Tempol (a radical scavenger). The rate of diphenyl-1-pyrenoylphosphonium fluorescence increase with Cu2+-Asc exposure was markedly attenuated in C17 and C48 cells as compared with H441. Annexin V-Cy3 was used to detect phosphatidylserine translocation from the inner to outer leaflet of the plasma membrane. Cu2+-Asc treatment induced phosphatidylserine translocation within 2 h in H441 cells but none was observed in C48 cells up to 24 h. These results indicate that 1-cysPrx can scavenge peroxides but in addition can reduce peroxidized membrane phospholipids. Thus, the enzyme can protect cells against oxidant-induced plasma membrane damage, thereby playing an important role in cellular defense against oxidant stress.

Identification of a human cDNA clone for lysosomal type Ca2+-independent phospholipase A2 and properties of the expressed protein.

A Ca2+-independent phospholipase A2 (PLA2) maximally active at pH 4 and specifically inhibited by the transition-state analogue 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) was isolated from rat lungs. The sequence for three internal peptides (35 amino acids) was used to identify a 1653-base pair cDNA clone (HA0683) from a human myeloblast cell line. The deduced protein sequence of 224 amino acids contained a putative motif (GXSXG) for the catalytic site of a serine hydrolase, but showed no significant homology to known phospholipases. Translation of mRNA produced from this clone in both a wheat germ system and Xenopus oocytes showed expression of PLA2 activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA2 with dipalmitoylphosphatidylcholine as substrate were Km = 0.25 mM and Vmax = 1.89 nmol/h. Activity with alkyl ether phosphatidylcholine as substrate was decreased significantly compared with diacylphosphatidylcholine. Significant lysophospholipase, phospholipase A1, or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase activity was not observed. Enzyme activity was insensitive to p-bromophenacyl bromide, bromoenol lactone, trifluoromethylarachidonoyl ketone, mercaptoethanol, and ATP, but was inhibited by MJ33 and diethyl p-nitrophenyl phosphate, a serine protease inhibitor. SDS-polyacrylamide gel electrophoresis with autoradiography of the translated [35S]methionine-labeled protein confirmed a molecular mass of 25.8 kDa, in good agreement with the enzyme isolated from rat lung. By Northern blot analysis, mRNA corresponding to this clone was present in both rat lung and isolated rat granular pneumocytes. These results represent the first molecular cloning of a cDNA for the lysosomal type Ca2+-independent phospholipase A2 group of enzymes.

An antisense oligonucleotide to 1-cys peroxiredoxin causes lipid peroxidation and apoptosis in lung epithelial cells.

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, reduces phospholipid hydroperoxides as well as organic peroxides and H2O2. To determine the physiological function(s) of 1-cysPrx, we have used an antisense strategy to suppress endogenous 1-cysPrx in L2 cells, a rat lung epithelial cell line. A 25-base antisense morpholino oligonucleotide was designed to bind a complementary sequence overlapping the translational start site (−18 to +7) in the rat 1-cysPrx mRNA, blocking protein synthesis. Treatment with an antisense oligonucleotide for 48 h resulted in approximately 60% suppression of the 1-cysPrx protein content as measured by immunoblot analysis and an approximately 44% decrease of glutathione peroxidase activity as compared with random oligonucleotide treated and control (vehicle only) cells. Accumulation of phosphatidylcholine hydroperoxide in plasma membranes was demonstrated by high pressure liquid chromatography assay for conjugated dienes (260 pmol/106cells for antisense versus 70 pmol/106 cells for random oligonucleotide and control cells) and by fluorescence of diphenyl-1-pyrenylphosphine, a probe for lipid peroxidation. The percentage of cells showing positive staining for annexin V and propidium iodide after antisense treatment was 40% at 28 h and 80% at 48 h. TdT-mediated dUTP nick end labeling assay at 48 h indicated DNA fragmentation in antisense-treated cells that was blocked by prior infection with adenovirus encoding 1-cysPrx or by pretreatment with a vitamin E analogue. The results indicate that 1-cysPrx can function in the intact cell as an antioxidant enzyme to reduce the accumulation of phospholipid hydroperoxides and prevent apoptotic cell death.

Oxidant stress stimulates expression of the human peroxiredoxin 6 gene by a transcriptional mechanism involving an antioxidant response element

Peroxiredoxin 6 (Prdx6) is a unique antioxidant enzyme that can reduce phospholipid and other hydroperoxides. A549 cells, a human lung-derived cell line, express both Prdx6 and Nrf2, a transcription factor that binds to antioxidant-response elements (AREs) and promotes expression of antioxidant genes. Treatment of A549 cells with 500 microM H(2)O(2) increased Prdx6 mRNA levels 2.5-fold, whereas treatment with 400 microM H(2)O(2) or 200 microM tert-butylhydroquinone (t-BHQ) triggered a corresponding 2.5-fold increase in reporter gene activity in A549 cells transfected with the pSEAP2:Basic vector (BD Bioscience), containing 1524 nucleotides of the human Prdx6 promoter region. Deletion of a consensus ARE sequence present between positions 357 and 349 before the start of transcription led to a striking decrease in both basal and H(2)O(2)- or t-BHQ-induced activation in A549 cells and H(2)O(2)-induced activation in primary rat alveolar type II cells. Cotransfection with Nrf2 stimulated the Prdx6 promoter in an ARE-dependent manner, whereas it was negatively regulated by Nrf3. siRNA targeting Nrf2 down-regulated reporter gene expression, whereas siRNA targeting the Nrf2 repressor, Keap1, up-regulated it. Binding of Nrf2 to the ARE sequence in chromatin was confirmed by PCR after chromatin immunoprecipitation. These data demonstrate that the ARE within the Prdx6 promoter is a key regulator of basal transcription of the Prdx6 gene and of its inducibility under conditions of oxidative stress.

Peroxiredoxin 6 as an antioxidant enzyme: protection of lung alveolar epithelial type II cells from H2O2-induced oxidative stress.

We evaluated the antioxidant role of peroxiredoxin 6 (Prdx6) in primary lung alveolar epithelial type II cells (AEC II) that were isolated from wild type (WT), Prdx6−/−, or Prdx6 transgenic (Tg) overexpressing mice and exposed to H2O2 at 50–500 µM for 1–24 h. Expression of Prdx6 in Tg AEC II was sevenfold greater than WT. Prdx6 null AEC II exposed to H2O2 showed concentration‐dependent cytotoxicity indicated by decreased “live/dead” cell ratio, increased propidium iodide (PI) staining, increased annexin V binding, increased DNA fragmentation by TUNEL assay, and increased lipid peroxidation by diphenylpyrenylphosphine (DPPP) fluorescence. Compared to Prdx6 null cells, oxidant‐mediated damage was significantly less in WT AEC II and was least in Prdx6 Tg cells. Thus, Prdx6 functions as an antioxidant enzyme in mouse AEC II. Prdx6 has been shown previously to reduce phospholipid hydroperoxides and we postulate that this activity is a major mechanism for the effectiveness of Prdx6 as an antioxidant enzyme. J. Cell. Biochem. 104: 1274–1285, 2008.

Peroxiredoxin 6 phosphorylation and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages.

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA2) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA2 activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47phox, cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA2 activity, blocked agonist-induced PLA2 activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA2s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA2 active site (S32A, H26A, and D140A), “rescued” Ang II-induced PLA2 activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA2 activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA2 activity facilitates assembly of the NOX2 complex and activation of the oxidase.

Cloning and expression of rat lung acidic Ca2+-independent PLA2 and its organ distribution

A clone for a rat acidic Ca2+-independent phospholipase A2(aiPLA2) was isolated from a cDNA library prepared from rat granular pneumocytes with a probe based on the human aiPLA2 sequence (T. S. Kim, C. S. Sundaresh, S. I. Feinstein, C. Dodia, W. R. Skach, M. K. Jain, T. Nagase, N. Seki, K. Ishikawa, N. Nomura, and A. B. Fisher. J. Biol. Chem. 272: 2542-2550, 1997). In addition, a consensus sequence for mouse aiPLA2 was constructed from several mouse cDNA clones in the GenBank and dbEST databases. Each sequence codes for a 224-amino acid protein with 88% identity of the amino acids among the three species and conservation of a putative lipase motif (GDSWG). Translation of mRNA produced from the rat clone in a wheat germ system resulted in expression of PLA2 activity with properties similar to those of the human enzyme, i.e., acidic pH optimum and Ca2+ independence. The localization of aiPLA2 in rat tissues was studied with the human cDNA probe, polyclonal and monoclonal antibodies, and aiPLA2activity. aiPLA2 is present in the lung as evidenced by high levels of mRNA and protein expression and by enzymatic activity that is inhibited by anti-PLA2 antibody and by the transition state analog 1-hexadecyl-3-trifluoroethylglycero- sn-2-phosphomethanol (MJ33). Immunocytochemistry showed the presence of aiPLA2 in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium. In the brain, heart, liver, kidney, spleen, and intestine, aiPLA2 mRNA content was <50% of that in the lung, immunoreactive protein was not detectable, and enzymatic activity was not inhibited by MJ33 or aiPLA2 antibody. These results show marked enrichment of aiPLA2in the lung compared with the other organs and suggest translational control of aiPLA2 expression.

Interaction of Surfactant Protein A with Peroxiredoxin 6 Regulates Phospholipase A2 Activity

Peroxiredoxin 6 (Prdx6) is a “moonlighting” protein with both GSH peroxidase and phospholipase A2 (PLA2) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA2 activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA2 activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA2 activity) showed Km0.35 mm and Vmax 138 nmol/min/mg of protein. SP-A inhibited PLA2 activity non-competitively with Ki 10 μg/ml and was Ca2+ -independent. Activity at pH 7.4 was ∼50% less, and inhibition by SP-A was partially dependent on Ca2+. Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni2 -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca2+ dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA2 activity of Prdx6 by SP-A.

1-Cys peroxiredoxin knock-out mice express mRNA but not protein for a highly related intronless gene

1‐Cys peroxiredoxin (1‐cysPrx), a member of the peroxiredoxin family with a single conserved cysteine, is a unique antioxidant enzyme. We have generated mice in which the 1‐cysPrx gene has been inactivated; they are viable and fertile. Mice have a highly related intronless gene (1‐cysPrx‐P1, GenBank accession number AF085220) with the same length of open reading frame (224 aa) as 1‐cysPrx but located on a different chromosome. Since the product of this gene possibly could mimic 1‐cysPrx function, we compared expression of 1‐cysPrx and 1‐cysPrx‐P1 in mouse tissues by real‐time polymerase chain reaction and Western blot. 1‐cysPrx mRNA and protein were expressed in all mouse tissues that were examined with the highest expression level in lung. 1‐cysPrx‐P1 mRNA was expressed only in testis. In the 1‐cysPrx ‘knock‐out’ mouse, 1‐cysPrx‐P1 mRNA expression level was similar to the wild type but protein expression was not detected. Thus, mouse 1‐cysPrx‐P1 is an mRNA‐expressed pseudogene that does not result in detectable protein in vivo.