Chloroplast NADPH-Dependent Thioredoxin Reductase from <em>Chlorella vulgaris</em> Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

<div><p>Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in <em>Chlorella vulgaris</em>. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from <em>C. vulgaris</em> and investigated the involvement of <em>Chlorella</em> NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced <em>Chlorella</em> 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature <em>C. vulgaris</em> NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature <em>C. vulgaris</em> 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, <em>tert</em>-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in <em>Chlorella</em>. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of <em>mCvNTRC</em>, and the elevated tolerances were more significant when both <em>mCvNTRC</em> and <em>mCvPrx</em> were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.</p> </div

Stress tolerances of yeast against freezing (top), heat (middle), and menadione-induced oxidative (bottom) stresses.

<p>Values are mean ± SD obtained from three or four independent experiments. Significance against pESC is indicated as *<i>p</i><0.01; **<i>p</i><0.001).</p

Superoxide generated in yeast cells during menadione treatment.

<p>Upper photos were taken with phase contrast microscopy, and lower photos were taken with fluorescence microscopy. Intensity of red fluorescence, which is derived from dihydroethidium, indicates superoxide levels in cells.</p

Peroxide reduction activity of His-mCvNTRC and His-mCvPrx proteins expressed in <i>E. coli</i>.

<p>(A) Five nucleotides in <i>mCvPrx</i> were modified for efficient expression of mCvPrx in <i>E. coli</i>. The original sequence is shown above, and the modified sequence is shown below. (B) His-mCvPrx was expressed in <i>E. coli</i> and purified by Ni²⁺-affinity chromatography. Lane 1, soluble protein extract of <i>E. coli</i>; lane 2, expressed protein purified by affinity chromatography. The arrowhead indicates a band for the His-mCvPrx. (C) <i>(left)</i> Representative kinetic data of peroxide reduction activities of mCvNTRC/mCvPrx against three peroxide substrates and decomposed peroxynitrite (PNdec) were shown. <i>(right)</i> Peroxide reduction activity of mCvNTRC/mCvPrx was shown as specific activity. Values are mean ± SD obtained from four independent experiments.</p

Autoimmune disease mouse model exhibits pulmonary arterial hypertension

<div><p>Background</p><p>Pulmonary arterial hypertension is often associated with connective tissue disease. Although there are some animal models of pulmonary hypertension, an autoimmune disease-based model has not yet been reported. MRL/lpr mice, which have hypergammaglobulinemia, produce various autoimmune antibodies, and develop vasculitis and nephritis spontaneously. However, little is known about pulmonary circulation in these mice. In the present study, we examined the pulmonary arterial pressure in MRL/lpr mice.</p><p>Methods and results</p><p>We used female MRL/lpr mice aged between 12 and 14 weeks. Fluorescent immunostaining showed that there was no deposition of immunoglobulin or C3 in the lung tissue of the MRL/lpr mice. Elevation of interferon-γ and interleukin-6 was recognized in the lung tissue of the MRL/lpr mice. Right ventricular systolic pressure, Fulton index and the ratio of right ventricular weight to body weight in the MRL/lpr mice were significantly higher than those in wild type mice with same background (C57BL/6). The medial smooth muscle area and the proportion of muscularized vessels in the lung tissue of the MRL/lpr mice were larger than those of the C57BL/6 mice. Western blot analysis demonstrated markedly elevated levels of prepro-endothelin-1 and survivin as well as decreased endothelial nitric oxide synthase phosphorylation in the lung tissue of the MRL/lpr mice. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling assay showed the resistance against apoptosis of pulmonary arterial smooth muscle cells in the MRL/lpr mice.</p><p>Conclusion</p><p>We showed that MRL/lpr mice were complicated with pulmonary hypertension. MRL/lpr mice appeared to be a useful model for studying the mechanism of pulmonary hypertension associated with connective tissue diseases.</p></div

Expression of mCvNTRC and mCvPrx in yeast.

<p>Structure of plasmids for expression of (A) <i>mCvNTRC</i>, (B) <i>mCvPrx</i>, and (C) both genes in yeast. (D) Expression of mCvNTRC and mCvPrx proteins was checked by Western blot analyses. The arrowheads indicate bands for the proteins of interest in each blot.</p

Sequence analyses of the full-length cDNA corresponding to <i>CvPrx</i>.

<p>(A) The phylogenetic tree was constructed with the full-length deduced amino acid sequences of <i>Prx</i> genes from <i>Arabidopsis</i> and <i>Chlorella</i> using the Clustal W program with bootstrapping, and was made visible with the TreeView program. Bar, 0.1 amino acid substitutions per site. (B) The amino acid sequence deduced from <i>CvPrx</i> was compared with the sequences deduced from <i>Arabidopsis Prx</i> cDNAs. The arrows indicate two conserved cysteine residues found in 2-Cys Prx. The asterisks indicate the N-terminal sequence of predicted <i>Chlorella</i> Prx described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045988#pone.0045988-Machida1" target="_blank">[13]</a>.</p

The levels of survivin expression and apoptosis of pulmonary arterial smooth muscle cells in the lung tissue of MRL/lpr mice.

<p>Survivin expression in the lung tissues was determined by western blotting (A). Representative immunoblots (upper panels) and graph (bottom panel) are shown. Bars are mean ± S.D. of quantitative densitometric analyses; n = 5 for each experimental group; *P<0.05 vs. C57BL/6 mice. Representative microphotographs of the pulmonary arteries in C57BL/6 mice and MRL/lpr mice stained by TUNEL staining (B). TUNEL-positive cells were indicated by red arrows. The graph showed the percentage of TUNEL-positive nuclei in medial smooth muscle layer (open bar; C57BL/6 mice, solid bar; MRL/lpr mice). Results are expressed as mean ± S.D. of 5 animals. *P<0.05 vs. C57BL/6 mice.</p

Cytokines in the lung tissue of MRL/lpr mice.

<p>The levels of cytokines in the lung tissue were assessed as described in the Methods. (open bar; C57BL/6 mice, solid bar; MRL/lpr mice). Results are expressed as mean ± S.D. of 9 to 11 animals. *P<0.05 vs. C57BL/6 mice.</p

Decreased eNOS activation and increased prepro-ET-1 expression in the lung tissue of MRL/lpr mice.

<p>eNOS expression and phosphorylation (A), and prepro-ET-1 expression (B) were determined by western blotting. Representative immunoblots (upper panels) and graphs (bottom panels) are shown. Bars are mean ± S.D. of quantitative densitometric analyses; n = 5 for each experimental group; *P<0.05 vs. C57BL/6 mice.</p