Hexameric oligomerization of mitochondrial peroxiredoxin PrxIIF and formation of an ultrahigh affinity complex with its electron donor thioredoxin Trx-o

Mitochondria from plants, yeast, and animals each contain at least one peroxiredoxin (Prx) that is involved in peroxide detoxification and redox signalling. The supramolecular dynamics of atypical type II Prx targeted to the mitochondrion was addressed in pea. Microcalorimetric (ITC) titrations identified an extremely high-affinity binding between the mitochondrial PsPrxIIF and Trx-o with a K(D) of 126+/-14 pM. Binding was driven by a favourable enthalpy change (DeltaH= -60.6 kcal mol(-1)) which was counterbalanced by unfavourable entropy changes (TDeltaS= -47.1 kcal mol(-1)). This is consistent with the occurrence of large conformational changes during binding which was abolished upon site-directed mutaganesis of the catalytic C59S and C84S. The redox-dependent interaction was confirmed by gel filtration of mitochondrial extracts and co-immunoprecipitation from extracts. The heterocomplex of PsPrxIIF and Trx-o reduced peroxide substrates more efficiently than free PsPrxIIF suggesting that Trx-o serves as an efficient and specific electron donor to PsPrxIIF in vivo. Other Trx-s tested by ITC analysis failed to interact with PsPrxIIF indicating a specific recognition of PsPrxIIF by Trx-o. PsPrxIIF exists primarily as a dimer or a hexamer depending on the redox state. In addition to the well-characterized oligomerization of classical 2-Cys Prx the results also show that atypical Prx undergo large structural reorganization with implications for protein-protein interaction and function.     

Glutathionylation of peroxiredoxin I induces decamer to dimers dissociation with concomitant loss of chaperone activity

Reversible protein glutathionylation, a redox-sensitive regulatory mechanism, plays a key role in cellular regulation and cell signaling. Peroxiredoxins (Prxs), a family of peroxidases that is involved in removing H(2)O(2) and organic hydroperoxides, are known to undergo a functional change from peroxidase to molecular chaperone upon overoxidation of its catalytic cysteine. The functional change is caused by a structural change from low molecular weight oligomers to high molecular weight complexes that possess molecular chaperone activity. We reported earlier that Prx I can be glutathionylated at three of its cysteine residues, Cys52, -83, and -173 [Park et al. (2009) J. Biol. Chem., 284, 23364]. In this study, using analytical ultracentrifugation analysis, we reveal that glutathionylation of Prx I, WT, or its C52S/C173S double mutant shifted its oligomeric status from decamers to a population consisting mainly of dimers. Cys83 is localized at the putative dimer-dimer interface, implying that the redox status of Cys83 may play an important role in stabilizing the oligomeric state of Prx I. Studies with the Prx I (C83S) mutant show that while Cys83 is not essential for the formation of high molecular weight complexes, it affects the dimer-decamer equilibrium. Glutathionylation of the C83S mutant leads to accumulation of dimers and monomers. In addition, glutathionylation of Prx I, both the WT and C52S/C173S mutants, greatly reduces their molecular chaperone activity in protecting citrate synthase from thermally induced aggregation. Together, these results reveal that glutathionylation of Prx I promotes changes in its quaternary structure from decamers to smaller oligomers and concomitantly inactivates its molecular chaperone function.     

Peroxiredoxin 3 has a crucial role in the contractile function of skeletal muscle by regulating mitochondrial homeostasis

Antioxidant systems against reactive oxygen species (ROS) are important factors in regulating homeostasis in various cells, tissues, and organs. Although ROS are known to cause to muscular disorders, the effects of mitochondrial ROS in muscle physiology have not been fully understood. Here, we investigated the effects of ROS on muscle mass and function using mice deficient in peroxiredoxin 3 (Prx3), which is a mitochondrial antioxidant protein. Ablation of Prx3 deregulated the mitochondrial network and membrane potential of myotubes, in which ROS levels were increased. We showed that the DNA content of mitochondria and ATP production were also reduced in Prx3-KO muscle. Of note, the mitofusin 1 and 2 protein levels decreased in Prx3-KO muscle, a biochemical evidence of impaired mitochondrial fusion. Contractile dysfunction was examined by measuring isometric forces of isolated extensor digitorum longus (EDL) and soleus muscles. Maximum absolute forces in both the EDL and the soleus muscles were not significantly affected in Prx3-KO mice. However, fatigue trials revealed that the decrease in relative force was greater and more rapid in soleus from Prx3-KO compared to wild-type mice. Taken together, these results suggest that Prx3 plays a crucial role in mitochondrial homeostasis and thereby controls the contractile functions of skeletal muscle.     

Reversible oxidation of the active site cysteine of peroxiredoxins to cysteine sulfinic acid. Immunoblot detection with antibodies specific for the hyperoxidized cysteine-containing sequence

We previously suggested that oxidation of the active site cysteine of peroxiredoxin (Prx) I or Prx II to cysteine sulfinic acid in H2O2-treated cells is reversible (Woo, H. A., Chae, H. Z., Hwang, S. C., Yang, K.-S., Kang, S. W., Kim, K., and Rhee, S. G. (2003) Science 300, 653-656). In contrast, it was recently proposed that sulfinylation of Prx II, but not that of Prx I or Prx III, is reversible (Chevallet, M., Wagner, E., Luche, S., van Dorssealaer, A., Leize-Wagner, E., and Rabilloud, T. (2003) J. Biol. Chem. 278, 37146-37153). The detection of sulfinylated proteins in both of these previous studies relied on complex proteomics analysis. We now describe a simple immunoblot assay for the detection of sulfinylated Prx enzymes that is based on antibodies produced in response to a sulfonylated peptide modeled on the conserved active site sequence. These antibodies recognized both sulfinic and sulfonic forms of Prx equally well and allowed the detection of sulfinylated Prx enzymes in H2O2-treated cells with high sensitivity and specificity. With the use of these antibodies, we demonstrated that not only the cytosolic enzymes Prx I and Prx II but also the mitochondrial enzyme Prx III undergo reversible sulfinylation. The generation of antibodies specific for sulfonylated peptides should provide insight into protein function similar to that achieved with antibodies to peptides containing phosphoserine or phosphothreonine.     

Evidence for the presence of ferritin in plant mitochondria.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.     

Immunological evidence for the presence of peroxiredoxin in pea leaf peroxisomes and response to oxidative stress conditions

Peroxiredoxins (Prxs) constitute a group of thiol-specific antioxidant enzymes which are present in bacteria, yeasts, and in plant and animal cells. Although Prxs are mainly localized in the cytosol, they are also present in mitochondria, chloroplasts, and nuclei, but there is no evidence of the existence of Prxs in plant peroxisomes. Using soluble fractions (matrices) of peroxisomes purified from leaves of pea (Pisum sativum L.) plants, the immunological analysis with affinity-purified IgG against yeast Prx1 revealed the presence of an immunoreactive band of about 50 kDa. The apparent molecular mass of the peroxisomal Prx was not sensitive to oxidizing and reducing conditions what could be a mechanism of protection against the oxidative environment existing in peroxisomes. Postembedment, EM immunocytochemical analysis with affinity-purified IgG against yeast Prx1 antibodies, confirmed that this protein was present in the peroxisomal matrix, mitochondria, and chloroplasts. In pea plants grown under oxidative stress conditions, the protein level of peroxisomal Prx was differentially modulated, being slightly induced by growth of plants with 50 µM CdCl₂, but being significantly reduced by treatment with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The presence in the matrix of peroxisomes of a protein immunorelated to Prx of about 50 kDa, which is in the range of molecular mass of the dimeric form of other Prxs, opens new questions on the molecular properties of Prxs, but also on their function in the metabolism of reactive oxygen and nitrogen species (ROS/RNS) in these plant cell organelles, where they could be involved in the regulation of hydrogen peroxide and/or peroxynitrite.     

Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria

Mitochondrial ribosomal protein 20 (Mrp20) is a component of the yeast mitochondrial large (54S) ribosomal subunit and is homologous to the bacterial L23 protein, located at the ribosomal tunnel exit site. The carboxy-terminal mitochondrial-specific domain of Mrp20 was found to have a crucial role in the assembly of the ribosomes. A new, membrane-bound, ribosomal-assembly subcomplex composed of known tunnel-exit-site proteins, an uncharacterized ribosomal protein, MrpL25, and the mitochondrial peroxiredoxin (Prx), Prx1, accumulates in an mrp20ΔC yeast mutant. Finally, data supporting the idea that the inner mitochondrial membrane acts as a platform for the ribosome assembly process are discussed.     

Cloning and characterization of a 2-Cys peroxiredoxin from Pisum sativum

A cDNA sequence coding for a pea (Pisum sativum L.) 2-Cys peroxiredoxin (2-Cys Prx) has been cloned. The deduced amino acid sequence showed a high sequence homology to the 2-Cys Prx enzymes of Phaseolus vulgaris (86%), Arabidopsis thaliana (75%), and Spinacia oleracea (75%), and contained a chloroplast target sequence at its N-terminus. The mature enzyme, without the transit peptide, has a molecular mass of 22 kDa as well as two cysteine residues (Cys-53 and Cys-175) which are well conserved among proteins of this group. The protein was expressed in a heterologous system using the expression vector pET3d, and was purified to homogeneity by three sequential chromatographic steps. The enzyme exhibits peroxidase activity on hydrogen peroxide (H(2)O(2)) and t-butyl hydroperoxide (TBHP) with DTT as reducing agent. Although both pea Trxs f and m reduce oxidized 2-Cys Prx, Trx m is more efficient. The precise conditions for oligomerization of 2-Cys Prx through extensive gel filtration studies are also reported. The transition dimer-decamer produced in vitro between pH 7.5 and 8.0 and the influence of DTT suggest that a great change in the enzyme quaternary structure of 2-Cys Prx may take place in the chloroplast during the dark-light transition. In addition, the cyclophilin-dependent reduction of chloroplast 2-Cys Prx is shown.     

The dual-targeted plant sulfiredoxin retroreduces the sulfinic form of atypical mitochondrial peroxiredoxin

Sulfiredoxin (Srx) couples the energy of ATP hydrolysis to the energetically unfavorable process of reducing the inactive sulfinic form of 2-cysteine peroxiredoxins (Prxs) to regenerate its active form. In plants, Srx as well as typical 2-cysteine Prx have been considered as enzymes with exclusive chloroplast localization. This work explores the subcellular localization of Srx in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana). Immunocytochemistry, analysis of protein extracts from isolated intact organelles, and cell-free posttranslational import assays demonstrated that plant Srx also localizes to the mitochondrion in addition to plastids. The dual localization was in line with the prediction of a signal peptide for dual targeting. Activity tests and microcalorimetric data proved the interaction between Srx and its mitochondrial targets Prx IIF and thioredoxin. Srx catalyzed the retroreduction of the inactive sulfinic form of atypical Prx IIF using thioredoxin as reducing agent. Arabidopsis Srx also reduced overoxidized human Prx V. These results suggest that plant Srx could play a crucial role in the regulation of Prx IIF activity by controlling the regeneration of its overoxidized form in mitochondria, which are sites of efficient reactive oxygen species production in plants.     

Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation.     

The function of peroxiredoxins in plant organelle redox metabolism

In 1996, cDNA sequences referred to as plant peroxiredoxins (Prx), i.e. a 1-Cys Prx and a 2-Cys Prx, were reported from barley. Ten years of research have advanced our understanding of plant Prx as thiol-based peroxide reductases with a broad substrate specificity, ranging from hydrogen peroxide to alkyl hydroperoxides and peroxinitrite. Prx have several features in common. (i) They are abundant proteins that are routinely detected in proteomics approaches. (ii) They interact with proteins such as glutaredoxins, thioredoxins, and cyclophilins as reductants, but also non-dithiol-disulphide exchange proteins. By work with transgenic plants, their activity was shown to (iii) affect metabolic integrity, (iv) protect DNA from damage in vitro and as shown here in vivo, and (v) modulate intracellular signalling related to reactive oxygen species and reactive nitrogen species. (vi) In all organisms Prx are encoded by small gene families that are of particular complexity in higher plants. A comparison of the Prx gene families in rice and Arabidopsis thaliana supports previous suggestions on Prx function in specific subcellular and metabolic context. (vii) Prx gene expression and activity are subjected to complex regulation realized by an integration of various signalling pathways. 2-Cys Prx expression depends on redox signals, abscisic acid, and protein kinase cascades. Besides these general properties, the chloroplast Prx have acquired specific roles in the context of photosynthesis. The thioredoxin-dependent peroxidase activity can be measured in crude plant extracts and contributes significantly to the overall H(2)O(2) detoxification capacity. Thus organellar Prx proteins enable an alternative water-water cycle for detoxification of photochemically produced H(2)O(2), which acts independently from the ascorbate-dependent Asada-Halliwell-Foyer cycle. 2-Cys Prx and Prx Q associate with thylakoid membrane components. The mitochondrial PrxII F is essential for root growth under stress. Following a more general introduction, the paper summarizes present knowledge on plant organellar Prx, addressing Prx in signalling, and also suggests some lines for future research.     

Alcohol induced hepatic fibrosis: role of acetaldehyde

Alcohol abuse is one of the major causes of liver fibrosis worldwide. Although the pathogenesis of liver fibrosis is a very complex phenomenon involving different molecular and biological mechanisms, several lines of evidence established that the first ethanol metabolite, acetaldehyde, plays a key role in the onset and maintenance of the fibrogenetic process. This review briefly summarizes the molecular mechanisms underlying acetaldehyde pro-fibrogenic effects. Liver fibrosis represents a general wound-healing response to a variety of insults. Although mortality due to alcohol abuse has been constantly decreasing in the past 20 years in Southern Europe and North America, in several Eastern-European countries and Great Britain Alcoholic Liver Disease (ALD) shows a sharply increasing trend [Bosetti, C., Levi, F., Lucchini, F., Zatonski, W.A., Negri, E., La, V.C., 2007. Worldwide mortality from cirrhosis: an update to 2002. J. Hepatol. 46, 827-839]. ALD has a complex pathogenesis, in which acetaldehyde (AcCHO), the major ethanol metabolite, plays a central role. Ethanol is mainly metabolized in the liver by two oxidative pathways. In the first one ethanol is oxidized to acetaldehyde by the cytoplasmic alcohol dehydrogenase enzyme (ADH), acetaldehyde is then oxidized to acetic acid by the mitochondrial acetaldehyde dehydrogenase (ALDH). The second pathway is inducible and involves the microsomal ethanol-oxidizing system (MEOS), in which the oxidation of ethanol to acetaldehyde and acetic acid also leads to generation of reactive oxygen species (ROS). Chronic ethanol consumption significantly inhibits mitochondrial ALDH activity while the rate of ethanol oxidation to acetaldehyde is even enhanced, resulting in a striking increase of tissue and plasma acetaldehyde levels [Lieber, C.S., 1997. Ethanol metabolism, cirrhosis and alcoholism. Clin. Chim. Acta 257, 59-84]. This review will focus on the molecular mechanisms by which acetaldehyde promote liver fibrosis.     

Mitochondrial peroxiredoxin 3 is rapidly oxidized in cells treated with isothiocyanates

Isothiocyanates are phytochemicals with anti-cancer properties that include the ability to trigger apoptosis. A substantial body of evidence suggests that reaction of the electrophilic isothiocyanate moiety with cysteine residues in cellular proteins and glutathione accounts for their biological activity. In this study we investigated the effect of several different isothiocyanates on the redox states of the cysteine-dependent peroxiredoxins (Prx) in Jurkat T lymphoma cells, and compared this to known effects on the selenoprotein thioredoxin reductase, glutathione reductase and intracellular GSH levels. Interestingly, oxidation of mitochondrial Prx3 could be detected as early as 5 min after exposure of cells to phenethyl isothiocyanate, with complete oxidation occurring at doses that only had small inhibitory effects on total cellular thioredoxin reductase and glutathione reductase activities. Peroxiredoxin oxidation was specific to the mitochondrial isoform with cytoplasmic Prx1 and Prx2 maintained in their reduced forms at all analyzed time points and concentrations of isothiocyanate. Phenethyl isothiocyanate could react with purified Prx3 directly, but it did not oxidize Prx3 or promote its oxidation by hydrogen peroxide. A selection of aromatic and alkyl isothiocyanates were tested and while all lowered cellular GSH levels, only the isothiocyanates that caused Prx3 oxidation were able to trigger cell death. We propose that pro-apoptotic isothiocyanates selectively disrupt mitochondrial redox homeostasis, as indicated by Prx3 oxidation, and that this contributes to their pro-apoptotic activity.     

Isolation of the ATP synthase subunit 6 and sequence of the mitochondrial ATP6 gene of the yeast Candida parapsilosis.

The mitochondrially translated product called subunit 6 was extracted from the yeast Candida parapsilosis mitochondria using an organic solvent mixture and purified by reverse-phase HPLC. The partial N-terminal sequence of subunit 6 reveals a post-translational cleavage site as in Saccharomyces cerevisiae. The structural mitochondrial gene ATP6 was isolated form a mitochondrial DNA library using the oligonucleotide probe procedure. The gene and the surrounding regions were cloned into M13tg130 and M13tg131 phage vectors. The insert contained an open reading frame 738-bp encoding a 246-amino-acid polypeptide. Mature subunit 6 contains 243 amino acid residues and the predicted molecular mass is 26,511 Da. The subunit shows 52% similarity with ATP synthase subunit 6 of the yeast S. cerevisiae. Comparison between protein and DNA sequences shows that the CUN codon family codes for a leucine in C. parapsilosis mitochondria.     

Biochemical characterization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells.

Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.