Structural and biochemical analyses reveal ubiquitin C-terminal hydrolase-L1 as a specific client of the peroxiredoxin II chaperone.

Peroxiredoxins (Prxs) play dual roles as both thiol-peroxidases and molecular chaperones. Peroxidase activity enables various intracellular functions, however, the physiological roles of Prxs as chaperones are not well established. To study the chaperoning function of Prx, we previously sought to identify heat-induced Prx-binding proteins as the clients of a Prx chaperone. By using His-tagged Prx I as a bait, we separated ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a heat-induced Prx I binding protein from rat brain crude extracts. Protein complex immunoprecipitation with HeLa cell lysates revealed that both Prx I and Prx II interact with UCH-L1. However, Prx II interacted considerably more favorably with UCH-L1 than Prx I. Prx II exhibited more effective molecular chaperone activity than Prx I when UCH-L1 was the client. Prx II interacted with UCH-L1 through its C-terminal region to protect UCH-L1 from thermal or oxidative inactivation. We found that chaperoning via interaction through C-terminal region (specific-client chaperoning) is more efficient than that involving oligomeric structural change (general-client chaperoning). Prx II binds either thermally or oxidatively unfolding early intermediates of specific clients and thereby shifted the equilibrium towards their native state. We conclude that this chaperoning mechanism provides a very effective and selective chaperoning activity.

Anti-inflammatory effects of sucrose-derived oligosaccharides produced by a constitutive mutant L. mesenteroides B-512FMCM dextransucrase in high fat diet-fed mice.

Oligosaccharide (OS) is used as a sugar replacement as well as an ingredient in functional foods because of its beneficial effects, mainly on reducing calorie content and promoting intestinal health. By contrast, the effects of OS on inflammation are less well investigated. The purpose of this study was to investigate the effects of sucrose-derived OS on glucose control and inflammation in high fat (HF) diet-fed mice. Male C57BL6 mice were randomly assigned to six treatment groups (n = 10-14 mice per group): 1) lean control (CON), 2) HF control, 3) HF-low sucrose (LS, 100 mg/kg/day), 4) HF-high sucrose (HS, 1000 mg/kg/day), 5) HF-low OS (LOS, 100 mg/kg/day), and 6) HF-high OS (HOS, 1000 mg/kg/day). PBS (vehicle), sucrose, and OS were administered by stomach gavage. Body weight, food intake, and markers of liver function (activities of aspartate aminotransferase and alanine aminotransferase) were not affected by the treatments. HOS treatment decreased levels of serum glucose, insulin, and homeostasis model assessment-insulin resistance compared with sucrose treatment. However, serum adiponectin levels of the HOS group were higher than those of the sucrose groups. Serum levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and fetuin-A were lower in the HOS group than in the sucrose groups. Hepatic gene expression levels of pro-inflammatory cytokines and related factors (fetuin-A, NF-κB, TLR4, TNF-alpha, and IL-6) were decreased and the levels of insulin signaling-related molecules (sirtuin 1, insulin receptor, and Akt) were increased in HOS-treated mice as compared with sucrose-treated mice. These results demonstrate that OS treatment is effective in improving glucose control and inflammation in high fat diet-fed mice.

Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling.

The observation that purified yeast glutamine synthetase is rapidly inactivated in a thiol-containing buffer yet retains activity in crude extracts containing the same thiol led to our discovery of an enzyme that protects against oxidation in a thiol-containing system. This novel antioxidant enzyme was shown to reduce hydroperoxides and, more recently, peroxynitrite with the use of electrons provided by a physiological thiol like thioredoxin. It defined a family of proteins, present in organisms from all kingdoms, that was named peroxiredoxin (Prx). All Prx enzymes contain a conserved Cys residue that undergoes a cycle of peroxide-dependent oxidation and thiol-dependent reduction during catalysis. Mammalian cells express six isoforms of Prx (Prx I to VI), which are classified into three subgroups (2-Cys, atypical 2-Cys, and 1-Cys) based on the number and position of Cys residues that participate in catalysis. The relative abundance of Prx enzymes in mammalian cells appears to protect cellular components by removing the low levels of peroxides produced as a result of normal cellular metabolism. During catalysis, the active site cysteine is occasionally overoxidized to cysteine sulfinic acid. Contrary to the general belief that oxidation to the sulfinic state is an irreversible process in cells, studies on the fate of the overoxidized Prx species revealed a mechanism by which the catalytically active thiol form is recovered. This sulfinic reduction is a slow, ATP-dependent process that is specific to 2-Cys Prx isoforms. This reversible overoxidation may represent an adaptation unique to eukaryotic cells that accommodates the intracellular messenger function of H(2)O(2), but experimental validation of such speculation is yet to come.

Screening Molecular Chaperones Similar to Small Heat Shock Proteins in Schizosaccharomyces pombe.

To screen molecular chaperones similar to small heat shock proteins (sHsps), but without α-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an α-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at 70℃ for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no α-crystalline domain in their sequences.

Stress-dependent regulation of the gene encoding thioredoxin reductase from the fission yeast.

The unique putative gene for thioredoxin reductase (TrxR) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The determined DNA sequence carries 3125 bp, and encodes the plausible 322 amino acid sequence of TrxR with a molecular mass of 34,618 Da. The S. pombe cells harboring the cloned TrxR gene contain increased TrxR activity, and shows higher survivals on solid media with mercuric chloride or aluminum chloride. The 1526 bp upstream region was fused into promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid. The synthesis of beta-galactosidase from the fusion plasmid pYUTR10 was enhanced by menadione, mercuric chloride, hydrogen peroxide, aluminium chloride and sodium selenite. Menadione significantly enhanced the TrxR mRNA level in the S. pombe cells, which was detected by RT-PCR. Induction of the S. pombe TrxR gene by menadione and mercuric chloride occurs through the mediation of the transcription factor Pap1. These results suggest that the S. pombe TrxR gene is one of the stress response-related genes.

Distinct functional roles of peroxiredoxin isozymes and glutathione peroxidase from fission yeast, Schizosaccharomyces pombe.

To investigate the differences in the functional roles of peroxiredoxins (Prxs) and glutathione peroxidase (GPx) of Schizosaccharomyces pombe, we examined the peroxidase and molecular chaperone properties of the recombinant proteins. TPx (thioredoxin peroxidase) exhibited a capacity for peroxide reduction with the thioredoxin system. GPx also showed thioreoxin-dependent peroxidase activity rather than GPx activity. The peroxidase activity of BCP (bacterioferritin comigratory protein) was similar to that of TPx. However, peroxidase activity was not observed for PMP20 (peroxisomal membrane protein 20). TPx, PMP20, and GPx inhibited thermal aggregation of citrate synthase at 43(o)C, but BCP failed to inhibit the aggregation. The chaperone activities of PMP20 and GPx were weaker than that of TPx. The peroxidase and chaperone properties of TPx, BCP, and GPx of the fission yeast are similar to those of Saccharomyces cerevisiae. The fission yeast PMP20 without thioredoxin-dependent peroxidase activity may act as a molecular chaperone.

Aspartyl aminopeptidase of Schizosaccharomyces pombe has a molecular chaperone function.

To screen chaperone proteins from Schizosaccharomyce pombe (S. pombe), we prepared recombinant citrate synthase of the fission yeast as a substrate of anti-aggregation assay. Purified recombinant citrate synthase showed citrate synthase activity and was suitable for the substrate of chaperone assay. Several heat stable proteins including aspartyl aminopeptidase (AAP) for candidates of chaperone were screened from the supernatant fraction of heat-treated crude extract of S. pombe. The purified AAP migrated as a single band of 47 kDa on SDS-polyacrylamide gel electrophoresis. The native size of AAP was estimated as 200 kDa by a HPLC gel permeation chromatography. This enzyme can remove the aspartyl residue at N-terminus of angiotensin I. In addition, AAP showed the heat stability and protected the aggregation of citrate synthase caused by thermal denaturation. This study showed that S. pombe AAP is a moonlight protein that has aspartyl aminopeptidase and chaperone activities.

Novel protective mechanism against irreversible hyperoxidation of peroxiredoxin: Nalpha-terminal acetylation of human peroxiredoxin II.

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), cycles between thiol (C(P)-SH) and disulfide (-S-S-) states via a sulfenic (C(P)-SOH) intermediate. Hyperoxidation of the C(P) thiol to its sulfinic (C(P)-SO(2)H) derivative has been shown to be reversible, but its sulfonic (C(P)-SO(3)H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C(P)-SO(3)H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that N(alpha)-terminal acetylation (N(alpha)-Ac) occurred exclusively on Prx II after demethionylation. N(alpha)-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N(alpha)-acetylated and N(alpha)-terminal acetylated Prx II revealed that N(alpha)-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T(m) from 59.6 to 70.9 degrees C. These findings suggest that the structural maintenance of Prx II by N(alpha)-Ac may be responsible for preventing its hyperoxidation to form C(P)-SO(3)H.

Irreversible oxidation of the active-site cysteine of peroxiredoxin to cysteine sulfonic acid for enhanced molecular chaperone activity.

The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO(2)H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO(3)H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H(2)O(2) concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO(3)H) by mass spectrometry. Tsa1p-SO(3)H was not an autoxidation product of Tsa1p-SO(2)H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO(3)H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO(3)H multimer lost its peroxidase activity, it gained approximately 4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO(3)H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO(3)H is a marker of cumulative oxidative stress in cells.

Characterization of a second gene encoding gamma-glutamyl transpeptidase from Schizosaccharomyces pombe.

The first gene encoding gamma-glutamyl transpeptidase (GGTI) of the fission yeast has previously been characterized, and its expression was found to be regulated by various oxidative stress-inducing agents. In this work, a second gene, encoding GGTII, was cloned and characterized from the fission yeast Schizosaccharomyces pombe. The structural gene encoding GGTII was amplified from the genomic DNA of the fission yeast and ligated into the shuttle vector pRS316 to generate the recombinant plasmid pPHJ02. The determined sequence contains 3040 bp and is able to encode the putative 611 amino acid sequence of GGTII, which resembles the counterparts of Saccharomyces cerevisiae, Homo sapiens, Rattus norvegicus, and Escherichia coli. The DNA sequence also contains 940-bp upstream and 289-bp downstream regions of the GGTII gene. The Schizosaccharomyces pombe cells harboring plasmid pPHJ02 showed about 4-fold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGTII gene is functional. The S. pombe cells containing the cloned GGTII gene were found to contain higher levels of both intracellular glutathione (GSH) content and GSH uptake. The S. pombe cells harboring plasmid pPHJ02 showed increased survival on solid media containing hydrogen peroxide, diethylmaleate, aluminum chloride, cadmium chloride, or mercuric chloride. The GGTII mRNA level was significantly elevated by treatment with GSH-depleting diethylmaleate. These results imply that the S. pombe GGTII gene produces functional GGTII protein and is involved in the response to oxidative stresses in S. pombe cells.

Escherichia coli periplasmic thiol peroxidase acts as lipid hydroperoxide peroxidase and the principal antioxidative function during anaerobic growth.

To clarify the enzymatic property of Escherichia coli periplasmic thiol peroxidase (p20), the specific peroxidase activity toward peroxides was compared with other bacterial thiol peroxidases. p20 has the most substrate preference and peroxidase activity toward organic hydroperoxide. Furthermore, p20 exerted the most potent lipid peroxidase activity. Despite that the mutation of p20 caused the highest susceptibility toward organic hydroperoxide and heat stress, the cellular level of p20 did not respond to the exposure of oxidative stress. Expression level of p20 during anaerobic growth was sustained at the approximately 50% level compared with that of the aerobic growth. Viability of aerobic p20Delta without glucose was reduced to the approximately 65% level of isogenic strains, whereas viability of aerobic p20Delta with 0.5% glucose supplement was sustained. The deletion of p20 resulted in a gradual loss of the cell viability during anaerobic growth. At the stationary phase, the viability of p20Delta was down to approximately 10% level of parent strains. An analysis of the protein carbonyl contents of p20Delta as a marker for cellular oxidation indicates that severe reduction of viability of anaerobic p20Delta was caused by cumulative oxidative stress. P20Delta showed hypersensitivity toward membrane-soluble organic hydroperoxides. An analysis of protein carbonyl and lipid hydroperoxide contents in the membrane of the stress-imposed p20Delta demonstrates that the severe reduction of viability was caused by cumulative oxidative stress on the membrane. Taken together, present data uncover in vivo function for p20 as a lipid hydroperoxide peroxidase and demonstrate that, as the result, p20 acts as the principal antioxidant in the anaerobic habitats.

Characterization and regulation of the gamma-glutamyl transpeptidase gene from the fission yeast Schizosaccharomyces pombe.

The structural gene for the putative gamma-glutamyl transpeptidase (GGT) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The determined sequence contained 3324 bp and encoded the predicted 630 amino acid sequence of GGT, which resembles counterparts in Homo sapiens, Rattus norvegicus, Saccharomyces cerevisiae, and Escherichia coli. The S. pombe cells harboring the cloned GGT gene showed about twofold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGT gene was functional. To monitor the expression of the S. pombe GGT gene, we fused the fragment 1085 bp upstream of the cloned GGT gene into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pGT98. The synthesis of beta-galactosidase from the fusion plasmid in S. pombe cells was enhanced by treatments with NO-generating sodium nitroprusside (SN), L-buthionine-(S,R)-sulfoximine (BSO), and glycerol. The GGT mRNA level in the S. pombe cells was increased by SN and BSO. Involvement of Pap1 in the induction of the GGT gene by SN and BSO was observed.

Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid.

By following peroxiredoxin I (Prx I)-dependent NADPH oxidation spectrophotometrically, we observed that Prx I activity decreased gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by two-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Cys(51)-SH to Cys(51)-SO(2)H. Thus, Cys(51)-SOH generated as an intermediate during catalysis appeared to undergo occasional further oxidation to Cys(51)-SO(2)H, which cannot be reversed by thioredoxin. The presence of H(2)O(2) alone was not sufficient to cause oxidation of Cys(51) to Cys(51)-SO(2)H. Rather, the presence of complete catalytic components (H(2)O(2), thioredoxin, thioredoxin reductase, and NADPH) was necessary, indicating that such hyperoxidation occurs only when Prx I is engaged in the catalytic cycle. Likewise, hyperoxidation of Cys(172)/Ser(172) mutant Prx I required not only H(2)O(2), but also a catalysis-supporting thiol (dithiothreitol). Kinetic analysis of Prx I inactivation in the presence of a low steady-state level (<1 microm) of H(2)O(2) indicated that Prx I was hyperoxidized at a rate of 0.072% per turnover at 30 degrees C. Hyperoxidation of Prx I was also detected in HeLa cells treated with H(2)O(2).

Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation.

The active-site cysteine of peroxiredoxins is selectively oxidized to cysteine sulfinic acid during catalysis, which leads to inactivation of peroxidase activity. This oxidation was thought to be irreversible. However, by metabolic labeling of mammalian cells with 35S, we show that the sulfinic form of peroxiredoxin I, produced during the exposure of cells to H2O2, is rapidly reduced to the catalytically active thiol form. The mammalian cells' ability to reduce protein sulfinic acid might serve as a mechanism to repair oxidatively damaged proteins or represent a new type of cyclic modification by which the function of various proteins is regulated.

Regulation of thioredoxin peroxidase activity by C-terminal truncation.

Thioredoxin peroxidase is a member of peroxiredoxin (Prx) family, which uses a thioredoxin (Trx) as an immediate electron donor for the reduction of peroxide. We have identified C-terminal truncated TPx from Schizosaccharomyces pombe and also have found the truncated form is significantly tenacious against the inactivation of H2O2 than the intact form. Peroxidase assay of a series of recombinant C-terminal truncation mutants (Delta192, Delta191, Delta188, Delta184, Delta176, and Delta165) revealed that TPx could be inactivated (Delta192), reactivated (Delta191-Delta176) and reinactivated (Delta165) by serial truncation from C-terminus. We did not find any significant kinetic difference among reactivated forms; however, distinctive loss of affinity to H2O2 (K(m) = 5 microM) than that of the intact form (<<5 microM, undeterminable) was monitored. Characterization of a series of Lys(191) point mutants manifested that the loss of affinity caused by a deprivation of positive charge born in Lys(191) and the loss of affinity resulted in the resistibility to H2O2. Disk inhibition assay with S. pombe cells overexpressing wild-type, Delta192 and Delta191 mutants evidenced that the truncated forms functioning in vitro as well as in vivo.

The tetrameric structure of Haemophilus influenza hybrid Prx5 reveals interactions between electron donor and acceptor proteins.

Cellular redox control is often mediated by oxidation and reduction of cysteine residues in the redox-sensitive proteins, where thioredoxin and glutaredoxin (Grx) play as electron donors for the oxidized proteins. Despite the importance of protein-protein interactions between the electron donor and acceptor proteins, there has been no structural information for the interaction of thioredoxin or Grx with natural target proteins. Here, we present the crystal structure of a novel Haemophilus influenza peroxiredoxin (Prx) hybrid Prx5 determined at 2.8-A resolution. The structure reveals that hybrid Prx5 forms a tightly associated tetramer where active sites of Prx and Grx domains of different monomers interact with each other. The Prx-Grx interface comprises specific charge interactions surrounded by weak interactions, providing insight into the target recognition mechanism of Grx. The tetrameric structure also exhibits a flexible active site and alternative Prx-Grx interactions, which appear to facilitate the electron transfer from Grx to Prx domain. Differences of electron donor binding surfaces in Prx proteins revealed by an analysis based on the structural information explain the electron donor specificities of various Prx proteins.