Inactivation of peroxiredoxin I by phosphorylation allows localized H(2)O(2) accumulation for cell signaling.

Despite its toxicity, H(2)O(2) is produced as a signaling molecule that oxidizes critical cysteine residues of effectors such as protein tyrosine phosphatases in response to activation of cell surface receptors. It has remained unclear, however, how H(2)O(2) concentrations above the threshold required to modify effectors are achieved in the presence of the abundant detoxification enzymes peroxiredoxin (Prx) I and II. We now show that PrxI associated with membranes is transiently phosphorylated on tyrosine-194 and thereby inactivated both in cells stimulated via growth factor or immune receptors in vitro and in those at the margin of healing cutaneous wounds in mice. The localized inactivation of PrxI allows for the transient accumulation of H(2)O(2) around membranes, where signaling components are concentrated, while preventing the toxic accumulation of H(2)O(2) elsewhere. In contrast, PrxII was inactivated not by phosphorylation but rather by hyperoxidation of its catalytic cysteine during sustained oxidative stress.

Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1ß mRNA Expression.

LPS induces inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, and causes an inflammatory response. The development of small molecules that have suppressive effect on those inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases. We synthesized 12 novel compounds with 4-amino-N-(4-(benzo[d]oxazol-2-ylamino)phenyl)butanamide moiety and evaluated their biological activities. Among them, 4 compounds (compound 5d, 5c, 5f, 5m and synthetic intermediate 4d) showed potent inhibition activities on IL-1ß and IL-6 mRNA expression in vitro. Further, in vivo activity was evaluated with two compounds (5f and 4d) and mRNA levels of IL-1ß, IL-6, and TNF-α were significantly decreased without hepatotoxicity. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective for suppression of representative inflammatory cytokines.

Novel Small Molecule Inhibitors Targeting the IL-6/STAT3 Pathway or IL-1ß.

Development of small molecules that inhibit inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases such as rheumatoid arthritis (RA). Following up a previous study, we synthesized 10 novel compounds with a 2,5-diaminobenzoxazole moiety and evaluated their biological activities. Among them, compound 3e showed potent inhibitory activity on Interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling inhibition (71.5%), and 3a showed excellent inhibitory activity on Interleukin 1 (IL-1ß) (92.1%). To test in vivo anti-inflammatory activity, compounds 3a and 3e were administered by intraperitoneal (IP) injection after subcutaneous (SC) injection of zymosan A into the right footpad of mice. Inflammation on the footpad was reduced after administration of compounds 3a and 3e. Especially, compound 3a showed a significant ameliorative effect on zymosan-induced inflammation. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective on the RA animal model through inhibition of the IL-6/STAT3 signaling pathway. Since drugs developed with small molecule inhibitors have several advantages over biological drugs, further study on these compounds is needed for the development of potent SMI drugs on RA.

Peroxiredoxin-1 Tyr194 phosphorylation regulates LOX-dependent extracellular matrix remodelling in breast cancer.

BACKGROUND: Peroxiredoxin 1 (PRDX1) belongs to an abundant family of peroxidases whose role in cancer is still unresolved. While mouse knockout studies demonstrate a tumour suppressive role for PRDX1, in cancer cell xenografts, results denote PRDX1 as a drug target. Probably, this phenotypic discrepancy stems from distinct roles of PRDX1 in certain cell types or stages of tumour progression. METHODS: We demonstrate an important cell-autonomous function for PRDX1 utilising a syngeneic mouse model (BALB/c) and mammary fibroblasts (MFs) obtained from it. RESULTS: Loss of PRDX1 in vivo promotes collagen remodelling known to promote breast cancer progression. PRDX1 inactivation in MFs occurs via SRC-induced phosphorylation of PRDX1 TYR194 and not through the expected direct oxidation of CYS52 in PRDX1 by ROS. TYR194-phosphorylated PRDX1 fails to bind to lysyl oxidases (LOX) and leads to the accumulation of extracellular LOX proteins which supports enhanced collagen remodelling associated with breast cancer progression. CONCLUSIONS: This study reveals a cell type-specific tumour suppressive role for PRDX1 that is supported by survival analyses, depending on PRDX1 protein levels in breast cancer cohorts.

Feedback control of adrenal steroidogenesis via H2O2-dependent, reversible inactivation of peroxiredoxin III in mitochondria.

Certain members of the peroxiredoxin (Prx) family undergo inactivation through hyperoxidation of the catalytic cysteine to sulfinic acid during catalysis and are reactivated by sulfiredoxin; however, the physiological significance of this reversible regulatory process is unclear. We now show that PrxIII in mouse adrenal cortex is inactivated by H(2)O(2) produced by cytochrome P450 enzymes during corticosterone production stimulated by adrenocorticotropic hormone. Inactivation of PrxIII triggers a sequence of events including accumulation of H(2)O(2), activation of p38 mitogen-activated protein kinase, suppression of steroidogenic acute regulatory protein synthesis, and inhibition of steroidogenesis. Interestingly, levels of inactivated PrxIII, activated p38, and sulfiredoxin display circadian oscillations. Steroidogenic tissue-specific ablation of sulfiredoxin in mice resulted in the persistent accumulation of inactive PrxIII and suppression of the adrenal circadian rhythm of corticosterone production. The coupling of CYP11B1 activity to PrxIII inactivation provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis.

Some Biological Consequences of the Inhibition of Na,K-ATPase by Translationally Controlled Tumor Protein (TCTP).

Na,K-ATPase is an ionic pump that regulates the osmotic equilibrium and membrane potential of cells and also functions as a signal transducer. The interaction of Na,K-ATPase with translationally controlled tumor protein (TCTP) results, among others, in the inhibition of the former's pump activity and in the initiation of manifold biological and pathological phenomena. These phenomena include hypertension and cataract development in TCTP-overexpressing transgenic mice, as well as the induction of tumorigenesis signaling pathways and the activation of Src that ultimately leads to cell proliferation and migration. This review attempts to collate the biological effects of Na,K-ATPase and TCTP interaction and suggests that this interaction has the potential to serve as a possible therapeutic target for selected diseases.

Minor phenolics from Angelica keiskei and their proliferative effects on Hep3B cells.

A new coumarin, (-)-cis-(3'R,4'R)-4'-O-angeloylkhellactone-3'-O-ß-d-glucopyranoside (1) and two new chalcones, 3'-[(2E)-5-carboxy-3-methyl-2-pentenyl]-4,2',4'-trihydroxychalcone (4) and (±)-4,2',4'-trihydroxy-3'-{2-hydroxy-2-[tetrahydro-2-methyl-5-(1-methylethenyl)-2-furanyl]ethyl}chalcone (5) were isolated from the aerial parts of Angelica keiskei (Umbelliferae), together with six known compounds: (R)-O-isobutyroyllomatin (2), 3'-O-methylvaginol (3), (-)-jejuchalcone F (6), isoliquiritigenin (7), davidigenin (8), and (±)-liquiritigenin (9). The structures of the new compounds were determined by interpretation of their spectroscopic data including 1D and 2D NMR data. All known compounds (2, 3, and 6-9) were isolated as constituents of A. keiskei for the first time. To identify novel hepatocyte proliferation inducer for liver regeneration, 1-9 were evaluated for their cell proliferative effects using a Hep3B human hepatoma cell line. All isolates exhibited cell proliferative effects compared to untreated control (DMSO). Cytoprotective effects against oxidative stress induced by glucose oxidase were also examined on Hep3B cells and mouse fibroblast NIH3T3 cells and all compounds showed significant dose-dependent protection against oxidative stress.

Circadian rhythm of hyperoxidized peroxiredoxin II is determined by hemoglobin autoxidation and the 20S proteasome in red blood cells.

The catalytic cysteine of the typical 2-Cys Prx subfamily of peroxiredoxins is occasionally hyperoxidized to cysteine sulfinic acid during the peroxidase catalytic cycle. Sulfinic Prx (Prx-SO2H) is reduced back to the active form of the enzyme by sulfiredoxin. The abundance of Prx-SO2H was recently shown to oscillate with a period of ∼24 h in human red blood cells (RBCs). We have now investigated the molecular mechanism and physiological relevance of such oscillation in mouse RBCs. Poisoning of RBCs with CO abolished Prx-SO2H formation, implicating H2O2 produced from hemoglobin autoxidation in Prx hyperoxidation. RBCs express the closely related PrxI and PrxII isoforms, and analysis of RBCs deficient in either isoform identified PrxII as the hyperoxidized Prx in these cells. Unexpectedly, RBCs from sulfiredoxin-deficient mice also exhibited circadian oscillation of Prx-SO2H. Analysis of the effects of protease inhibitors together with the observation that the purified 20S proteasome degraded PrxII-SO2H selectively over nonhyperoxidized PrxII suggested that the 20S proteasome is responsible for the decay phase of PrxII-SO2H oscillation. About 1% of total PrxII undergoes daily oscillation, resulting in a gradual loss of PrxII during the life span of RBCs. PrxII-SO2H was detected in cytosolic and ghost membrane fractions of RBCs, and the amount of membrane-bound PrxII-SO2H oscillated in a phase opposite to that of total PrxII-SO2H. Our results suggest that membrane association of PrxII-SO2H is a tightly controlled process and might play a role in the tuning of RBC function to environmental changes.

Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody.

Signal transducer and activator of transcription 3 (STAT3) is a multifunctional protein that participates in signaling pathways initiated by various growth factors and cytokines. It exists in multiple forms including those phosphorylated on Tyr(705) (pYSTAT3) or Ser(727) (pSSTAT3) as well as the unphosphorylated protein (USTAT3). In addition to the canonical transcriptional regulatory role of pYSTAT3, both USTAT3 and pSSTAT3 function as transcriptional regulators by binding to distinct promoter sites and play signaling roles in the cytosol or mitochondria. The roles of each STAT3 species in different biological processes have not been readily amenable to investigation, however. We have now prepared an intrabody that binds specifically and with high affinity to the tyrosine-phosphorylated site of pYSTAT3. Adenovirus-mediated expression of the intrabody in HepG2 cells as well as mouse liver blocked both the accumulation of pYSTAT3 in the nucleus and the production of acute phase response proteins induced by interleukin-6. Intrabody expression did not affect the overall accumulation of pSSTAT3 induced by interleukin-6 or phorbol 12-myristate 13-acetate (PMA), the PMA-induced expression of the c-Fos gene, or the PMA-induced accumulation of pSSTAT3 specifically in mitochondria. In addition, it had no effect on interleukin-6-induced expression of the gene for IFN regulatory factor 1, a downstream target of STAT1. Our results suggest that the engineered intrabody is able to block specifically the downstream effects of pYSTAT3 without influencing those of pSSTAT3, demonstrating the potential of intrabodies as tools to dissect the cellular functions of specific modified forms of proteins that exist as multiple species.

Bacteroides fragilis Enterotoxin Upregulates Heme Oxygenase-1 in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase- and NF-κB-Dependent Pathway, Leading to Modulation of Apoptosis.

The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although expression of heme oxygenase-1 (HO-1) is associated with regulation of inflammatory responses, little is known about HO-1 induction in ETBF infection. This study was conducted to investigate the effect of BFT on HO-1 expression in intestinal epithelial cells. Stimulation of intestinal epithelial cells with BFT resulted in upregulated expression of HO-1. BFT activated transcription factors such as NF-κB, AP-1, and Nrf2 in intestinal epithelial cells. Upregulation of HO-1 in intestinal epithelial cells was dependent on activated IκB kinase (IKK)-NF-κB signals. However, suppression of Nrf2 or AP-1 signals in intestinal epithelial cells did not result in significant attenuation of BFT-induced HO-1 expression. HO-1 induction via IKK-NF-κB in intestinal epithelial cells was regulated by p38 mitogen-activated protein kinases (MAPKs). Furthermore, suppression of HO-1 activity led to increased apoptosis in BFT-stimulated epithelial cells. These results suggest that a signaling pathway involving p38 MAPK-IKK-NF-κB in intestinal epithelial cells is required for HO-1 induction during exposure to BFT. Following this induction, increased HO-1 expression may regulate the apoptotic process in responses to BFT stimulation.

Crude Preparations of Helicobacter pylori Outer Membrane Vesicles Induce Upregulation of Heme Oxygenase-1 via Activating Akt-Nrf2 and mTOR-IκB Kinase-NF-κB Pathways in Dendritic Cells.

Helicobacter pylori sheds outer membrane vesicles (OMVs) that contain many surface elements of bacteria. Dendritic cells (DCs) play a major role in directing the nature of adaptive immune responses against H. pylori, and heme oxygenase-1 (HO-1) has been implicated in regulating function of DCs. In addition, HO-1 is important for adaptive immunity and the stress response. Although H. pylori-derived OMVs may contribute to the pathogenesis of H. pylori infection, responses of DCs to OMVs have not been elucidated. In the present study, we investigated the role of H. pylori-derived crude OMVs in modulating the expression of HO-1 in DCs. Exposure of DCs to crude H. pylori OMVs upregulated HO-1 expression. Crude OMVs obtained from a cagA-negative isogenic mutant strain induced less HO-1 expression than OMVs obtained from a wild-type strain. Crude H. pylori OMVs activated signals of transcription factors such as NF-κB, AP-1, and Nrf2. Suppression of NF-κB or Nrf2 resulted in significant attenuation of crude OMV-induced HO-1 expression. Crude OMVs increased the phosphorylation of Akt and downstream target molecules of mammalian target of rapamycin (mTOR), such as S6 kinase 1 (S6K1). Suppression of Akt resulted in inhibition of crude OMV-induced Nrf2-dependent HO-1 expression. Furthermore, suppression of mTOR was associated with inhibition of IκB kinase (IKK), NF-κB, and HO-1 expression in crude OMV-exposed DCs. These results suggest that H. pylori-derived OMVs regulate HO-1 expression through two different pathways in DCs, Akt-Nrf2 and mTOR-IKK-NF-κB signaling. Following this induction, increased HO-1 expression in DCs may modulate inflammatory responses in H. pylori infection.

EW-7197 inhibits hepatic, renal, and pulmonary fibrosis by blocking TGF-ß/Smad and ROS signaling.

Fibrosis is an inherent response to chronic damage upon immense apoptosis or necrosis. Transforming growth factor-beta1 (TGF-ß1) signaling plays a key role in the fibrotic response to chronic liver injury. To develop anti-fibrotic therapeutics, we synthesized a novel small-molecule inhibitor of the TGF-ß type I receptor kinase (ALK5), EW-7197, and evaluated its therapeutic potential in carbon tetrachloride (CCl4) mouse, bile duct ligation (BDL) rat, bleomycin (BLM) mouse, and unilateral ureteral obstruction (UUO) mouse models. Western blot, immunofluorescence, siRNA, and ChIP analysis were carried out to characterize EW-7197 as a TGF-ß/Smad signaling inhibitor in LX-2, Hepa1c1c7, NRK52E, and MRC5 cells. In vivo anti-fibrotic activities of EW-7197 were examined by microarray, immunohistochemistry, western blotting, and a survival study in the animal models. EW-7197 decreased the expression of collagen, α-smooth muscle actin (α-SMA), fibronectin, 4-hydroxy-2, 3-nonenal, and integrins in the livers of CCl4 mice and BDL rats, in the lungs of BLM mice, and in the kidneys of UUO mice. Furthermore, EW-7197 extended the lifespan of CCl4 mice, BDL rats, and BLM mice. EW-7197 blocked the TGF-ß1-stimulated production of reactive oxygen species (ROS), collagen, and α-SMA in LX-2 cells and hepatic stellate cells (HSCs) isolated from mice. Moreover, EW-7197 attenuated TGF-ß- and ROS-induced HSCs activation to myofibroblasts as well as extracellular matrix accumulation. The mechanism of EW-7197 appeared to be blockade of both TGF-ß1/Smad2/3 and ROS signaling to exert an anti-fibrotic activity. This study shows that EW-7197 has a strong potential as an anti-fibrosis therapeutic agent via inhibition of TGF-ß-/Smad2/3 and ROS signaling.

Ablation of peroxiredoxin II attenuates experimental colitis by increasing FoxO1-induced Foxp3+ regulatory T cells.

Peroxiredoxin (Prx) II is an intracellular antioxidant molecule that eliminates hydrogen peroxide, employing a high substrate-binding affinity. PrxII deficiency increases the levels of intracellular reactive oxygen species in many types of cells, which may increase reactive oxygen species-mediated inflammation. In this study, we investigated the susceptibility of PrxII knockout (KO) mice to experimentally induced colitis and the effects of PrxII on the immune system. Wild-type mice displayed pronounced weight loss, high mortality, and colon shortening after dextran sulfate sodium administration, whereas colonic inflammation was significantly attenuated in PrxII KO mice. Although macrophages were hyperactivated in PrxII KO mice, the amount of IFN-γ and IL-17 produced by CD4(+) T cells was substantially reduced. Foxp3(+) regulatory T (Treg) cells were elevated, and Foxp3 protein expression was increased in the absence of PrxII in vitro and in vivo. Restoration of PrxII into KO cells suppressed the increased Foxp3 expression. Interestingly, endogenous PrxII was inactivated through hyperoxidation during Treg cell development. Furthermore, PrxII deficiency stabilized FoxO1 expression by reducing mouse double minute 2 homolog expression and subsequently activated FoxO1-mediated Foxp3 gene transcription. PrxII overexpression, in contrast, reduced FoxO1 and Foxp3 expression. More interestingly, adoptive transfer of naive CD4(+) T cells from PrxII KO mice into immune-deficient mice attenuated T cell-induced colitis, with a reduction in mouse double minute 2 homolog expression and an increase in FoxO1 and Foxp3 expression. These results suggest that inactivation of PrxII is important for the stability of FoxO1 protein, which subsequently mediates Foxp3(+) Treg cell development, thereby attenuating colonic inflammation.

A new 9,10-dihydrophenanthrene and cell proliferative 3,4-δ-dehydrotocopherols from Stemona tuberosa.

A new compound, 9,10-dihydro-5-methoxy-8-methyl-2,7-phenanthrenediol (1), was isolated from the roots of Stemona tuberosa Lour. (Stemonaceae) together with two new optically active compounds, (2S,4'R,8'R)-3,4-δ-dehydrotocopherol (2) and (2R,4'R,8'R)-3,4-δ-dehydrotocopherol (3). The structures of compounds 1-3 were determined on the basis of spectroscopic data analysis. Compounds 2 and 3 were each purified from a stereoisomeric mixture of 2 and 3 by preparative HPLC using a chiral column for the first time. The absolute configurations at C-2 of 2 and 3 were determined by Circular Dichroism (CD) experiments. As a part of the research to find natural wound healing agents, all isolates and the mixture of 2 and 3 were evaluated for their cell proliferative effects using a mouse fibroblast NIH3T3 and a HeLa human cervical cancer cell line. As a result, 1, 2, 3, or the mixture of 2 and 3 showed 41.6%, 78.4%, 118.6%, 38.2% increases of cell proliferation in the mouse fibroblast NIH3T3 respectively, compared to 28.4% increase of δ-tocopherol. Moreover, none of them induced cancer cell proliferation. Therefore, 3,4-δ-dehydrotocopherols, especially pure isomers 2 and 3 can be suggested as potential wound healing agents.

Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides.

Peroxiredoxins (Prxs) contain an active site cysteine that is sensitive to oxidation by H(2)O(2). Mammalian cells express six Prx isoforms that are localized to various cellular compartments. The oxidized active site cysteine of Prx can be reduced by a cellular thiol, thus enabling Prx to function as a locally constrained peroxidase. Regulation of Prx via phosphorylation in response to extracellular signals allows the local accumulation of H(2)O(2) and thereby enables its messenger function. The fact that the oxidation state of the active site cysteine of Prx can be transferred to other proteins that are less intrinsically susceptible to H(2)O(2) also allows Prx to function as an H(2)O(2) sensor.

The Role of Oxidative Inactivation of Phosphatase PTEN and TCPTP in Fatty Liver Disease.

Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are becoming increasingly prevalent worldwide. Despite the different etiologies, their spectra and histological feature are similar, from simple steatosis to more advanced stages such as steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Studies including peroxiredoxin knockout models revealed that oxidative stress is crucial in these diseases, which present as consequences of redox imbalance. Protein tyrosine phosphatases (PTPs) are a superfamily of enzymes that are major targets of reactive oxygen species (ROS) because of an oxidation-susceptible nucleophilic cysteine in their active site. Herein, we review the oxidative inactivation of two tumor suppressor PTPs, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and T-cell protein tyrosine phosphatase (TCPTP), and their contribution to the pathogenicity of ALD and NAFLD, respectively. This review might provide a better understanding of the pathogenic mechanisms of these diseases and help develop new therapeutic strategies to treat fatty liver disease.

Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver.

UNLABELLED: Peroxiredoxins (Prxs) are peroxidases that catalyze the reduction of reactive oxygen species (ROS). The active site cysteine residue of members of the 2-Cys Prx subgroup (Prx I to IV) of Prxs is hyperoxidized to cysteine sulfinic acid (Cys-SO(2) ) during catalysis with concomitant loss of peroxidase activity. Reactivation of the hyperoxidized Prx is catalyzed by sulfiredoxin (Srx). Ethanol consumption induces the accumulation of cytochrome P450 2E1 (CYP2E1), a major contributor to ethanol-induced ROS production in the liver. We now show that chronic ethanol feeding markedly increased the expression of Srx in the liver of mice in a largely Nrf2-dependent manner. Among Prx I to IV, only Prx I was found to be hyperoxidized in the liver of ethanol-fed wildtype mice, and the level of Prx I-SO(2) increased to ≈30% to 50% of total Prx I in the liver of ethanol-fed Srx(-/-) mice. This result suggests that Prx I is the most active 2-Cys Prx in elimination of ROS from the liver of ethanol-fed mice and that, despite the up-regulation of Srx expression by ethanol, the capacity of Srx is not sufficient to counteract the hyperoxidation of Prx I that occurs during ROS reduction. A protease protection assay revealed that a large fraction of Prx I is located together with CYP2E1 at the cytosolic side of the endoplasmic reticulum membrane. The selective role of Prx I in ROS removal is thus likely attributable to the proximity of Prx I and CYP2E1. CONCLUSION: The pivotal functions of Srx and Prx I in protection of the liver in ethanol-fed mice was evident from the severe oxidative damage observed in mice lacking either Srx or Prx I.

Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins.

Hydrogen peroxide (H2O2) accumulates transiently in various cell types stimulated with peptide growth factors and participates in receptor signaling by oxidizing the essential cysteine residues of protein tyrosine phosphatases and the lipid phosphatase PTEN. The reversible inactivation of these phosphatases by H2O2 is likely required to prevent futile cycles of phosphorylation-dephosphorylation of proteins and phosphoinositides. The accumulation of H2O2 is possible even in the presence of large amounts of the antioxidant enzymes peroxiredoxin I and II in the cytosol, probably because of a built-in mechanism of peroxiredoxin inactivation that is mediated by H2O2 and reversed by an ATP-dependent reduction reaction catalyzed by sulfiredoxin.

Mitochondrial Peroxiredoxin III Protects against Non-Alcoholic Fatty Liver Disease Caused by a Methionine-Choline Deficient Diet.

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common chronic liver disease worldwide. In addition, NAFLD may increase the risk of cardiovascular and liver-related diseases, and displays features of metabolic syndrome. In NAFLD, oxidative stress is primarily caused by excessive free fatty acids. The oxidation of fatty acids is usually caused by ß-oxidation of mitochondria under normal conditions, resulting in the production of energy. However, when the inflow of fatty acids in NAFLD becomes excessive, the ß-oxidation of mitochondria becomes saturated and the oxidation process increases at sites including peroxisomes and microsomes, thereby increasing production of reactive oxygen species (ROS). Thus, hepatic mitochondrial ROS play an important role in the pathogenesis of NAFLD. Eliminating mitochondrial ROS may improve NAFLD, but the underlying mechanism remains unclear. We examined the effect of mitochondrial ROS on NAFLD by focusing on peroxiredoxin (Prx), an antioxidant protein that can remove hydrogen peroxide. The protective effect and pathological phenomenon of mitochondrial peroxiredoxin in methionine-choline deficient diet (MCD)-induced liver injury was assessed in a mouse model of NAFLD. In these mice, mitochondrial peroxiredoxin deficiency significantly increased hepatic steatosis and fibrosis. In addition, ablation of Prx III enhances susceptibility to MCD diet-induced oxidative stress and exacerbates NAFLD progression by promoting inflammation. The binding assay results also showed that Prx III-deficient mice had more severe liver damage than Prx III-abundant mice in MCD diet liver injury models. The present data suggest that mitochondrial peroxiredoxin III could be a therapeutic target for preventing and suppressing diet-induced NAFLD.

Maturation of Mitochondrially Targeted Prx V Involves a Second Cleavage by Mitochondrial Intermediate Peptidase That Is Sensitive to Inhibition by H2O2.

Prx V mRNA contains two in-frame AUG codons, producing a long (L-Prx V) and short form of Prx V (S-Prx V), and mouse L-Prx V is expressed as a precursor protein containing a 49-amino acid N-terminal mitochondria targeting sequence. Here, we show that the N-terminal 41-residue sequence of L-Prx V is cleaved by mitochondrial processing peptidase (MPP) in the mitochondrial matrix to produce an intermediate Prx V (I-Prx V) with a destabilizing phenylalanine at its N-terminus, and further, that the next 8-residue sequence is cleaved by mitochondrial intermediate peptidase (MIP) to convert I-Prx V to a stabilized mature form that is identical to S-Prx V. Further, we show that when mitochondrial H2O2 levels are increased in HeLa cells using rotenone, in several mouse tissues by deleting Prx III, and in the adrenal gland by deleting Srx or by exposing mice to immobilized stress, I-Prx V accumulates transiently and mature S-Prx V levels decrease in mitochondria over time. These findings support the view that MIP is inhibited by H2O2, resulting in the accumulation and subsequent degradation of I-Prx V, identifying a role for redox mediated regulation of Prx V proteolytic maturation and expression in mitochondria.