Hee-Young Yang

Roles of peroxiredoxin II in the regulation of proinflammatory responses to LPS and protection against endotoxin-induced lethal shock

Mammalian 2-Cys peroxiredoxin II (Prx II) is a cellular peroxidase that eliminates endogenous H2O2. The involvement of Prx II in the regulation of lipopolysaccharide (LPS) signaling is poorly understood. In this report, we show that LPS induces substantially enhanced inflammatory events, which include the signaling molecules nuclear factor κB and mitogen-activated protein kinase (MAPK), in Prx II–deficient macrophages. This effect of LPS was mediated by the robust up-regulation of the reactive oxygen species (ROS)–generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and the phosphorylation of p47phox. Furthermore, challenge with LPS induced greater sensitivity to LPS-induced lethal shock in Prx II–deficient mice than in wild-type mice. Intravenous injection of Prx II–deficient mice with the adenovirus-encoding Prx II gene significantly rescued mice from LPS-induced lethal shock as compared with the injection of a control virus. The administration of catalase mimicked the reversal effects of Prx II on LPS-induced inflammatory responses in Prx II–deficient cells, which suggests that intracellular H2O2 is attributable, at least in part, to the enhanced sensitivity to LPS. These results indicate that Prx II is an essential negative regulator of LPS-induced inflammatory signaling through modulation of ROS synthesis via NADPH oxidase activities and, therefore, is crucial for the prevention of excessive host responses to microbial products.

RtxA1-Induced Expression of the Small GTPase Rac2 Plays a Key Role in the Pathogenicity of Vibrio vulnificus

Infection with the human pathogen Vibrio vulnificus leads to the generation of reactive oxygen species (ROS) via NAD(P)H oxidase (Nox) in host cells. In the present study, we employed mutant V. vulnificus strains to identify an essential virulence factor responsible for this ROS generation. We found that repeats-in-toxin A1 (RtxA1) expressed by V. vulnificus acts via Nox1 to induce significant ROS generation in the intestine epithelial cells, which ultimately results in cell death. Furthermore, RtxA1 modulates the small GTPase Rac2, which is known to play an important role in the activation of Nox. When mice were infected by the oral method, in contrast with the wild-type bacteria, an RtxA1-deficient V. vulnificus mutant was unable to induce ROS generation within the intestine and failed to cause death. These findings strongly suggest that RtxA1-induced Rac2 expression is a critical step underlying the pathogenicity of V. vulnificus.

Antioxidant enzymes as redox-based biomarkers: a brief review.

The field of redox proteomics focuses to a large extent on analyzing cysteine oxidation in proteins under different experimental conditions and states of diseases. The identification and localization of oxidized cysteines within the cellular milieu is critical for understanding the redox regulation of proteins under physiological and pathophysiological conditions, and it will in turn provide important information that are potentially useful for the development of novel strategies in the treatment and prevention of diseases associated with oxidative stress. Antioxidant enzymes that catalyze oxidation/reduction processes are able to serve as redox biomarkers in various human diseases, and they are key regulators controlling the redox state of functional proteins. Redox regulators with antioxidant properties related to active mediators, cellular organelles, and the surrounding environments are all connected within a network and are involved in diseases related to redox imbalance including cancer, ischemia/reperfusion injury, neurodegenerative diseases, as well as normal aging. In this review, we will briefly look at the selected aspects of oxidative thiol modification in antioxidant enzymes and thiol oxidation in proteins affected by redox control of antioxidant enzymes and their relation to disease. [BMB Reports 2015; 48(4): 200-208]

Proteomic Analysis of Protein Expression Affected by Peroxiredoxin V Knock-Down in Hypoxic Kidney

Peroxiredoxin V, an atypical thioredoxin peroxidase, is widely expressed in mammalian tissues. In addition, Prdx V is localized in mitochondria, peroxisome, cytosol, and the nucleus. Prdx V has been reported to protect a wide range of cellular environments as an antioxidant enzyme, and its dysfunctions may be implicated in several diseases, such as cancer, inflammation, and neurodegenerative disease. Identification and relative quantification of proteins affected by Prdx V may help identify novel signaling mechanisms that are important for oxidative stress response. However, the role of Prdx V in the modulation of hypoxia-related cellular response is not studied yet. To examine the function of endogenous Prdx V in hypoxic condition in vivo, we generated a transgenic mouse model with Prdx V siRNA expression controlled by U6 promoter. Of many tissues, the knockdown of Prdx V expression was displayed in the kidney, lung, and liver but not the spleen and skin. We conducted on the basis of nano-UPLC-MS(E) proteomic study to identify the Prdx V-affected protein networks in hypoxic kidneys. In this study, we identified protein networks associated with oxidative stress, fatty acid metabolism, and mitochondrial dysfunction. Our results indicated that Prdx V affected to regulation of kidney homeostasis under hypoxia stress.

Comparative proteomic analysis for the insoluble fractions of colorectal cancer patients

We used label-free quantitative proteomics with the insoluble fractions from colorectal cancer (CRC) patients to gain further insight into the utility of profiling altered protein expression as a potential biomarker for cancer. The insoluble fractions were prepared from paired tumor/normal biopsies from 13 patients diagnosed with CRC (stages I to IV). Fifty-six proteins identified in data pooled from the 13 cases were differentially expressed between the tumor and adjacent normal tissue. The connections between these proteins are involved in reciprocal networks related to tumorigenesis, cancer incidence based on genetic disorder, and skeletal and muscular disorders. To assess their potential utility as biomarkers, the relative expression levels of the proteins were validated using personal proteomics and a heat map to compare five individual CRC samples with five normal tissue samples. Further validation of a panel of proteins (KRT5, JUP, TUBB, and COL6A1) using western blotting confirmed the differential expression. These proteins gave specific network information for CRC, and yielded a panel of novel markers and potential targets for treatment. It is anticipated that the experimental approach described here will increase our understanding of the membrane environment in CRC, which may provide direction for making diagnoses and prognoses through molecular biomarker targeting.

Peroxiredoxin V selectively regulates IL-6 production by modulating the Jak2–Stat5 pathway

Mammalian peroxiredoxin V (PrdxV) is a multifunctional protein that protects cells from DNA damage and inhibits stress-induced apoptosis. However, PrdxV is also known to be involved in modulating lipopolysaccharide (LPS)-induced host cell signaling, but its precise role is not fully understood. In this study, we used stably transfected RAW264.7 cells and transiently transfected 293-mTLR4-MD2-CD14 cells expressing wild-type (WT) or mutant (C48S) PrdxV to characterize the function and mechanism of action of PrdxV in LPS-induced immune responses. We found that PrdxV selectively reduces production of interleukin 6 (IL-6) by inhibiting activation of signal transducer and activator of transcription 5 (Stat5) through interaction with Jak2. Notably, this activity of PrdxV was dependent on its catalytic Cys48 residue, but not its peroxidase activity. The binding of to Jak2 effectively inhibited Jak2 phosphorylation, but PrdxV did not act as efficiently as SOCS1 (suppressor of cytokine signaling 1). Our results suggest that PrdxV is a key mediator contributing to the regulation of LPS/TLR4-induced immune responses.

Comparative proteomic analysis of cysteine oxidation in colorectal cancer patients

Oxidative stress promotes damage to cellular proteins, lipids, membranes and DNA, and plays a key role in the development of cancer. Reactive oxygen species disrupt redox homeostasis and promote tumor formation by initiating aberrant activation of signaling pathways that lead to tumorigenesis. We used shotgun proteomics to identify proteins containing oxidation-sensitive cysteines in tissue specimens from colorectal cancer patients. We then compared the patterns of cysteine oxidation in the membrane fractions between the tumor and non-tumor tissues. Using nano-UPLC-MSE proteomics, we identified 31 proteins containing 37 oxidation-sensitive cysteines. These proteins were observed with IAM-binding cysteines in non-tumoral region more than tumoral region of CRC patients. Then using the Ingenuity pathway program, we evaluated the cellular canonical networks connecting those proteins. Within the networks, proteins with multiple connections were related with organ morphology, cellular metabolism, and various disorders. We have thus identified networks of proteins whose redox status is altered by oxidative stress, perhaps leading to changes in cellular functionality that promotes tumorigenesis.

The role of peroxiredoxin V in (-)-epigallocatechin 3-gallate-induced multiple myeloma cell death.

(-)-Epigallocatechin 3-gallate (EGCG) is a potent antioxidant polyphenol in green tea that acts as an anticancer agent via both direct and indirect pathways. Although the relationship between EGCGs anticancer effects and its antioxidant activity is not fully understood, it is known that EGCG stimulates production of reactive oxygen species (ROS), which induce oxidative stress leading to cell death. In IM9 multiple myeloma cells, EGCG acted in a dose- and time-dependent manner to induce apoptotic cell death. Among the antioxidant enzymes expressed in IM9 cells, levels of peroxiredoxin V (PrdxV) were selectively and significantly reduced by EGCG. Moreover, the ROS scavenger NAC completely inhibited EGCG-induced apoptosis and PrdxV reduction, while overexpression of PrdxV, but not a Prdx(VC48S) mutant, protected IM9 cells from EGCG-induced apoptosis. EGCG-induced reductions in cell viability and PrdxV levels were also observed in primary CD138+ multiple myeloma cells from patients. These results suggest that PrdxV is a key target via which EGCG mediates its anticancer effects.

In‐depth analysis of cysteine oxidation by the RBC proteome: Advantage of peroxiredoxin II knockout mice

Peroxiredoxin II (Prdx II, a typical 2‐Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Mice lacking Prdx II proteins had heinz bodies in their peripheral blood, and morphologically abnormal cells were detected in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). In this study, a labeling experiment with the thiol‐modifying reagent biotinylated iodoacetamide (BIAM) in Prdx II−/− mice revealed that a variety of RBC proteins were highly oxidized. To identify oxidation‐sensitive proteins in Prdx II−/− mice, we performed RBC comparative proteome analysis in membrane and cytosolic fractions by nano‐UPLC‐MSE shotgun proteomics. We found oxidation‐sensitive 54 proteins from 61 peptides containing cysteine oxidation, and analyzed comparative expression pattern in healthy RBCs of Prdx II+/+ mice, healthy RBCs of Prdx II−/− mice, and abnormal RBCs of Prdx II−/− mice. These proteins belonged to cellular functions related with RBC lifespan maintain, such as cytoskeleton, stress‐induced proteins, metabolic enzymes, signal transduction, and transporters. Furthermore, protein networks among identified oxidation‐sensitive proteins were analyzed to associate with various diseases. Consequently, we expected that RBC proteome might provide clues to understand redox‐imbalanced diseases.

Gene Expression Profiling Related to the Enhanced Erythropoiesis in Mouse Bone Marrow Cells

Peroxiredoxin II knockout (Prdx II−/−) mice had a spontaneous phenotype of hemolytic anemia. In this study, we found that Ter‐119+CD71+ cells increased in Prdx II−/− mice bone marrow (BM) at 8 weeks of age. We examined the differential expression profiles to bone marrow cells (BMCs) between Prdx II+/+ and Prdx II−/− mice using a cDNA microarray. We identified the 136 candidates were differentially expressed a greater twofold increase or decrease than EPO receptor. In this study, we focused on the up‐regulated NBPs during erythropoietic differentiation. According to cDNA microarray results, six NBPs except zfp‐127 were up‐regulated during erythropoiesis in Prdx II−/− mice. Among the six candidates, eIF3‐p44, hnRNPH1, G3bp, and Zfpm‐1 were dramatically increased at day 7 of the in vitro erythropoietic differentiation of human CD34+ cells. However, DJ‐1 and Rbm3 were slightly increased only at day 12. Our results suggest that up‐regulated NBPs might be involved during erythropoietic differentiation. J. Cell. Biochem. 104: 295–303, 2008.