Kyu Hyung Han

Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells

Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been considered to have a beneficial effect against various diseases mediated by reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against the oxidative stresses, the lack of their transduction ability into cells resulted in limited ability to detoxify intracellular ROS. To render the catalase enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant catalase proteins were generated. A human liver catalase gene was cloned and fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) and arginine-rich peptides (RRRRRRRRR) in a bacterial expression vector to produce genetic in-frame Tat-CAT and 9Arg-CAT fusion proteins, respectively. The expressed and purified fusion proteins can be transduced into mammalian cells (HeLa and PC12 cells) in a time- and dose-dependent manner when added exogenously in culture medium, and transduced fusion proteins were enzymatically active and stable for 60 h. When exposed to H(2)O(2), the viability of HeLa cells transduced with Tat-CAT or 9Arg-CAT fusion proteins was significantly increased. In combination with transduced SOD, transduced catalase also resulted in a cooperative increase in cell viability when the cells were treated with paraquat, an intracellular antioxide anion generator. We then evaluated the ability of the catalase fusion proteins to transduce into animal skin. This analysis showed that Tat-CAT and 9Arg-CAT fusion proteins efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin, as judged by immunohistochemistry and specific enzyme activities. These results suggest that Tat-CAT and 9Arg-CAT fusion proteins can be used in protein therapy for various disorders related to this antioxidant enzyme.

PEP-1–SIRT2 inhibits inflammatory response and oxidative stress-induced cell death via expression of antioxidant enzymes in murine macrophages

Sirtuin 2 (SIRT2), a member of the sirtuin family of proteins, plays an important role in cell survival. However, the biological function of SIRT2 protein is unclear with respect to inflammation and oxidative stress. In this study, we examined the protective effects of SIRT2 on inflammation and oxidative stress-induced cell damage using a cell permeative PEP-1-SIRT2 protein. Purified PEP-1-SIRT2 was transduced into RAW 264.7 cells in a time- and dose-dependent manner and protected against lipopolysaccharide- and hydrogen peroxide (H₂O₂)-induced cell death and cytotoxicity. Also, transduced PEP-1-SIRT2 significantly inhibited the expression of cytokines as well as the activation of NF-κB and mitogen-activated protein kinases (MAPKs). In addition, PEP-1-SIRT2 decreased cellular levels of reactive oxygen species (ROS) and of cleaved caspase-3, whereas it elevated the expression of antioxidant enzymes such as MnSOD, catalase, and glutathione peroxidase. Furthermore, topical application of PEP-1-SIRT2 to 12-O-tetradecanoylphorbol 13-acetate-treated mouse ears markedly inhibited expression levels of COX-2 and proinflammatory cytokines as well as the activation of NF-κB and MAPKs. These results demonstrate that PEP-1-SIRT2 inhibits inflammation and oxidative stress by reducing the levels of expression of cytokines and ROS, suggesting that PEP-1-SIRT2 may be a potential therapeutic agent for various disorders related to ROS, including skin inflammation.

Transduced PEP-1-ribosomal protein S3 (rpS3) ameliorates 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice

This study investigated the preventive effect of ribosomal protein S3 (rpS3) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. A cell permeable expression vector PEP-1-rpS3 was constructed. Topical application of the vector markedly inhibited TPA-induced expression levels of cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines. Application of PEP-1-rpS3 also resulted in a significant reduction in the activation of nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) in TPA-treated ears. These results indicate that PEP-1-rpS3 inhibits inflammatory response cytokines and enzymes by blocking NF-kB and MAPK, prompting the suggestion that PEP-1-rpS3 can be used as a therapeutic agent against skin inflammation.

Protective effects of PEP-1-Catalase on stress-induced cellular toxicity and MPTP-induced Parkinson's disease.

Parkinson’s disease (PD) is a neurodegenerative disability caused by a decrease of dopaminergic neurons in the substantia nigra (SN). Although the etiology of PD is not clear, oxidative stress is believed to lead to PD. Catalase is antioxidant enzyme which plays an active role in cells as a reactive oxygen species (ROS) scavenger. Thus, we investigated whether PEP-1-Catalase protects against 1-methyl-4-phenylpyridinium (MPP+) induced SH-SY5Y neuronal cell death and in a 1-methyl-4-phenyl-1,2,3,6-trtrahydropyridine (MPTP) induced PD animal model. PEP-1-Catalase transduced into SH-SY5Y cells significantly protecting them against MPP+-induced death by decreasing ROS and regulating cellular survival signals including Akt, Bax, Bcl-2, and p38. Immunohistochemical analysis showed that transduced PEP-1-Catalase markedly protected against neuronal cell death in the SN in the PD animal model. Our results indicate that PEP-1-Catalase may have potential as a therapeutic agent for PD and other oxidative stress related diseases. [BMB Reports 2015; 48(7): 395-400]

PEP-1–metallothionein-III protein ameliorates the oxidative stress-induced neuronal cell death and brain ischemic insults

BACKGROUND Oxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult. METHODS A human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1-MT-III protein. PEP-1-MT-III protein was purified using affinity chromatograph. Transduced PEP-1-MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry. RESULTS Purified PEP-1-MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1-MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1-MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1-MT-III protein protects against oxidative stress induced cell death in vitro and in vivo. GENERAL SIGNIFICANCE Transduced PEP-1-MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1-MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimers disease, and Parkinsons disease.

Antitumor activity of methyl gallate by inhibition of focal adhesion formation and Akt phosphorylation in glioma cells

BACKGROUND Methyl gallate (MG) possesses a wide range of biological properties that include anti-oxidant, anti-inflammatory, and anti-microbial activities. However, its anti-tumor activity has not been extensively examined in cancer cells. Thus, we examined the effect of MG in both glutamate-induced rat C6 and human U373 glioma cell proliferation and migration. METHODS MG was isolated from the stem bark of Acer barbinerve. Cell viability and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch wound-healing assay, respectively. Focal adhesion formation was detected with immunofluorescence. RESULTS Treatment of C6 and U373 glioma cells with MG significantly reduced cell viability, migration, and Akt phosphorylation level. Glutamate stimulation markedly increased the level of ERK1/2 phosphorylation. However, cells treated with MG displayed decreased ERK1/2 phosphorylation. Inhibition of ERK1/2 by MG or MEK1/2 inhibitor significantly inhibited paxillin phosphorylation at Ser(83) and focal adhesion turn-over produced inefficient glioma cell migration. In addition, activation of Akt and ERK1/2 upon glutamate stimulation was independently regulated by Ca(2+) and protein kinase C activity, respectively, via the α-amino-3-hydroxy-5-methy-4-isoxazolepropionate acid glutamate receptor and metabotropic glutamate receptor. GENERAL SIGNIFICANCE Our results clearly indicate that MG has a strong anti-tumor effect through the down-regulation of the Akt and ERK1/2 signaling pathways. Thus, methyl gallate is a potent anti-tumor and novel therapeutic agent for glioma.

Transduced Tat-DJ-1 protein protects against oxidative stress-induced SH-SY5Y cell death and Parkinson disease in a mouse model

Parkinson’s disease (PD) is a well known neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compact (SN). Although the exact mechanism remains unclear, oxidative stress plays a critical role in the pathogenesis of PD. DJ-1 is a multifunctional protein, a potent antioxidant and chaperone, the loss of function of which is linked to the autosomal recessive early onset of PD. Therefore, we investigated the protective effects of DJ-1 protein against SH-SY5Y cells and in a PD mouse model using a cell permeable Tat-DJ-1 protein. Tat-DJ-1 protein rapidly transduced into the cells and showed a protective effect on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death by reducing the reactive oxygen species (ROS). In addition, we found that Tat-DJ-1 protein protects against dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-induced PD mouse models. These results suggest that Tat-DJ-1 protein provides a potential therapeutic strategy for against ROS related human diseases including PD.

Transduced Tat-glyoxalase protein attenuates streptozotocin-induced diabetes in a mouse model.

Diabetes mellitus (DM) is characterized by hyperglycemia. Glyoxalase 1 (GLO) has considerable potential as a possible therapeutic agent for DM. However, the precise action of GLO remains unclear in DM. In this study, we examined the protective effects of GLO protein in a streptozotocin (STZ)-induced diabetes animal model using cell-permeable Tat-GLO protein. Purified Tat-GLO protein was efficiently transduced into RINm5F cells in a time- and dose-dependent manner and protected cells against sodium nitroprusside (SNP)-induced cell death and DNA fragmentation. Furthermore, Tat-GLO protein significantly inhibited blood glucose levels and altered the serum biochemical parameters in STZ-induced diabetic mice. These results demonstrate that transduced Tat-GLO protein protects pancreatic cells by the inhibition of STZ-mediated toxicity. Therefore, Tat-GLO protein could be useful as a therapeutic agent against DM.

PEP-1-PON1 Protein Regulates Inflammatory Response in Raw 264.7 Macrophages and Ameliorates Inflammation in a TPA-Induced Animal Model

Paraoxonase 1 (PON1) is an antioxidant enzyme which plays a central role in various diseases. However, the mechanism and function of PON1 protein in inflammation are poorly understood. Since PON1 protein alone cannot be delivered into cells, we generated a cell permeable PEP-1-PON1 protein using protein transduction domains, and examined whether it can protect against cell death in lipopolysaccharide (LPS) or hydrogen peroxide (H2O2)-treated Raw 264.7 cells as well as mice with 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced skin inflammation. We demonstrated that PEP-1-PON1 protein transduced into Raw 264.7 cells and markedly protected against LPS or H2O2-induced cell death by inhibiting cellular reactive oxygen species (ROS) levels, the inflammatory mediator’s expression, activation of mitogen-activated protein kinases (MAPKs) and cellular apoptosis. Furthermore, topically applied PEP-1-PON1 protein ameliorates TPA-treated mice skin inflammation via a reduction of inflammatory response. Our results indicate that PEP-1-PON1 protein plays a key role in inflammation and oxidative stress in vitro and in vivo. Therefore, we suggest that PEP-1-PON1 protein may provide a potential protein therapy against oxidative stress and inflammation.

PEP-1-HO-1 prevents MPTP-induced degeneration of dopaminergic neurons in a Parkinson's disease mouse model.

Heme oxygenase-1 (HO-1) degrades heme to carbon dioxide, biliverdin, and Fe2+, which play important roles in various biochemical processes. In this study, we examined the protective function of HO-1 against oxidative stress in SH-SY5Y cells and in a Parkinson’s disease mouse model. Western blot and fluorescence microscopy analysis demonstrated that PEP-1-HO-1, fused with a PEP-1 peptide can cross the cellular membranes of human neuroblastoma SH-SY5Y cells. In addition, the transduced PEP-1-HO-1 inhibited generation of reactive oxygen species (ROS) and cell death caused by 1-methyl-4-phenylpyridinium ion (MPP+). In contrast, HO-1, which has no ability to transduce into SH-SY5Y cells, failed to reduce MPP+-induced cellular toxicity and ROS production. Furthermore, intraperitoneal injected PEP-1-HO-1 crossed the blood-brain barrier in mouse brains. In a PD mouse model, PEP-1-HO-1 significantly protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity and dopaminergic neuronal death. Therefore, PEP-1-HO-1 could be a useful agent in treating oxidative stress induced ailments including PD. [BMB Reports 2014; 47(10): 569-574]