Tat-DJ-1 inhibits oxidative stress-mediated RINm5F cell death through suppression of NF-κB and MAPK activation.

Oxidative stress is highly involved in the development of diabetes mellitus by destruction of pancreatic ß-cells. DJ-1 is an antioxidant protein and DJ-1 expression levels are known to be reduced in diabetes mellitus. Thus, we examined the effects of DJ-1 protein against oxidative stress-induced pancreatic ß-cell (RINm5F) death using cell permeable wild-type and mutant-type (C106A) Tat-DJ-1 proteins, which both efficiently transduced into RINm5F cells. Intracellular stability of wild-type Tat-DJ-1 persisted two times longer than C106A Tat-DJ-1. Wild-type Tat-DJ-1 protein markedly protected cells from hydrogen peroxide-induced toxicities such as cell death, reactive oxygen species generation, and DNA fragmentation. Further, wild-type Tat-DJ-1 protein significantly inhibited hydrogen peroxide-induced activation of mitogen-activated protein kinases and NF-κB signaling. On the other hand, C106A Tat-DJ-1 protein did not show the same protective effects. These results indicate that wild-type Tat-DJ-1 inhibits oxidative stress-induced cellular toxicity and activation of mitogen-activated protein kinases and NF-κB signals in RINm5F cells. These results suggest that wild-type Tat-DJ-1 protein may be a potential therapeutic agent against diabetes mellitus or toward the prevention of pancreatic ß-cell destruction.

Tat-CBR1 inhibits inflammatory responses through the suppressions of NF-κB and MAPK activation in macrophages and TPA-induced ear edema in mice.

Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E2 (PGE2) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB) and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases.

Introducing transglycosylation activity in Bacillus licheniformis α-amylase by replacement of His235 with Glu.

To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM(-1)s(-1) for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM(-1)s(-1) for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235-located on a wide open groove near subsite +1-is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.

Tat-Thioredoxin-like protein 1 attenuates ischemic brain injury by regulation of MAPKs and apoptosis signaling.

Thioredoxin-like protein 1 (TXNL1), one of the thioredoxin superfamily known as redox-regulator, plays an essential in maintaining cell survival via various antioxidant and anti-apoptotic mechanisms. It is well known that relationship between ischemia and oxidative stress, however, the role of TXNL1 protein in ischemic damage has not been fully investigated. In the present study, we aimed to determine the protective role of TXNL1 against on ischemic injury in vitro and in vivo using cell permeable Tat-TXNL1 fusion protein. Transduced Tat-TXNL1 inhibited ROS production and cell death in H2O2-exposed hippocampal neuronal (HT-22) cells and modulated MAPKs and Akt activation, and pro-apoptotic protein expression levels in the cells. In an ischemia animal model, Tat-TXNL1 markedly decreased hippocampal neuronal cell death and the activation of astrocytes and microglia. These findings indicate that cell permeable Tat-TXNL1 protects against oxidative stress in vitro and in vivo ischemic animal model. Therefore, we suggest Tat-TXNL1 can be a potential therapeutic protein for ischemic injury. [BMB Reports 2023; 56(4): 234-239].

Utilization of an Intracellular Calcium Mobilization Assay for the Screening of Transduced FK506-Binding Proteins.

FK506-binding proteins (FKBPs) belong to the immunophilin family and are linked to various disease states, including the inflammatory response. The inhibition of cytokine and chemokine expression in addition to positive effects of FKBPs on corneal inflammation in animal models suggests that they may be used for ophthalmic delivery in the treatment of dry eye disease. To pass the effective barriers protecting eye tissues, testing the transduction domains of FKBPs is essential. However, monitoring their transduction efficiencies is not a simple task. The quantitative measurement of FKBP interactions was performed using a cell model with a specific G protein-coupled receptor, as FKBPs had been known to act at the inositol 1,4,5-trisphosphate receptor (IP3R) leading to the inhibition of intracellular calcium mobilization. Because of its luminescence amplitude and stability, human urotensin II receptor was expressed in aequorin parental cells to measure the action of selected FKBPs. This luminescence-based functional assay platform exhibited a high signal-to-background ratio of more than 100 and a Z' factor at 0.6204. As expected, changes in the sequence of the transduction domain affected the function of the FKBPs. The intracellular calcium mobilization assay with selected FKBPs represented a robust and reliable platform to screen initial candidates. Although the precise nature of the control that FKBPs exert on the IP3R is uncertain, this approach can be used to develop innovative anti-inflammatory treatments for dry eye disease by optimizing protein transduction domain sequences.

Transduced Tat-PRAS40 prevents dopaminergic neuronal cell death through ROS inhibition and interaction with 14-3-3σ protein.

Proline rich Akt substrate (PRAS40) is a component of mammalian target of rapamycin complex 1 (mTORC1) and activated mTORC1 plays important roles for cellular survival in response to oxidative stress. However, the roles of PRAS40 in dopaminergic neuronal cell death have not yet been examined. Here, we examined the roles of Tat-PRAS40 in MPP+- and MPTP-induced dopaminergic neuronal cell death. Our results showed that Tat-PRAS40 effectively transduced into SH-SY5Y cells and inhibited DNA damage, ROS generation, and apoptotic signaling in MPP+-induced SH-SY5Y cells. Further, these protective mechanisms of Tat-PRAS40 protein display through phosphorylation of Tat-PRAS40, Akt and direct interaction with 14-3-3σ protein, but not via the mTOR-dependent signaling pathway. In a Parkinson's disease animal model, Tat-PRAS40 transduced into dopaminergic neurons in mouse brain and significantly protected against dopaminergic cell death by phosphorylation of Tat-PRAS40, Akt and interaction with 14-3-3σ protein. In this study, we demonstrated for the first time that Tat-PRAS40 directly protects against dopaminergic neuronal cell death. These results indicate that Tat-PRAS40 may provide a useful therapeutic agent against oxidative stress-induced dopaminergic neuronal cell death, which causes diseases such as PD.

Tat-thioredoxin 1 reduces inflammation by inhibiting pro-inflammatory cytokines and modulating MAPK signaling.

Thioredoxin 1 (Trx1) serves a central role in redox homeostasis. It is involved in numerous other processes, including oxidative stress and apoptosis. However, to the best of our knowledge, the role of Trx1 in inflammation remains to be explored. The present study investigated the function and mechanism of cell permeable fused Tat-Trx1 protein in macrophages and a mouse model. Transduction levels of Tat-Trx1 were determined via western blotting. Cellular distribution of transduced Tat-Trx1 was determined by fluorescence microscopy. 2',7'-Dichlorofluorescein diacetate and TUNEL staining were performed to determine the production of reactive oxygen species and DNA fragmentation. Protein and gene expression were measured by western blotting and reverse transcription-quantitative PCR (RT-qPCR), respectively. Effects of skin inflammation were determined using hematoxylin and eosin staining, changes in ear weight and ear thickness, and RT-qPCR in ear edema animal models. Transduced Tat-Trx1 inhibited lipopolysaccharide-induced cytotoxicity and activation of NF-κB, MAPK and Akt. Additionally, Tat-Trx1 markedly reduced the production of inducible nitric oxide synthase, cyclooxygenase-2, IL-1ß, IL-6 and TNF-α in macrophages. In a 12-O-tetradecanoylphorbol-13-acetate-induced mouse model, Tat-Trx1 reduced inflammatory damage by inhibiting inflammatory mediator and cytokine production. Collectively, these results demonstrated that Tat-Trx1 could exert anti-inflammatory effects by inhibiting the production of pro-inflammatory mediators and cytokines and by modulating MAPK signaling. Therefore, Tat-Trx1 may be a useful therapeutic agent for diseases induced by inflammatory damage.

PEP-1-GLRX1 protein exhibits anti-inflammatory effects by inhibiting the activation of MAPK and NF-κB pathways in Raw 264.7 cells.

Glutaredoxin 1 (GLRX1) has been recognized as an important regulator of redox signaling. Although GLRX1 plays an essential role in cell survival as an antioxidant protein, the function of GLRX1 protein in inflammatory response is still under investigation. Therefore, we wanted to know whether transduced PEP-1-GLRX1 protein inhibits lipopolysaccharide (LPS)- and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced inflammation. In LPS-exposed Raw 264.7 cells, PEP-1-GLRX1 inhibited cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), activation of mitogen activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB) expression levels. In a TPA-induced mouse-ear edema model, topically applied PEP-1-GLRX1 transduced into ear tissues and significantly ameliorated ear edema. Our data reveal that PEP-1-GLRX1 attenuates inflammation in vitro and in vivo, suggesting that PEP-1-GLRX1 may be a potential therapeutic protein for inflammatory diseases. [BMB Reports 2020; 53(2): 106-111].

239+240Pu concentration and isotope ratio (240Pu/239Pu) in aerosols during high dust (Yellow Sand) period, Korea.

Concentration and isotope ratio of Pu were analyzed for aerosols collected at Anmyeondo located in the western coast of Korea using multiple collector inductively coupled plasma mass spectrometer equipped with desolvated micro-concentric nebulizer. Aerosols were collected from June 2001 to April 2002 using high volume air sampler. The samples consist of high dust samples (Yellow Sand), and also low dust samples; maximum Al concentration was 74.2 microg/m(3) and minimum was 0.17 microg/m(3). Pu was concentrated using 0.1 ml TEVA resin columns after conc. HNO(3) extraction. Isotope dilution using (242)Pu spike and mass bias correction using (233)U and (236)U mixed solution enabled the quantification of Pu and measurement of isotope ratio simultaneously. The contribution of (238)U from both spikes and samples was minimized by careful chemical separation and optimization of spike concentration. The (238)U(1)H and tail contribution on (239)Pu peak were about 0.75 x 10(-5) and 1 x 10(-5) of (238)U intensity, respectively, and they were corrected from (239)Pu using externally determined ((238)U(1)H + tailing)/(238)U ratio and (238)U measurement during acquisition. The detection limits of this analytical procedure were 0.61 fg/ml and 0.56 fg/ml for (239)Pu and (240)Pu, respectively (4 nBq/m(3) and 12 nBq/m(3) for (239)Pu and (240)Pu, respectively). The precision of isotope ratio measurement was better than 2% for larger quantity than 20 fg of (239)Pu. In spring, maximum concentration of 0.580 microBq/m(3) for (239)Pu and 0.404 microBq/m(3) for (240)Pu was observed when Al concentration was maximum, so called as Yellow Sand event. Pu concentrations in aerosols are well correlated with Al, a tracer of soil dust. The ratios of Pu/Al were 0.0082 (microBq/microg) and 0.0055 (microBq/microg) for (239)Pu/Al and (240)Pu/Al, respectively. Isotope ratios of Pu ((240)Pu/(239)Pu) in Yellow Sand samples show 0.191+/-0.014 close to those of global fallout. These facts indicate that Yellow Sand plays an important role in the behavior of Pu in the environment like other concomitant metals such as Al, Fe etc.

Transduced Tat-DJ-1 protein inhibits cytokines-induced pancreatic RINm5F cell death.

Loss of pancreatic ß-cells by oxidative stress or cytokines is associated with diabetes mellitus (DM). DJ-1 is known to as a multifunctional protein, which plays an important role in cell survival. We prepared cell permeable wild type (WT) and mutant type (M26I) Tat-DJ-1 proteins to investigate the effects of DJ-1 against combined cytokines (IL-1ß, IFN-γ and TNF-α)-induced RINm5F cell death. Both Tat-DJ-1 proteins were transduced into RINm5F cells. WT Tat-DJ-1 proteins significantly protected against cell death from cytokines by reducing intracellular toxicities. Also, WT Tat-DJ-1 proteins markedly regulated cytokines-induced pro- and anti-apoptosis proteins. However, M26I Tat-DJ-1 protein showed relatively low protective effects, as compared to WT Tat-DJ-1 protein. Our experiments demonstrated that WT Tat-DJ-1 protein protects against cytokine-induced RINm5F cell death by suppressing intracellular toxicities and regulating apoptosisrelated protein expression. Thus, WT Tat-DJ-1 protein could potentially serve as a therapeutic agent for DM and cytokine related diseases. [BMB Reports 2016; 49(5): 297-302].

Tat-ATOX1 inhibits streptozotocin-induced cell death in pancreatic RINm5F cells and attenuates diabetes in a mouse model.

Antioxidant 1 (ATOX1) functions as an antioxidant against hydrogen peroxide and superoxide, and therefore may play a significant role in many human diseases, including diabetes mellitus (DM). In the present study, we examined the protective effects of Tat-ATOX1 protein on streptozotocin (STZ)-exposed pancreatic insulinoma cells (RINm5F) and in a mouse model of STZ-induced diabetes using western blot analysis, immunofluorescence staining and MTT assay, as well as histological and biochemical analysis. Purified Tat-ATOX1 protein was efficiently transduced into RINm5F cells in a dose- and time-dependent manner. Additionally, Tat-ATOX1 protein markedly inhibited reactive oxygen species (ROS) production, DNA damage and the activation of Akt and mitogen activated protein kinases (MAPKs) in STZ-exposed RINm5F cells. In addition, Tat-ATOX1 protein transduced into mice pancreatic tissues and significantly decreased blood glucose and hemoglobin A1c (HbA1c) levels as well as the body weight changes in a model of STZ-induced diabetes. These results indicate that transduced Tat-ATOX1 protein protects pancreatic ß-cells by inhibiting STZ-induced cellular toxicity in vitro and in vivo. Based on these findings, we suggest that Tat-ATOX1 protein has potential applications as a therapeutic agent for oxidative stress-induced diseases including DM.

Tat-NOL3 protects against hippocampal neuronal cell death induced by oxidative stress through the regulation of apoptotic pathways.

Oxidative stress-induced apoptosis is associated with neuronal cell death and ischemia. The NOL3 [nucleolar protein 3 (apoptosis repressor with CARD domain)] protein protects against oxidative stress-induced cell death. However, the protective mechanism responsible for this effect as well as the effects of NOL3 against oxidative stress in ischemia remain unclear. Thus, we examined the protective effects of NOL3 protein on hydrogen peroxide (H2O2)-induced oxidative stress and the mechanism responsible for these effects in hippocampal neuronal HT22 cells and in an animal model of forebrain ischemia using Tat-fused NOL3 protein (Tat-NOL3). Purified Tat-NOL3 protein transduced into the H2O2-exposed HT22 cells and inhibited the production of reactive oxygen species (ROS), DNA fragmentation and reduced mitochondrial membrane potential (ΔΨm). In addition, Tat-NOL3 prevented neuronal cell death through the regulation of apoptotic signaling pathways including Bax, Bcl-2, caspase-2, -3 and -8, PARP and p53. In addition, Tat-NOL3 protein transduced into the animal brains and significantly protected against neuronal cell death in the CA1 region of the hippocampus by regulating the activation of microglia and astrocytes. Taken together, these findings demonstrate that Tat-NOL3 protein protects against oxidative stress-induced neuronal cell death by regulating oxidative stress and by acting as an anti-apoptotic protein. Thus, we suggest that Tat-NOL3 represents a potential therapeutic agent for protection against ischemic brain injury.

The effects of PEP-1-FK506BP on dry eye disease in a rat model.

As FK506 binding proteins (FK506BPs) are known to play an important role in the regulation of a variety of biological processes related to cell survival, this study was designed to examined the protective effects of FK506 binding protein 12 (FK506BP) on low humidity air flow induced dry eye in a rat model using transduced PEP-1-FK506BP. After the topical application of PEP-1-FK506BP, tear volumes were markedly increased and significant prevention of cornea damage was observed compared with dry eye rats. Further, immunohistochemical analysis demonstrated that PEP-1-FK506BP markedly prevented damage to the cornea, the bulbar conjunctiva, and the palpebral conjunctiva epithelial lining compared with dry eye rats. In addition, caspase-3 and PARP expression levels were found to be decreased. These results demonstrated that topical application of PEP-1-FK506BP significantly ameliorates dry eye injury in an animal model. Thus, we suggest that PEP-1-FK506BP can be developed as a new ophthalmic drop to treat dry eye diseases.

Effects of low doses of Tat-PIM2 protein against hippocampal neuronal cell survival.

Oxidative stress is considered a major factor in various neuronal diseases including ischemia-reperfusion injury. Proviral Integration Moloney 2 (PIM2) proteins, one of the families of PIM kinases, play crucial roles in cell survival. However, the functions of PIM2 protein against ischemia are not understood. Therefore, the protective effects of PIM2 against oxidative stress-induced hippocampal HT22 cell death and brain ischemic injury were evaluated using Tat-PIM2, a cell permeable fusion protein. Tat-PIM2 protein transduced into hippocampal HT22 cells. Low doses of transduced Tat-PIM2 protein protected against oxidative stress-induced cell death including DNA damage and markedly inhibited the activation of mitogen activated protein kinase (MAPKs), NF-κB and the expression levels of Bax protein. Furthermore, Tat-PIM2 protein transduced into the CA1 region of the hippocampus and significantly prevented neuronal cell death in an ischemic insult animal model. These results indicated that low doses of Tat-PIM2 protein protects against oxidative stress-induced neuronal cell death, suggesting low doses of Tat-PIM2 protein provides a potential therapeutic agent against oxidative stress-induced neuronal diseases including ischemia.

Down-regulation of MAPK/NF-κB signaling underlies anti-inflammatory response induced by transduced PEP-1-Prx2 proteins in LPS-induced Raw 264.7 and TPA-induced mouse ear edema model.

Excessive reactive oxygen species (ROS) production plays a crucial role in causing various diseases, including inflammatory disorders. The activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling is implicated in stimulating inflammatory response and cytokines. Peroxiredoxin 2 (Prx2) is a 2-cysteine (Cys) peroxiredoxin capable of removing endogenous hydrogen peroxide (H2O2). PEP-1 peptide, a protein transduction domain, consists of three domains which are used to transduce exogenous therapeutic proteins into cells. The correlation between effectively transduced PEP-1-Prx2 and ROS-mediated inflammatory response is not clear. In the present study, we investigated the protective effects of cell permeable PEP-1-Prx2 on oxidative stress-induced inflammatory activity in Raw 264.7 cells and in a mouse ear edema model after exposure to lipopolysaccharides (LPS) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Transduced PEP-1-Prx2 suppressed intracellular ROS accumulation and inhibited the activity of MAPKs and NF-κB signaling that led to the suppression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and cytokines in LPS-induced Raw 264.7 cells and TPA-induced mouse ear edema model. Given these results, we propose that PEP-1-Prx2 has therapeutic potential in the prevention of inflammatory disorders.