• Korean Journal of Microbiology
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• v.45 no.4
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• pp.291-299
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• 2009

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

• Nuclear Engineering and Technology
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• v.39 no.2
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• pp.103-110
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• 2007

The High Temperature Gas-cooled Reactor (HTGR) possesses inherent safety features and is recognized as a representative advanced nuclear system for the future. Based on the success of the HTR-10, the long-time operation test and safety demonstration tests were carried out. The long-time operation test verifies that the operation procedure and control method are appropriate for the HTR-10 and the safety demonstration test shows that the HTR-10 possesses inherent safety features with a great margin. Meanwhile, two new projects have been recently launched to further develop HTGR technology. One is a prototype modular plant, denoted as HTR-PM, to demonstrate the commercial capability of the HTGR power plant. The HTR-PM is designed as $2{\times}250$ MWt, pebble bed core with a steam turbine generator that serves as an energy conversion system. The other is a gas turbine generator system coupled with the HTR-10, denoted as HTR-10GT, built to demonstrate the feasibility of the HTGR gas turbine technology. The gas turbine generator system is designed in a single shaft configuration supported by active magnetic bearings (AMB). The HTR-10GT project is now in the stage of engineering design and component fabrication. R&D on the helium turbocompressor, a key component, and the key technology of AMB are in progress.

• Journal of Life Science
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• v.18 no.4
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• pp.522-529
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• 2008

High-temperature requirement A2(HtrA2) has been known as a human homologue of bacterial HtrA that has a molecular chaperone function. HtrA2 is mitochondrial serine protease that plays a significant role in regulating the apoptosis; however, the physiological function of HtrA2 still remains elusive. To establish experimental system for the investigation of new insights into the function of HtrA2 in mammalian cells, we first obtained $HtrA2^{+/+}$ and $HtrA2^{-/-}$ MEF cells lines and identified those cells based on the expression pattern and subcellular localization of HtrA2, using immunoblot and biochemical assays. Additionally, we observed that the morphological characteristics of $HtrA2^{-/-}$ MEF cells are different form those of $HtrA2^{+/+}$ MEF cells, showing a rounded shape instead of a typical fibroblast-like shape. Growth rate of $HtrA2^{-/-}$ MEF cells was also 1.4-fold higher than that of $HtrA2^{+/+}$ MEF cells at 36 hours. Furthermore, we verified both MEF cell lines induced caspsase-dependent cell death in response to apoptotic stimuli such as heat shock, staurosporine, and rotenone. The relationship between HtrA2 and heat shock-induced cell death is the first demonstration of the research field of HtrA2. Our study suggests that those MEF cell lines are suitable reagents to further investigate the molecular mechanism by which HtrA2 regulates the balance between cell death and survival.

• Journal of Life Science
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• v.16 no.4
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• pp.571-578
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• 2006

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.

• BMB Reports
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• v.38 no.3
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• pp.266-274
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• 2005

High temperature requirement A (HtrA) and its homologues constitute the HtrA familiy proteins, a group of heat shock-induced serine proteases. Bacterial HtrA proteins perform crucial functions with regard to protein quality control in the periplasmic space, functioning as both molecular chaperones and proteases. In contrast to other bacterial quality control proteins, including ClpXP, ClpAP, and HslUV, HtrA proteins contain no regulatory components or ATP binding domains. Thus, they are commonly referred to as ATP-independent chaperone proteases. Whereas the function of ATP-dependent chaperone-proteases is regulated by ATP hydrolysis, HtrA exhibits a PDZ domain and a temperature-dependent switch mechanism, which effects the change in its function from molecular chaperone to protease. This mechanism is also related to substrate recognition and the fine control of its function. Structural and biochemical analyses of the three HtrA proteins, DegP, DegQ, and DegS, have provided us with clues as to the functional regulation of HtrA proteins, as well as their roles in protein quality control at atomic scales. The objective of this brief review is to discuss some of the recent studies which have been conducted regarding the structure and function of these HtrA proteins, and to compare their roles in the context of protein quality control.

• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1133-1140
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• 2006

Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low $460{\mu}g$ for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was $42^{\circ}C$ instead of $37^{\circ}C$. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of $5{\mu}M\;of\;{\beta}-casein$ during 3 hr incubation at $37^{\circ}C$. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.

• Journal of Life Science
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• v.16 no.5
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• pp.716-722
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• 2006

Superoxide dismutase (SOD) is physiologically important in regulating cellular homeostasis and apoptotic cell death, and its mutations are the cause of familial amyotrophic lateral sclerosis (FALS). Mitochondrial serine protease HtrA2 has a pro-apoptotic function and has known to be associated with neurodegenerative disorders. To investigate the relationship between genes associated with apoptotic cell death, such as HtrA2 and SOD1, we utilized the pGEX expression system to develop a simple and rapid method for purifying wild-type and ALS-associated mutant SOD1 proteins in a suitable form for biochemical studies. We purified SOD1 and SOD1 (G93A) proteins to approximately 90% purity with relatively high yields (3 mg per liter of culture). Consistent with the result in mammalian cells, SOD1 (G93A) was more insoluble than wild-type SOD1 in E. coli, indicating that research on the aggregate formation of SOD1 may be possible using this pGEX expression system in E. coli. We investigated the HtrA2 serine protease activity on SOD1 to assess the relationship between two proteins. Not only wild-type SOD1 but also ALS-associated mutant SOD1 (G93A) were cleaved by HtrA2, resulting in the production of the 19 kDa and 21 kDa fragments that were specific for anti-SOD1 antibody. Using protein gel electrophoresis and immunoblot assay, we compared the relative molecular masses of thrombin-cleaved GST-SOD1 and HtrA2-cleaved SOD1 fragments and can predict that the HtrA2-cleavage sites within SOD1 are the peptide bonds between leucine 9-lysine 10 (L9-K10) and glutamine 23-lysine 24 (Q23-K24). Our study indicates that SOD1 is one of the substrate for HtrA2, suggesting that both HtrA2 and SOD1 may be important for modulating the HtrA2-SOD1-mediated apopotic cell death that is associated with the pathogenesis of neurodegenerative disorder.

• Biomolecules & Therapeutics
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• v.25 no.5
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• pp.497-503
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• 2017

Recent reports claimed that glucosylsphingosine (GS) is highly accumulated and specifically evoking itch-scratch responses in the skins of atopic dermatitis (AD) patients. However, it was unclear how GS can trigger itch-scratch responses, since there were no known molecular singling pathways revealed yet. In the present study, it was verified for the first time that GS can activate mouse serotonin receptor 2a (mHtr2a) and 2b (mHtr2b), but not 2c (mHtr2c) that are expressed in HEK293T cells. Specifically, effects of GS on all mouse serotonin receptor 2 subfamily were evaluated by calcium imaging techniques. The GS-induced intracellular calcium increase was dose-dependent, and antagonists such as ketanserin (Htr2a antagonist) and RS-127445 (Htr2b antagonist) significantly blocked the GS-induced responses. Moreover, the proposed GS-induced responses appear to be mediated by phospholipase C (PLC), since pretreatment of a PLC inhibitor U-73122 abolished the GS-induced responses. Additionally, the GS-induced calcium influx is probably mediated by endogenous TRPC ion channels in HEK293T cells, since pretreatment of SKF-96365, an inhibitor for TRPC, significantly suppressed GS-induced response. In conclusion, the present study revealed for the first time that GS can stimulate mHtr2a and mHtr2b to induce calcium influx, by utilizing PLC-dependent pathway afterwards. Considering that GS is regarded as a pruritogen in AD, the present study implicates a novel GS-induced itch signaling pathway.

• Biomolecules & Therapeutics
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• v.28 no.1
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• pp.83-91
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• 2020

Tryptamines are monoamine alkaloids with hallucinogenic properties and are widely abused worldwide. To hasten the regulations of novel substances and predict their abuse potential, we designed and synthesized four novel synthetic tryptamine analogs: Pyrrolidino tryptamine hydrochloride (PYT HCl), Piperidino tryptamine hydrochloride (PIT HCl), N,N-dibutyl tryptamine hydrochloride (DBT HCl), and 2-Methyl tryptamine hydrochloride (2-MT HCl). Then, we evaluated their rewarding and reinforcing effects using the conditioned place preference (CPP) and self-administration (SA) paradigms. We conducted an open field test (OFT) to determine the effects of the novel compounds on locomotor activity. A head-twitch response (HTR) was also performed to characterize their hallucinogenic properties. Lastly, we examined the effects of the compounds on 5-HTR1a and 5-HTR2a in the prefrontal cortex using a quantitative real-time polymerase chain reaction (qRT-PCR) assay. None of the compounds induced CPP in mice or initiated SA in rats. PYT HCl and PIT HCl reduced the locomotor activity and elevated the 5-HTR1a mRNA levels in mice. Acute and repeated treatment with the novel tryptamines elicited HTR in mice. Furthermore, a drug challenge involving a 7-day abstinence from drug use produced higher HTR than acute and repeated treatments. Both the acute treatment and drug challenge increased the 5-HTR2a mRNA levels. Ketanserin blocked the induced HTR. Taken together, the findings suggest that PYT HCl, PIT HCl, DBT HCl, and 2-MT HCl produce hallucinogenic effects via 5-HTR2a stimulation, but may have low abuse potential.

• Nuclear Engineering and Technology
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• v.41 no.3
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• pp.307-318
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• 2009

KAERI (Korea Atomic Energy Research Institute) has developed the GAMMA+ code for a thermo-fluid and safety analysis of a VHTR (Very High Temperature Gas-Cooled Reactor). A key safety issue of the VHTR design is to demonstrate its inherent safety features for an automatic reactor power trip and power stabilization during an anticipated transient without scram (ATWS) accident such as a loss of forced cooling by a trip of the helium circulator (LOFC) or a reactivity insertion by a control rod withdrawal (CRW). This paper intends to show the ATWS assessment capability of the GAMMA+ code which can simulate the reactor power response by solving the point-kinetic equations with six-group delayed neutrons, by considering the reactivity changes due to the effects of a core temperature variation, xenon transients, and reactivity insertions. The present benchmark calculations are performed by using the safety demonstration experiments of the 10 MW high temperature gas cooled-test module (HTR-10) in China. The calculation results of the power response transients and the solid core temperature behavior are compared with the experimental data of a LOFC ATWS test and two CRW ATWS tests by using a 1mk-control rod and a 5mk-control rod, respectively. The GAMMA+ code predicts the power response transients very well for the LOFC and CRW ATWS tests in HTR-10.