인간 유래 ChaC 단백질의 glutathione 대사에서 구조밎 기능이 대한 연구

학위논문(박사)--서울대학교 대학원 :약학대학 약학과,2020. 2. Byung Woo Han.Glutathione (GSH) degradation plays an essential role not only in the GSH homeostasis but also in housekeeping functions, which regulate cell survival especially in cancer cells. Among human GSH degradation enzymes, cytosolic ChaC1 and ChaC2 enzymes act on GSH to form 5-L-oxoproline and Cys-Gly specifically in cytosol and share 60% sequence similarities. Here, I report the crystal structures of ChaC2 in two different conformations and compare structural features with other known γ-glutamylcyclotransferase enzymes. The unique domain-swapped loop of ChaC2 seems to function as a gate to achieve specificity for GSH binding and regulate the constant GSH degradation rate. Intensive structural and biochemical analyses of ChaC2 revealed that Glu74 and Glu83 play crucial roles in directing the assembly of the swapping dimer and in modulating the enzyme activity. Based on the docking study of GSH to ChaC2 and binding assay, I propose a substrate binding mode and catalytic mechanism. I also found that overexpression of ChaC2, but not ChaC2 E74Q or ChaC2 E83Q, significantly promoted breast cancer cell line proliferation, suggesting that the GSH degradation role of ChaC protein plays a role in breast cancer cell growth. My structural and functional analyses of ChaC2 will contribute to the development of modulators in the ChaC family, which could effectively regulate the progression of GSH degradation-related diseases and cancers.Glutathione(GSH)의 분해는 GSH의 항상성을 유지할 뿐만 아니라, 암 세포에서 세포 생존에 중요한 역할을 한다고 알려져 있다. 특히 GSH 분해를 담당하는 효소들 중 ChaC1과 ChaC2는 공통적으로 GSH를 분해하여 5-L-oxoproline 과 Cys-Gly 펩타이드를 만드는 역할을 하는데, 서로의 아미노산 서열은 60% 정도 유사하다고 알려져 있다. 본 연구에서는 ChaC2의 각각 다른 두 가지 형태의 결정 구조를 밝혔고, 이 구조들을 기존에 알려져 있던 다른 γ-glutamylcyclotransferase 계열의 효소들과 비교하였다. ChaC2 결정 구조에서 발견되는 domain-교환 loop은 GSH와의 결합 특이성을 부여하는 부분으로, GSH 분해 속도를 조절하는 데 중요한 역할을 하는 것으로 보인다. 여러 생화학적인 실험 결과와 단백질 구조 분석을 통하여 Glu74와 Glu83이 이합체의 형성과 단백질 활성의 조절에 중요한 역할을 한다는 것을 알 수 있었고, GSH와 ChaC2 사이의 docking 연구와 결합 실험을 통해 GSH와 ChaC2의 사이의 결합 방식과 ChaC2의 반응 메커니즘을 제시하였다. 뿐만 아니라, ChaC2의 과발현이 E74Q와 E83Q 변이 단백질들에 비해 유방암 세포의 생존을 증가시키는 실험 결과를 토대로 ChaC2에 의한 GSH의 분해가 유방암의 발달에 특정 역할을 할 것이라 제시할 수 있었다. 결과적으로, ChaC2의 구조와 기능에 대한 본 연구는 ChaC 계열 단백질들을 조절할 수 있는 분자의 개발에 기여하고, 더 나아가 GSH 분해와 관련된 질병과 암을 효율적으로 조절할 수 있는 치료의 기반을 제공한다.Abstract i Table of Contents ii List of Tables iv List of Figures v Abbreviations vi I. Introduction １ 1. Study Background １ 2. Purpose of Research ６ II. Materials and methods ８ 1. Bioinformatics analysis ８ 2. Cloning and protein production ８ 2.1. Cloning and protein production of ChaC2 ８ 2.2. Cloning and protein production of ChaC1 10 3. Mutagenesis 13 4. Crystallization 13 4.1. Crystallization of ChaC2 13 4.2. Crystallization of ChaC1 15 5. X-ray diffraction data collection, structure determination 18 6. Preparation of ChaC2-overexpressing cells 20 7. GSH degradation activity of ChaC enzymes by LC-MS 21 8. GSH assays 22 9. Viability assay (MTT assay) 22 10. Colony-forming assay 23 11. Immunoblotting 23 12. Docking study 23 13. Surface plasmon resonance experiment 23 14. Statistical analysis 24 15. Accession numbers 23 III. Results 25 1. ChaC2 structure was determined through rational Met 25 2. The overall structure of ChaC2 adopts the GGCT fold 31 3. Domain-swapping structure of the ChaC2 homodimer. 33 4. Single mutations of E74Q and E83Q induced conformational 36 5. Structural comparison of human ChaC2 and other GGCT 47 6. Purified ChaC proteins have GGCT activity in vitro 51 7. ChaC2 E74Q and ChaC2 E83Q mutation significantly reduced 53 8. ChaC2 overexpression promotes cell proliferation in MCF-7 55 IV. Discussion 58 1. Role of domain-swapped ChaC2 homodimer conformation 58 2. Proposed mechanism of substrate recognition and GSH... 61 3. Correlation of ChaC2, GSH degradation, and breast cancer 68 Reference 70 Abstract in Korean 73 Acknowledgements 74Docto

IMPROVEMENT OF CO2 PURIFYING SYSTEM BY PHOTOCATALYST FOR APPLICATION IN MICROALGAE CULTURE TECHNOLOGY

By reactive grinding method Vanadium-doped rutile TiO2 nanoparticle material was obtained with an average particle size of 20‐40nm, the Brunauer–Emmet–Teller (BET) specific surface area about 20 m2g−1 and it absorbed strongly in the UV region and increased at the visible wavelength of 430 – 570 nm. This study focused on the improvement of exhaust gas treatment from coal-fired flue gas of the traditional adsorption-catalysis system (Modular System for Treating Flue Gas - MSTFG) by using the V2O5/TiO2 Rutile as photocatalyst. The results showed that integrating both catalytic systems mentioned above increased the gas treatment efficiency: CO from 77 % to over 98 %, NOx from 50 % to 93 %, SO2 was absent as opposed to the input gas component. Also it showed that V2O5/TiO2 Rutile integrated with MSTFG has got high efficiency of CO treatment, also secured the high obtained CO2 concentration as a valuable carbon source for microagal mass culture as well as saving energy and simplifying devices

Reproductive Biology of Moonlight Gourami (Trichopodus microlepis) in U Minh Thuong and U Minh Ha National Parks

This study aimed to provide information about the reproductive biology of moonlight gourami (Trichopodus microlepsis) distributed in U Minh National Parks. Fish samples were collected monthly from January to October 2019 at two locations: U Minh Ha (UMH, Ca Mau, n=462) and U Minh Thuong (UMT, Kien Giang, n=635). Results showed that the maturation-stage ratios of females were similar between the two fish populations. Mature females (with stage IV-ovaries) and the increase in their gonadal somatic index (GIS) were found from June to October, indicating that this period is their spawning season. GSI reached the highest values in August with 8.04% in UMH and 6.44% in UMT. Female fish of the UMH population showed higher average fecundity (7,483±3,008 eggs/female) and larger egg diameter (762±55 µm) than those of UMT (6,898±2,952 eggs/female and 754±43 µm, respectively), but these differences were not significant (p>0.05)

Effects of maturity stages and fermentation of cocoa beans on total phenolic contents and antioxidant capacities in raw cocoa powder

Consumption of cocoa (Theobroma cacao L.) and cocoa products is associated with numerous health benefits due to their high levels of polyphenols and antioxidant capacities. In this study, changes of total phenolic contents (TPC) and antioxidant capacities (AC) of raw cocoa powder at four maturity stages, under different fermentation methods and fermentation duration of cocoa beans were investigated. The TPC and AC were measured using Folin–Ciocalteu and Trolox equivalent antioxidant capacity (TEAC)/ABTS assay, respectively. In term of maturity stages, the powder of unfermented cocoa beans harvested from the stage one contained significantly lower levels of TPC (6.39 ± 0.02 g CE/100 g DM) and AC (26.82 ± 0.13 mol TE/100 g DM) than those from the beans harvested from the stage two, three and four. For all maturity stages studied, fermenting cocoa beans increased antioxidant capacities of the raw cocoa powder. Cocoa fermentation using the commercial enzyme Pectinex® Ultra SP-L resulted in lower TPC but higher AC in comparison to those treated without enzymes, however prolonged fermentation time in cocoa beans significantly reduced TPC and AC in the powder. Strong correlations between TPC and AC of fermented samples with (R = 0.923) and without enzyme supplement (R = 0.942) were obtained. Two-way anova analyses showed that changes of TPC and AC of cocoa beans were dependent on maturity stages, fermentation methods and fermentation duration. As a conclusion, fermentation of cocoa beans harvested at the maturity stage two and three was found to be optimum for the high levels of AC and TPC attainment; fermented beans with commercial enzyme could be utilized to reduce labor cost by shortening the fermentation duration

Incidence of dengue and chikungunya viruses in mosquitoes and human patients in border provinces of Vietnam

Background: Dengue virus remains a major threat in Vietnam, while chikungunya virus is expected to become one. Surveillance was conducted from 2012 to 2014 in Vietnam to assess the presence of dengue and chikungunya viruses in patients hospitalized with acute fever in five Vietnam provinces neighboring Lao PDR and Cambodia. Surveillance was extended to mosquitoes present in the vicinity of the patients' households. Results: A total 558 human serum samples were collected along with 1104 adult mosquitoes and 12,041 larvae from 2250 households. Dengue virus was found in 17 (3%) human serum samples and in 9 (0.8%) adult mosquitoes. Chikungunya virus was detected in 2 adult mosquitoes (0.18%) while no chikungunya virus was detected in humans. Differing densities of mosquito populations were found, with the highest in the Long An Province border with Cambodia. Long An Province also displayed the lowest rate of infection, despite a very high Breteau Index, high human population density and presence of the main cross border road system. The highest incidence was found in Dac Nong Province, where the Breteau and Container indices were the second lowest. Dengue virus was detected in five Aedes albopictus, three Aedes aegypti and one Culex vishnui. Chikungunya virus was detected in two Ae. aegypti. All infected mosquitoes belonged to haplotypes described in other parts of the world and a number of novel haplotypes were found among uninfected mosquitoes. Conclusions: Dengue is considered to be regularly introduced to Vietnam from Cambodia, mostly through human movement. The data reported here provides a complementary picture. Due to intensive international trade, long-distance transportation of mosquito populations may play a role in the regular importation of dengue in Vietnam through Ho Chi Minh City. It is important to decipher the movement of mosquitoes in Vietnam, not only at the Lao PDR and Cambodia borders but also through international trade routes. Mosquito surveillance programs should address and follow mosquito populations instead of mosquito species

Pencil lead graphite electrochemically modified with polyglutamic acid as a sensor for detection of enrofloxacin in aqueous media

This study investigates the modification of pencil lead graphite electrodes with polyglutamic acid using an effective and fast static method to develop a sensor for the detection of enrofloxacin (ENR). The successful fabrication of pGA on the electrode surface was confirmed by scanning electron microscopy, energy dispersive X-ray analysis, and Fourier-transform infrared spectroscopy. The conditions of electrochemical modification, including the applied potentials and number of cycles in the potentiostatic process, were systematically investigated to determine their effects on the ENR electrochemical response. The pH of the electrolyte media was also explored to elucidate the electrochemical reaction mechanism of ENR. The developed electrochemical sensor was evaluated using square wave stripping voltammetry for ENR detection. Under optimal conditions, the sensor demonstrated good reproducibility with a relative standard deviation of 4.3% (from five measurements) for ENR signal detection. A linear relationship between ENR concentration and its peak current was observed in the concentration range of 0.1 to 5 µM, with a high correlation coefficient of 0.9988. The limit of detection for ENR using the sensor was 0.12 µM. Our findings provide valuable insights into the design and optimisation of pencil lead graphite electrode-based sensors for ENR detection in aqueous media

WATER QUALITY STATUS OF WHITE LEG SHRIMP FARMING AREAS IN BINH DAI DISTRICT, BEN TRE PROVINCE, VIETNAM

This study focused on water quality assessment of farmed shrimp ponds in Binh Dai District, Ben Tre Province. The goals of the study were to provide chemical parameters of pond water for farmers, to identify problems and causes in farmed shrimp related to water quality and disease, and to help managers find appropriate solutions to minimize damage to local shrimp farming. Water samples were collected monthly from January to June 2021 from 90 vannamei farmers in six different areas, including Vang Quoi Dong, Dinh Trung Binh Thoi, Phu Long, Phu Vang, and Binh Thang of Binh Dai District. Parameters investigated in this study included pH, alkalinity, NH4+, NH3, NO2- and three other minerals. Water quality parameters were analyzed based on APHA standard analysis methods. The results showed that the water quality of shrimp farming areas in Binh Dai fluctuated from time to time, and water quality parameters were suitable for vannamei shrimp farming. However, the concentration of NO2- was always higher than the permitted threshold. The results from cluster analysis and MANOVA testing showed that water quality of the shrimp farming areas in Binh Dai was significantly different (p < 0.05), especially NO2-, Ca, Mg and alkalinity. These findings suggest that farmers must apply proper solutions for water quality management in each farming area to maximize crop production

Transitions in diatom assemblages and pigments through dry and wet season conditions in the Red River, Hanoi (Vietnam)

Background and aims – Biomonitoring is an important tool for assessing river water quality, but is not routinely applied in tropical rivers. Marked hydrological changes can occur between wet and dry season conditions in the tropics. Thus, a prerequisite for ecological assessment is that the influence of ‘natural’ hydrological change on biota can be distinguished from variability driven by water quality parameters of interest. Here we aimed to (a) assess seasonal changes in water quality, diatoms and algal assemblages from river phytoplankton and artificial substrates through the dry-wet season transition (February–July 2018) in the Red River close to Hanoi and (b) evaluate the potential for microscopic counts and high-performance liquid chromatography (HPLC) analysis of chlorophyll and carotenoid pigments for biomonitoring in large tropical rivers.Methods – River water (phytoplankton) and biofilms grown on artificial glass substrates were sampled monthly through the dry (February–April) to wet (May–August) season transition and analysed via microscopic and HPLC techniques.Key results – All phototrophic communities shifted markedly between the dry and wet seasons. Phytoplankton concentrations were low (ca. thousands of cells/mL) and declined as the wet season progressed. The dominant phytoplankton taxa were centric diatoms (Aulacoseira granulata and Aulacoseira distans) and chlorophytes (Scenedesmus and Pediastrum spp.), with chlorophytes becoming more dominant in the wet season. Biofilm diatoms were dominated by Melosira varians, and areal densities declined in the wet season when fast-growing pioneer diatom taxa (e.g. Achnanthidium minutissimum, Planothidium lanceolatum) and non-degraded Chlorophyll a concentrations increased, suggesting active phytobenthos growth in response to scour damage. Otherwise, a-phorbins were very abundant in river seston and biofilms indicating in situ Chlorophyll a degradation which may be typical of tropical river environments. The very large range of total suspended solids (reaching > 120 mg L-1) and turbidity appears to be a key driver of photoautotrophs through control of light availability.Conclusions – Hydrological change and associated turbidity conditions exceed nutrient influences on photoautotrophs at inter-seasonal scales in this part of the Red River. Inter-seasonal differences might be a useful measure for biomonitoring to help track how changes in suspended solids, a major water quality issue in tropical rivers, interact with other variables of interest