살모넬라균 유래 VapBC 와 SehAB 독소-항독소 단백질의 구조적, 기능적 연구

학위논문(박사)--서울대학교 대학원 :약학대학 약학과,2020. 2. 이봉진.세균성의 독소-항독소 시스템은 영양부족, 항생제 처리, 산화스트레스 등의 세포가 힘든 환경에 처했을 때 세포의 유지에 필수적인 역할을 하는 것으로 알려져 주목받고 있다. 독소-항독소 시스템은 항독소의 독소 저해 방식 혹은 항독소의 형태에 따라 6가지 독소-항독소 시스템이 존재한다. 그 중에서 타입 2 독소-항독소 시스템은 가장 많이 연구가 진행되었다. 타입 2 독소-항독소 시스템은 상대적으로 불안정한 항독소 단백질과 안정적인 독소 단백질로 구성되어 있다. 세포가 안정적인 환경에서는 독소와 항독소는 복합체를 형성하고 있지만 세포가 산화스트레스, 온도변화, 영양부족, 항생제 처리 등의 척박한 환경에 놓이게 되면 특정 단백 분해 효소에 의해 상대적으로 불안정한 항독소가 분해되고 유리된 독소는 특정 RNA를 분해하는 등의 활성을 나타냄으로써 세포가 사멸하거나 혹은 생장이 멈춘다. 타입 2 독소-항독소 시스템의 항독소의 구조에는 DNA와 결합하는 도메인이 포함되어 있는데, DNA와 결합하는 도메인을 통해 프로모터 지역의 특정 서열과 결합하여 독소와 항독소의 전사를 조절한다. 이러한 타입 2 독소-항독소 시스템의 특성은 새로운 항생제 타겟으로 주목받고 있다. 병원성 균인 살모넬라 티피뮤리엄에는 최소 14개의 타입 2 독소-항독소 시스템이 존재하며 이제까지 밝혀진 구조는 없다. 우리는 이번 연구를 통해 VapBC 독소-항독소 복합체 구조를 결정화를 통해 DNA와 결합하지 않은 그리고 결합한 구조를 각각 2 Å 그리고 2.8 Å 으로 구하였다. VapC 독소는 구조 속에 PIN domain (pilT N-terminal domain) 을 가지고 있다. PIN domain을 가지고 있는 VapC 독소는 금속 의존적 RNA 분해 능을 가진다. 또한 구조적으로 음전하를 띤 잔기들을 활성 부위에 가지고 있다. 선행된 VapBC 연구에서 기존의 VapC 독소는 마그네슘 혹은 망간 의존적인 RNA 분해 능을 보인다고 보고되었다. 하지만 우리는 처음으로 칼슘 의존적인 RNA 분해 능을 보이는 VapC 독소 단백질을 발견하였다. VapB 항독소 단백질에는 N 말단에 Abr-B DNA 결합 domain을 가지고 있으며 이 DNA 결합 domain이 프로모터 지역의 특정 서열과 결합하여 VapBC 복합체의 전사를 조절한다. DNA와 결합된 VapBC 구조를 통하여 VapB 항독소가 DNA와 결합하는 형태와 결합에 관여하는 잔기를 관찰할 수 있었다. 또한 SehAB 복합체의 구조를 결정화를 통해 2.49 Å 으로 구하였다. SehA 독소는 RelE 독소 계열로 알려져 있으며, RelE 독소는 리보솜 의존적인 RNA분해능을 보인다. SehA 독소는 RelE 계열의 독소이므로 RNA 분해 능을 보일 것으로 예상되어, 형광-퀜처 실험을 이용하여 RNA 분해 능을 측정해본 결과 SehA 독소의 C 말단 알파 나선이 독소의 활성에 영향을 준다는 것을 밝힐 수 있었다. SehB 항독소는 C 말단에 DNA 결합 도메인인 Helix-Turn-Helix를 가지고 있다. SehAB 복합체의 구조를 통해서 SehB 항독소와 SehA 항독소의 결합에 관여하는 잔기들을 관찰 할 수 있었고, SehB 항독소가 SehA 독소를 제어하는 방식을 관찰할 수 있었다. 본 연구에서는 DNA와 결합한 VapBC와 결합하지 않은 VapBC 구조를 관찰할 수 있었고, 이 구조를 근거로 VapB 항독소의 VapC 독소 제어를 관찰할 수 있었다. DNA와 결합한 VapBC 복합체 구조를 바탕으로 여러 균주 유래의 VapBC를 포함한 VapBC 복합체의 전반적인 DNA와의 결합 양상 관찰할 수 있었다. 또한, 처음으로 칼슘 의존적인 RNA 분해 능을 보인 VapC 독소를 밝혀내었다. DNA와 결합한 VapBC 복합체 구조는 현재까지 두 개의 균주에서 유래한 구조만 보고되었으며, 또한 칼슘 의존적 RNA분해능을 보이는 VapC는 처음으로 보고되었다. 이번 연구에서 밝힌 결과들은 향후 VapBC 연구의 발전에 기여할 것이다. 또한, 결정화를 통해 구한 SehAB 복합체 구조를 구하였고 구해진 구조를 바탕으로 SehA 독소와 SehB 항독소의 결합에 관여하는 잔기들을 관찰할 수 있었다. 또한 RNA 분해 능 실험을 통해 SehA 독소의 RNA 분해 능에 영향을 끼치는 요소들을 규명할 수 있었다. SehAB는 유사체와 달리 SehB 항독소가 SehA 독소의 활성 부위와의 직접적인 결합 없이 SehA 독소의 활성을 제어하는 양상을 보이는데, 이러한 SehAB 만의 독특한 결합 양상은 SehAB 복합체가 연구 주제로서 충분한 의의를 지닌다는 것을 말해준다. 살모넬라 균은 현재까지도 경제적으로 발전이 더딘 국가에 널리 퍼져있으며 기존의 항생제에 대한 내성 또한 증가하고 있다. 그러므로 새로운 기전의 항생제 개발은 살모넬라균의 치료에 많은 도움을 줄 것이다. 이번 연구에서 밝힌 3가지 독소-항독소 복합체 구조는 독소-항독소의 간의 구조적, 기능적 정보를 제공하였고 또한 본 연구에서 밝힌 독소-항독소의 구조적 특징과 기능은 독소-항독소 구조를 이용한 새로운 항생 후보 물질 개발에 초석이 될 것이다.Bacterial toxin-antitoxin (TA) system has gained attention for its essential roles in cellular maintenance and survival under harsh environmental conditions such as nutrient deficiency, antibiotic treatment and oxidative stress. The TA systems are generally classified into six types, according to the structure of antitoxin or mode of inhibition toxin activity by antitoxin. Among them, type 2 TA systems are the most studied. The type II TA system is composed a labile antitoxin and a stable toxin. Under normal environments, the antitoxin protein binds to its cognate toxin and forms a stable complex to inhibit the toxic effects. However, under stressful environments accompanying with oxidative stress, temperature changes, starvation caused by malnutrition, and treatment of antibiotics, kinds of proteases which were produced by host cells degrade the antitoxin molecules and allow its cognate toxin to be released. This situation can lead to deaths or growth inhibition of the host cells. All type II TA operons are autoregulated at the transcriptional level by antitoxins, which bind to TA locus promoters. Type II antitoxin usually has a DNA-binding domain at the N-terminus of the protein, while the C-terminus of the antitoxin protein interacts with cognate toxin. This DNA-binding domain has a domain such as RHH (Ribbon-Helix-Helix), HTH (Helix-Turn-Helix), PhD like-, or AbrB-like domain. There are at least 14 type II TA systems in Salmonella enterica serovar Typhimurium LT2, one of pathogenic bacteria, and none of their structures have been determined. We determined the crystal structure of the VapBC TA complex from S. Typhimurium LT2 in DNA-free and –bound forms at 2 Å and 2.8 Å resolution, respectively. The VapC toxin possesses a PIN-domain ¬that shows metal dependent ribonuclease activity. The PIN domain has four acidic residues that form a negatively charged cavity. We were identified VapC from S. Typhimurium LT2 as a putative Ca2+-dependent ribonuclease using a fluorescence quenching assay, which is discriminated from previous data showing that VapC homologs have Mg2+ or Mn2+-dependent ribonuclease activities. The VapBLT2 possesses a Abr-B type DNA binding domain which is regulate transcription of VapBCLT2. Additionally, we revealed the crystal structure of the SehAB TA complex from S. Typhimurium at 2.49 Å resolution. SehA toxin shares RelE toxin ¬¬protein features that shows ribosome dependent ribonuclease activity. The SehA toxin is expected to show ribonuclease activity also. As a result of measuring ribonuclease activity of SehA toxin, we could reveal that C-terminal alpha helix of SehA toxin have a significant effect on toxin activity. The SehB antitoxin has a Helix Turn Helix–type DNA binding domain on the C-terminal. Based on the structure, we can see the way SehB antitoxin inhibits SehA toxin. The DNA-bound and –free VapBC structures revealed details of interaction mode between VapBC and the cognate promoter DNA, including the steric inhibition of the VapC active site by VapB and the linear conformation of bound DNA in the VapBC complex. It also revealed the first VapC toxin that showed Ca2+-dependent ribonuclease activity. We could observe the unique SehA toxin and SehB antitoxin binding mode through the SehAB complex structure and found the factors affecting the activity of SehA toxin. Salmonella has spread to many countries where economic development is slow to this day, and resistance to existing antibiotics is also increasing. Therefore, the development of new antibiotics is expected to be of great help in the treatment of Salmonella. The three toxin - antitoxin complexes identified in this study provided the binding and structural information of the toxin-antitoxin, and the structural features and functions of the toxin-antitoxin described in this study will be a cornerstone in the development of new antibiotic candidates.Introduction 1 Chapter 1. Crystal structure of DNA-free and bound VapBC from Salmonella Typhimurium LT2 and VapC as a putative Ca2+-depende0nt ribonuclease 6 1.1. Introduction 6 1.2. Materials and Methods 10 1.2.1. Cloning, expression, and protein purification 10 1.2.2. Crystallization and X-ray data collection 16 1.2.3. Structure determination and refinement 17 1.2.4. In vitro ribonuclease assay 20 1.2.5. Inductively coupled plasma mass spectrometry 21 1.2.6. Fluorescence polarization assay 22 1.3 Results and Discussion 23 1.3.1 Overall architecture of VapBCLT2 complex 23 1.3.2 Structure of VapCLT2 toxin 27 1.3.3 Identification of VapCLT2 as a Ca2+-dependent ribonuclease 35 1.3.4 Interaction between VapBLT2 and VapCLT2 41 1.3.5 Overall structure of VapBCLT2 bound to DNA 48 1.3.6 DNA-binding mode of VapBLT2 56 1.4 Conclusion 65 Chapter 2. Crystal structure of the novel toxin-antitoxin complex SehAB from Salmonella Typhimurium LT2: implication into pathogenicity with its unique interaction mode 67 2.1. Introduction 67 2.2. Materials and Methods 71 2.2.1. Gene cloning 71 2.2.2. Protein expression and purification 73 2.2.3. Crystallization and X-ray data collection 77 2.2.4. Structure determination, refinement, and analysis 78 2.2.5. in vitro ribonuclease assay 79 2.3. Results and discussions 81 2.3.1. Structure determination and model quality 81 2.3.2. Overall structure of S. Typhimurium SehAB complex 82 2.3.3 Structure of SehA toxin 85 2.3.4 SehA toxin as ribonuclease 89 2.3.5 SehB antitoxin 94 2.3.6 Interaction between SehA toxin and SehB antitoxin 99 2.4. Conclusion 104 Summary 106 Reference 111 국문 초록 121 감사의글 126Docto

Metal-slit assisted fresnel-lens design for optical couplingfocusing

학위논문(석사) --서울대학교 대학원 :전기. 컴퓨터공학부, 2009.2.Maste

Structural Study on Antitoxin protein from Mycobacterium tuberculosis H37 Rv by NMR spectroscopy

학위논문 (석사)-- 서울대학교 대학원 : 약학과, 2014. 2. 이봉진.Mycobacterium tuberculosis is a pathogenic bacterial species and the causative factor of tuberculosis. Tuberculosis is one of the most oldest disease which has human afflicted. Despite of people in the world use a live attenuated vaccine and several antibiotics, this disease make lots of death until now. They have already some kind of antibiotics and therapeutic tactic, but upcoming of multi drug resistance so we need to make new antibiotics. Toxin – Antitoxin system which is found in most of the bacteria and fungi was originally created for adapt extremely environmental conditions. Toxin – Antitoxin system constitute relatively stable toxin and labile antitoxin. Depend on type of antitoxin, they divide all of 3 types Toxin – Antitoxin system. Basically Toxin has lots of toxic effect such as Rnase, interrupt DNA gyrase and then cause of these toxic effect they killed cell or they interrupt cell growth. In Toxin – Antitoxin system, antitoxin bind to toxin tightly to inhibit toxin activities. Rv0599c, antitoxin protein included in Type2 Toxin - Antitoxin system, and it inhibit toxin`s toxic effect by binding to toxin. Under the several stress conditions, the protease which induced by stress will degrade antitoxin from the toxin, and then the free toxin has activity such as cell growth halt, and cell death. Type 2 Toxin – Antitoxin system composed of toxin protein and labile antitoxin protein. Type2 Toxin – Antitoxin system include VapBC, MazEF, RelAB and so on. Rv0599c is VapB antitoxin. Be known this protein has DNA binding function. Rv0599c has 78 amino acid and pI value is 5.0365. In order to determine tertiary structure of Rv0599c protein, we used E. coli expression system and purified hexa-histidine tagged Rv0599c by IMAC purification system. To get a more purified fractions, we used Ion – Exchange purification system, finally we used Size Exclusion purification system. And we also used Circular Dichroism to get a proper buffer condition for NMR spectroscopy. Using the buffer condition, We performed HSQC at various temperature condition, and we decided certain temperature. Finally, using buffer condition and temperature I finished backbone assignment. The secondary structure and tertiary structure of Rv0599c were predicted by TALOS server and CS23D server respectively. we found homologues by DALI server. Actually, Toxin – Antitoxin complex and antitoxin protein was known bind the certain DNA sequence. Also Rv0599c antitoxin protein, we made certain DNA sequence, and we performed NMR titration experiment to confirm antitoxin protein binding function. I think if we inhibit binding function of antitoxin to toxin, new antibiotic can be proper to M. tuberculosis by toxin activities.I. Introduction 1.1 Introduction of SBDD 1 1.2 Characteristics of Mycobacterium tuberculosis 1 1.3 Symptom of Tuberculosis 2 1.4 Treatment of tuberculosis 3 1.5 Toxin – Antitoxin System 3 1.6 Characteristics of Antitoxin protein (Rv0599c) 5 1.7 Purpose of the study 6 Ⅱ. Materials and Methods 2.1 . Materials 7 2.1.1. Reagents 7 2.1.2 Apparatus 7 2.2 Methods 8 2.2.1 Cloning of target protein 8 2.2.2 Over-expression and purification 9 2.2.3 Circular Dichroism spectroscopy 11 2.3 Structural and Functional studies by NMR spectroscopy 12 2.3.1 NMR spectroscopy 12 2.3.2 Backbone assignment 13 2.3.3 Secondary structure and Modeling structure 13 2.3.4 Search for Structural homologues of Rv0599c 14 2.3.5 DNA synthesis and preparation 14 Ⅲ. Result 3.1 Over-expression and purification 15 3.1.1 Over-expression and Solubility test 15 3.1.2. Purification 16 3.2 Circular Dichroism spectrum of Rv0599c 17 3.2.1 Effects of pH and Salinity on Rv0599c 17 3.3 NMR studies of 15N-13C labeled proteins 19 3.3.1 Measurement of 1H-15N HSQC 19 3.3.2 Sequential assignment 20 3.3.3 Secondary and Tertiary structure prediction 24 3.3.4. Structural homology search for Rv0599c 25 3.4 Functional study 26 Ⅳ. Disussion Ⅴ. Referenece 국문초록Maste

지구물리 탐사기법을 이용한 심해 Dmitri Donskoi호 확인

2

Toxicity of Persistent Organic Pollutions, PAHs and TBT, in Zooplankton and Influence on Their Viability

We conducted three experiments to estimate the toxicity of POPs(persistentorganic pollutants) on two copepod species ( Acartia erythraea and A. omorii ) andArtemia sp.; (1) 48 h-LC50 of A. omorii with the five PAHs [polycyclic aromatic hydro-carbons anthracene, benzo[a]pyrene, fluoranthene, phenanthrene, pyrene] which wereoften detected in the Gwangyang Bay, (2) toxicity of benzo[a]pyrene and TBT onArtemia in different temperatures (10 C, 15C, 20C), (3) effects of benzo[a]pyrene andTBT on egg production rate, hatching rate and fecal pellet production of two copepodspecies( A. erythraea and A. omorii ) fed on Heterocapsa triquetra (dinoflagellate) exposedin benzo[a]pyrene. Toxic chemicals which were most effective to A. omorii were fluo-ranthene (48 h -LC50 19.20 g L-1) and benzo[a]pyrene (48 h -LC50 29.89 g L-1). The toxi-city of chemicals to Artemia increased when temperature increased. The toxicity ofTBT was about 100 times higher than that of benzo[a]pyrene at 15 C. Food materials(Heterocapsa triquetra ) exposed in benzo[a]pyrene, affected negatively the rate of eggproduction, hatching rate and the fecal pellet production of the copepods at the highconcentration. It is suggested that an increase in the concentration of benzo[a]pyrenemight effect the production of copepods in marine ecosystems. This study suggests thatcopepods may be used as a indicator for early warning of the risk of POPs in marineecosystems.33Nkciothe

Seasonal Variation of Zooplankton Community in Gwangyang Bay, Korea

This study was conducted bimonthly from June 2001 to June 2003 to investi-gate the seasonal variation of the zooplankton community in Gwangyang Bay. Zoo-plankton were collected at 9 stations using a NORPAC net from surface layer. The zoo-plankton community consisted of 47 taxa and the mean abundance was 6,205 inds. m-3during the survey period. The maximum abundance was observed to be 26,060inds. m-3in June 2002 and the minimum in August 2001 with 630 inds. m-3. Copepods were thepredominant constituent, wihich comprised 4.6 ~ 84.1% (mean 38.2%) of the total zoo-plankton abundance. Dominant species of copepods were Acartia omorii, Acartiaerythraea, Centropages abdominalis, Paracalanus parvus. Paracalanus parvus domi-nated from June 2001 to December 2002. A red tide causative dinoflagellate, Noctilucascintillans , dominated from June 2002 to February 2003. Acartia omorii and Cen-tropages abdominalis dominated in winter and spring seasons. While, Acartia erythraeadominated in summer and fall seasons. In June and August, Cladocerans and Cirripedlarvae dominated. The abundance of zooplankton according to the tidal cycle showedconsiderable fluctuations with a range of 2,768 ~ 15,856inds. m-3 (≒ 5.7 times).33Nkciothe

(A) study on the wireless network access for the ocean observation sensors

한국해양연구

Control unit development of mass sampler for dissolved and particulated organic materials

한국해양연구

Effects of benzo[a]pyrene on Growth and Photosynthesis of Phytoplankton

We examined the impacts of anthropogenic pollutant (benzo[a]pyrene) onSkeletonemacostatum, Heterosigma akashiwo, Prorocentrum dentatum, P. minimum, Akashiwosanguinea ), which are dominant in Korean coastal water. After the 72 h exposure tobenzo[a]pyrene, the dramatic decrease in cell numbers was observed in the range of 1to 10 g L-1 for S. costatum, P. minimum, P. dentatum , whereas for A. sanguinea and H.akashiwo at the low concentrations 0.1 to 1 g L-1. Among the 5 phytoplankton species,the highest growth inhibition concentration (IC 50) was 6.20 g L-1 for P. minimum,followed by 2.14 g L-1 for P. dentatum , 1.68g L-1 for S. costatum, 0.74g L-1 for H.akashiwo , 0.10g L-1 for A. sanguinea . The five species exposed to the low concen-tration of 1 g L-1 were recovered after transferring to new media, but the speciesexposed to the high concentrations of 10 and 100 g L-1 were not recovered, with theexception of P. minimum . These results indicate that the thecate dinoflagellate P. mini-mum is most tolerant to the chemical and the athecate dinoflagellate A. sanguinea isnot. Generally, the cell -specific photosynthetic capacity of H. akashiwo exposed to thelow concentrations of 0.1 and 1 g L-1 was higher than that of the cells in the control,whereas the cells exposed to the high concentrations of 5 and 10 g L-1 showed thenegligible photosynthetic level by the first few days of the experiment. In the case ofthe cells exposed to the concentration of 5 g L-1, after 12 days of the experiment thephotosynthetic capacity was increased toward the end of the experiment. This indi-cates that H. akashiwo may utilize the benzo[a]pyrene as a carbon source for its growthwhen exposed to low concentrations. Results suggest that anthropogenic pollutantssuch as benzo[a]pyrene may have significant influence on the succession of phyto-plankton species composition and the primary production in coastal marine environ-ments.33Nkciothe