Influence of Biological Small Molecules on the Twister Ribozyme and Development of Methodologies to Study and Discover Small Riboyzmes

Messina, Kyle The Pennsylvania State University ProQuest Dissert 2019 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

The central dogma of molecular biology describes the distinct roles for the three major biological macromolecules: DNA, RNA and protein. In recent years, it has become increasingly apparent that the roles for DNA, RNA and proteins are more complex and less distinct than previously thought. This is especially true for RNA which has been found to be involved in a plethora of different biological activities. Of particular interest are functional RNAs such as riboswitches, RNA which undergo structural changes upon ligand binding, and ribozymes, RNAs which exhibit catalytic activity.Ribozymes are a relatively recent discovery with the earliest examples identified in the 1980’s with the discovery of the self-splicing RNA intron in Tetrahymena thermophila. Since then, a number of ribozymes have been identified among diverse organisms. Ribozymes are divided into two main classes, the large and small ribozymes, with a majority of ribozymes belonging to the small ribozyme class. The small ribozymes are typically 100 nucleotides or fewer in length and catalyze a site-specific phosphodiester cleavage reaction. A total of nine small self-cleaving ribozymes have been identified thus far with nearly half being identified in the last several years. Among the newly identified self-cleaving ribozymes is the tc, which has been intensely studied through several structural and mechanistic studies to elucidate its catalytic mechanism.The twister ribozyme is one of the most catalytically active self-cleaving ribozymes as it is purported to use multiple catalytic strategies. The twister ribozyme is hypothesized to utilize two ionizable residues, with pKas of 6.9 and 9.5, thought to be a general acid and general base. The higher pKa of 9.5 has been attributed to a guanine, which is hypothesized to serve as both a general base and electrostatically stabilize the phosphorane intermediate. The lower pKa of 6.9 has been attributed to the conserved A1 which is purported to act as a general acid. While crystallographic evidence supports the role of A1 as a general acid, the available experimental evidence is mixed with no clear answer. As such, one goal of this thesis is to further define the catalytic mechanism of the twister ribozyme. The other goal of this thesis is to develop new methodologies to investigate the structure and biological activity of small self-cleaving ribozymes.Chapter 2 is focused on characterizing and establishing the mechanism by which small biological molecules stimulate the self-cleavage activity of the twister ribozyme. We find that moderate buffer concentrations can stimulate the catalytic activity of the twister ribozyme up to 5-fold. The buffers are a range of small molecules including common laboratory buffers, and biological metabolites such as imidazole, amino acids, and amino sugars. Additionally, Bronsted plot analysis indicates that the small molecules assist in proton transfer, most likely general acid catalysis. Further, we observe that at biological Mg2+ concentrations and low pH, the self-cleavage activity of the twister ribozyme appears largely buffer independent while at biological Mg2+ concentrations and pH or at high Mg2+ concentrations, the self-cleavage activity of the twister ribozyme is buffer dependent. As such, we propose a multi-channel mechanism for the twister ribozyme consisting of a buffer independent and buffer dependent channels. This work establishes a simple way to overcome the limited chemical diversity of RNA and could apply to the catalytic mechanisms of many ribozymes in vivo.Chapter 3 is aimed at characterizing the role that the A1 residue plays in the catalytic mechanism of the twister ribozyme through a combination of chemical rescue and glycosidic conformational analysis experiments. We observe that inhibited twister ribozyme constructs containing an A1 N3 deaza or abasic A1 modification can be rescued over 100-fold using small protonatable molecules such as imidazole and histidine, similar to the chemical rescue effects observed in the antigenomic HDV ribozyme with a C76U mutation. Additionally, Bronsted plot analysis indicates that the small molecules rescue catalytic activity through proton transfer, suggesting that the wild type A1 residue is also involved in proton transfer, likely general acid catalysis. We also determine through glycosidic conformational analysis that an 8BrA1 modified twister ribozyme is up to 10-fold faster than a non-modified A1 residue in an appropriate background suggesting that the catalytic conformation is syn as suggested by multiple crystallographic studies. This study provides functional evidence that A1 is syn while conducting proton transfer.The goal of Chapter 4 is to develop a novel computational and experimental pipeline to identify and assay the in vitro catalytic activity of putative ribozymes en masse. To do this, we developed a computational pipeline, based on RNABOB, to identify ribozyme candidates of known ribozyme motifs with variant secondary structures. Four RNABOB descriptors were written based on the type III hammerhead ribozyme, the human HDV-like CPEB3 ribozyme and the type P1 twister ribozyme, both with and without the P3 stem, with loosened constraints identified nearly 23,500 ribozyme candidates among 18 different organisms. Additionally, we optimized an experimental pipeline to assay thousands of ribozymes at a time for in vitro catalytic activity by taking advantage of massively parallel oligo synthesis (MPOS) to produce the DNA templates. Currently, we have optimized a majority of the experimental pipeline and successfully identified all active ribozymes in a sample set of oligos.The goal of Chapter 5 is to develop a scaffold to artificially increase the size of small nucleic acid structures to a size that is amenable for cryo-EM visualization and single particle reconstruction. Thus far, we have developed a nucleic acid-based scaffold, dubbed a “Nanosprout Scaffold,” that can multimerize small nucleic acids into a larger structure of an appropriate size for cryo-EM visualization. The Nanosprout Scaffold consists of a DNA oligonucleotide, denoted DNA guide, that multiple nucleic acids of interest with 5’-extensions can multimerize to via complementary base pairing interactions. So far, we have designed multiple Nanosprout Constructs based on the env22 twister ribozyme, and the 10MD5/10MD5- AC DNAzymes with moderate success. We observe, in aqueous conditions, that Nanosprout Constructs assemble with moderate to high affinity and with catalytic activity approaching, or on par, with the native constructs. Additionally, we are able to visualize individual env22 twister ribozymes via TEM and cryo-EM, albeit several issues persist as the fully multimerized env22 Nanosprout Constructs either partially disassemble or do not form under these conditions. The Nanosprout Scaffold is a promising start to a scaffold system that can be adapted to small nucleic acids for cryo-EM structure determination.

Preparation of Activated Carbon from Petroleum Residue and Mesoporous Carbon by Catalytic Gasification for Capacitive Deionization

이송미 과학기술연합대학원대학교 2023 국내박사

Catalytic Activities of Manganese Oxides for Aldehydes VOCs Combustion : 알데히드류 VOCs 연소를 위한 망간산화물의 촉매 활성

마충곤 전남대학교 대학원 2007 국내석사

Volatile organic compounds such as acetaldehyde and propionaldehyde are the most common air pollutants emitted from chemical, petrochemical and allied industries. Such air pollutants can have a bad effect on health and environment. Therefore, governments, especially industrialized countries have taken various methods to reduce and control the VOCs emission. Catalytic oxidation is a relatively recently applied alternative for the treatment of VOCs in air streams. Among the catalysts we used, Mn oxides are recognised as being very active for total oxidation of hydrocarbons and VOCs and they are considered to be environment-friendly materials. In this research, we use the Mn oxide (MnO, MnO2, Mn2O3, Mn3O4) catalysts for acetaldehyde and propionaldehyde combustion and choose the XRD(X-ray Diffractometer) analysis for verifying the crystal changes of Mn oxide catalysts during the whole pretreatment process. The catalytic activity of Mn oxide catalysts by the order of MnO < MnO2 < Mn2O3 < Mn3O4 is confirmed during acetaldehyde and propionaldehyde combustion and Mn3O4 catalyst shows the near total oxidation of acetaldehyde and propionaldehyde at 260℃. Compared with other acetaldehyde and propionaldehyde combustion results, T50 and T90 of Mn3O4 are the lowest of all the catalysts and its have proven the best catalytic activity. At the same time, in case of propionaldehyde combustion, not only the Mn3O4 but also the other Mn oxide catalysts are shown the better catalytic activity than for acetaldehyde combustion. It is indicated that the crystal structure of Mn3O4 catalyst converted into the Mn2O3 phase by XRD analysis. Without considering the influence of other factors, Mn3O4 catalyst not only has an excellent catalytic activity but also has the lowest price in these four Mn oxide catalysts. We expect the Mn3O4 catalyst is using in many industrial activities for aldehydes VOCs combustion.

Selective Catalytic Reduction of Nitrogen Oxides with NH3 over VOx/TiO2 catalysts

윤승희 서울대학교 대학원 2018 국내박사

Nitrogen oxides (NOx) are pollutants promoting the photochemical smog, acid rain, ozone depletion and greenhouse effect. NOx are emitted from cars, trucks and buses, power plants, and off-road equipment. In addition, emission of N2O from mobile and off-road engine is now being currently regulated because of its 298 times larger greenhouse effect than CO2, thereby implying that N2O formation from the exhaust gas after-treatment system should be suppressed. Selective catalytic reduction (SCR) using vanadium supported TiO2 catalysts applied to reduce the emission of NOx from engines has been considered to be major source for N2O emission in the system. Recently, various promoters for commercial SCR catalysts are used to improve DeNOx activity at low temperature. Finding the optimum condition was aimed by changing promoters (W, Ce, Zr and Mn) in VOx/TiO2 catalyst, not only to improve SCR reactivity, but also to reduce N2O formation at high temperature. In addition, the order of impregnation between promoters and vanadium precursor on TiO2 support was changed to observe its effect on activity and N2O selectivity. It was found that W and Ce added VOx/TiO2 catalysts showed the most active DeNOx properties at low temperature. Additionally, the difference in the order of impregnation had an influence on the SCR activity. Advanced low temperature activity of the vanadium firstly added catalysts (W or Ce/V/TiO2) was attributed to the formation of more polymerized VOx on the sample. Based on the results described above, W and Ce were chosen as good promoters to improve selective catalytic reduction activity for VOx/TiO2 catalysts. Therefore, the optimum ratio and loading of W and Ce on VOx/TiO2 catalyst were investigated in order to improve SCR reactivity in low temperature region and to minimize N2O production in high temperature region. In addition, the order of impregnation between W and Ce precursors on VOx/TiO2 catalyst was changed during the preparation while observing its effect on SCR activity and N2 selectivity. Furthermore, it was found that W and Ce overloaded VOx/TiO2 catalyst such as W/Ce/V/TiO2 (15:15:1 wt%) showed the most remarkable DeNOx properties over the wide temperature region. Additionally, this catalyst significantly suppressed N2O formation during SCR reaction, especially at 350 – 400 oC. According to the characterization results, it was found that such promoted activity was originated from the improved reducibility and morphology of W and Ce species on VOx/TiO2 catalyst when they are incorporated together at high loading. Secondly, it was demonstrated that vanadium catalyst supported on microporous TiO2 obtained from the hydrothermal synthesis of anatase TiO2 in the presence of LiOH suppressed significantly N2O emission compared to conventional VOx/TiO2 catalysts. 5 wt% VOx/TiO2 catalysts supported on two types of TiO2 having distinctive pore structure, mesopore (DT-51) and micropore (microporous TiO2; micro) were applied to selective catalytic reduction of NOx with NH3 to investigate the effect of pore structure of TiO2 on sulfur poisoning. During the SCR reaction in the presence SO2 for 12 h, 5 wt% VT (DT-51) showed more drastic decrease in activity than 5 wt% VT (micro). Larger amount of SO2 was desorbed over the post-reaction 5 wt% VT (DT-51) sample during the temperature programmed decomposition which was consistent with the elemental analysis. Such larger amount of sulfate formation could be explained by the more active SO2 oxidation on the 5 wt% VT (DT-51) than 5 wt% VT (micro) because SO2 oxidation is the important step to generate sulfate species on the catalysts. It could be ascribed to the difference in the tendency of oxidation reaction affected by the vanadium species, since it was known that more V–O–V bonds existed on the surface of 5 wt% VT (DT-51) having bulk-like VOx species whereas V=O bonds were prevalent on 5 wt% VT (micro) having more dispersed VOx. In situ FT-IR results also provided the evidence about the formation of ammonium bisulfate through strong interaction between NH3 and SO3 on 5 wt% VT (DT-51), although 5 wt% VT (micro) did not. Consequently, the different vanadium species determined by the pore structure of TiO2 had a significant effect on the SO2 poisoning during SCR reaction. At last, sulfate solution was impregnated into two types of TiO2, P25 and microporous TiO2, to investigate the role of the bond of Ti–S in sulfur poisoning of VOx/TiO2. Among different kinds of sulfur sources, the introduction of 1.5 wt% of sulfur doped by H2SO4 solution showed the most significant effect on promoting SCR reactivity and N2O suppression. It was found that the improvement of NOx conversion was originated from the increase of acid sites of catalysts which was analyzed by NH3 TPD. In addition, the less decrease in NOx conversion was observed after being exposed to SO2 and H2O during SCR reaction for 12 h when TiO2 was pre-sulfated before VOx impregnation. According to the introduction of sulfur into TiO2, 1 wt% VOx supported by both P25 and microporous TiO2 showed minimized degradation of NOx conversion after sulfur poisoning by 0.19% and 0.68%, respectively. The formation of Ti–O–S bonds on VOx supported by sulfur doped TiO2 at the expense of Ti–OH bonds on the surface could explain the suppression of SO2 oxidation leading to the strong resistance to sulfur poisoning.

Engineering PH20 Hyaluronidase via Rational Design for Enhanced Catalytic Activity and Thermostability

임승옥 아주대학교 일반대학원 2025 국내석사

Development of New Carbon-Carbon Bond Formations and Application to the Total Synthesis of Biologically Active Compounds

김애진 성균관대학교 일반대학원 2013 국내석사

Part A. 탈탄산 반응을 통한 새로운 탄소–탄소 결합 반응의 개발 전이금속촉매를 이용한 교차반응은 구조적으로 다양한 유기 분자를 생산하기 위해 가장 유용한 반응으로 알려져 있다. 특히 비활성 탄소–수소 결합의 직접적인 조작을 통한 새로운 탄소–탄소 결합 반응의 개발은 다양한 유기화합물의 기본 골격을 직접적으로 합성할 수 있는 가장 매력적인 연구 분야이다. 최근 전이금속 촉매를 이용한 알데하이드 또는 알코올과 다양한 지향성기 (예., 피리딘, 옥심, 아세트아닐라이드, 인돌)를 가진 방향족 화합물의 산화적 아실화 반응이 보고되고 있다. 그러나, 알데히드 동등체인 α-oxocarboxylic acids를 이용한 전이 금속촉매 하 탈탄산 교차반응은 아직 많은 연구가 되어 있지 않다. 본 논문에서는 O-phenylcarbamates와 O-methyl ketoximes 의 팔라듐 촉매를 이용한 효과적인 산화적 ortho-아실화 반응을 개발하였다. 본 반응은 다양한 기질 내 ortho 탄소-수소 결합을 선택적으로 활성화하여 목적하는 아릴 케톤 화합물을 우수한 수율로 합성할 수 있다. 또한 oxime 지향기는 가수 분해 후 케톤 화합물로의 변환이 가능하며, carbamate 지향기는 가수 분해 후 phenol 화합물로의 변환이 용이하여 관련 생리활성 화합물의 전합성에 매우 용이하게 적용될 수 있다. 또한 상기 화학 반응은 생리활성 천연물의 중요 골격 구조로 알려져 있는 3-isochromanone화합물의 직접적인 합성에 적용될 수 있는 유용한 합성법이다. 현재 본 연구팀은 기 개발된 합성법을 이용하여 다양한 천연물 및 의약품의 전합성 연구를 진행 중에 있다. 주제어: 아실화, 탈탄산, 팔라듐, 탄소–수소 결합 활성화, α-Keto Acids Part B. 촉매 반응의 탄소–탄소 결합 형성을 통한 (+)-Fluvastatin 유도체와 (+)-trans-Aerangis Lactone의 전합성 3-Hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA) 환원 효소 억제제인 스타틴은 혈중 콜레스테롤 수치를 감소시켜 각종 심혈관 질환을 치료할 수 있는 물질로서 알려져 있다. 특히, fluvastatin (LescolTM)은 3-hydroxy-3-methylglutaric acid가 mevalonic acid로 전환되는 것을 억제시키는 약리 기전을 가지고 있으며, 20~40 mg을 매일 투여하면 저밀도 지단백 콜레스테롤 수치를 20~30% 낮추는 것으로 알려져 있다. 이러한 강력한 약리 효과와 흥미로운 구조적 특징때문에 스타틴은 매력적인 합성 대상으로 주목을 받고 있다. Lactone은 맛과 향기의 중요한 성분으로, 식품과 향수의 첨가제로 광범위하게 사용된다. 많은 lactone은 수분 작용과 종자 발아 자극을 유도함으로써 흥미로운 생물학적 활성을 나타내며, 페로몬, 방부제에도 포함되어 있는 물질이다. 특히, δ-lactones은 다양한 생물학적 활성을 나타내는 수많은 천연물의 주요 골격구조로 알려져 있다. δ-Lactone의 대표적인 예인 trans-aerangis lactone은 Aerangis confusa와 Aerangis kirkii의 향기 구성요소로서 1993년 Kaiser에 의해 최초 보고 되었다. 전이 금속 촉매를 이용한 탄소-탄소 결합 연구의 일환으로, 우리는 촉매적 접근 방법을 이용한 다양한 생리활성 화합물의 효율적인 합성법 개발을 연구하고 있다. 본 논문은 (+)-fluvastatin 유도체와 (+)-trans-aerangis lactone의 비대칭 전합성에 대한 새로운 촉매적 접근방법을 보고한다. 주요 합성단계로는 iridium 촉매를 이용한 비대칭 카보닐 알릴화 및 크로틸화 반응, ruthenium촉매를 이용한 올레핀화 반응, copper 촉매를 이용한 인돌의 위치선택적 C-3 아릴화 반응이 있다. 주제어: 스타틴, 플루바스타틴, 애란지스 락톤, 전합성, 촉매반응 Abstract: Part A Development of New Carbon–Carbon Bond Formation via Decarboxylative Cross-Coupling Reactions Transition metal-catalyzed cross-coupling reaction has emerged as a powerful tool available for synthetic chemists to produce structurally diverse organic molecules. In particular, carbon–carbon cross-coupling reactions involving selective activation of carbon–hydrogen bonds has become an attractive alternative to traditional cross-coupling reactions, because such methods avoid a multistep preparation of preactivated starting materials and a production of stoichiometric metallic waste. Thus, cross-coupling reactions via C–H bond activation can lead to an improved overall efficiency of the desired transformation. Recently, transition-metal-catalyzed oxidative acylation of sp2 C–H bonds in aromatic compounds with various directing groups, e.g., pyridines, oximes, acetanilides, and indole, with aldehydes or alcohols have been reported. However, transition metal-catalyzed decarboxylative cross-coupling reaction using aryl carboxylic acids as coupling partners is relatively unexplored. Herein we described a novel method for Pd-catalyzed oxidative ortho-acylation of O-phenylcarbamates and O-methyl ketoximes with α-oxocarboxylic acids under ammonium persulfate as a convenient oxidant via C–H bond activation. These transformations have been applied to a wide range of substrates, and typically proceed with excellent level of region- and chemoselectivity as well as with high functional group tolerance. Also, we demonstrated a palladium-catalyzed decarboxylative acylation of phenylacetamides with α-oxocarboxylic acids via C–H bond activation. This protocol provides efficient access to a range of ortho-acyl phenylacetamides, which can be easily converted to 3-isochromanone derivatives. Further applications of this method to the synthesis of biologically active compounds are in progress. Key words: Acylation, Decarboxylative, Palladium, C–H Activation, α-Keto Acids Abstract: Part B Total Synthesis of (+)-Fluvastatin Analogue and (+)-trans-Aerangis Lactone via Catalytic Carbon–Carbon Bond Formations Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have become the most frequently prescribed agent for the treatment of hypercholesterolemia due to the compelling evidence of their effect on reducing the rates of cardiovascular events. In particular, Fluvastatin (LescolTM) inhibits the enzyme that reduces 3-hydroxy-3-methylglutaric acid to mevalonic acid, thus blocking the biosynthesis of cholesterol and lowers low-density lipoprotein cholesterol levels by 20-30% at a daily dose of 20-40 mg. Because of their potent pharmacological effects and interesting structural features, statins have attracted considerable attention as powerful synthetic targets. Lactones are important flavor and aroma constituents that are extensively used as additives in food and perfume. Many lactones exhibit interesting biological activities as attractants for pollination and seed germination stimulants. They also act as allergens, pheromones, antiseptics, and cardiotonic agents. In particular, δ-lactones appear as a ubiquitous structural motif in a number of natural products that display a wide range of biological activity. As representative examples of δ-lactones, trans-aerangis lactones were first reported by Kaiser in 1993 as the main odoriferous components of the African moth orchids Aerangis confusa and Aerangis kirkii. As part of an ongoing research program directed toward the development of trasition metal-catalyzed carbon-carbon bond forming reactions, we have been interested in developing efficient routes to the asymmetric synthesis of biologically active compounds. In this paper, we present a new catalytic strategy for the asymmetric total synthesis of (+)–fluvsatatin analogue and (+)–trans-aerangis lactone, including catalytic carbon-carbon bond formations, i.e., iridium-catalyzed enantioselective carbonyl allylation or crotylation, and rhuthenium-catalyzed intermolecular metathesis, Cu-catalyzed C-3 arylation of indoles. Key words: Statins, Fluvastatin, Aerangis Lactone, Total Synthesis, Catalytic

Thermoacidophilic archeon인 Sulfolobus solfataricus P1으로부터 유래한 carboxylesterase의 binding site와 catalytic site에 관한 연구

이예나 강원대학교 대학원 2011 국내석사

Carboxylesterase (E.C 3.1.1.1)는 carboxylic ester의 가수분해를 촉매 하는 효소이다. 지난 실험에서 고온에서 서식하는 Sulfolobus solfataricus P1 으로 부터 열에 안정한 34 kDa의 monomeric carboxylesterase를 정제하여 성질조사를 수행하고 E. coli BL21 (DE3)에서 발현시키는 실험을 진행하였다 (BBA, 2006). 그 결과, 최적 pH와 최적 온도가 각각 ㏗ 5.0, 85 ℃ 라는 것을 확인하였고, 80 ℃에서 5일 동안 incubation 시켜도 효소활성이 41%나 남아있다는 것을 확인할 수 있었다. 또한 기질 특이성 실험결과 pNP-esters 중에서 pNP-caprylate (C_8)에 대해 가장 큰 활성을 나타내었으며, 그에 대한 specificity constant (kcat/㎞) 는 207.1 s^(-1)·㎛^(-1)이었다. Carboxylesterase는 유기용매나 detergent와 같은 여러 가지 denaturing agent에 대해 높은 안정성을 보였다. chemical modifier들을 이용하여 이 효소의 active site를 구성하는 아미노산들을 조사한 결과 p-chloromercuribenzoate, diethyl pyrocabonate, diisopropyl fluorophosphate, 그리고 phenylmetylsulfonyl fluoride에 의해 효소의 활성이 크게 저해되는 것을 확인할 수 있었고, 이 효소의 촉매작용에 cysteine, histidine, serine의 관여함을 확인할 수 있었다. 또한 다른 archaea 균주의 carboxylesterase와 아미노산 서열 동질성을 조사한 결과 active site에 Ser-His-Asp로 구성된 catalytic triad를 포함하고 있으며, 예상되는 위치가 Ser 151, Asp 244, 그리고 His 274이었다. 또한 chemical inhibitor에 의해 저해되면 효소활성에 영향을 주는, 이 효소에 포함되어 있는 4가지 Cys 94, 101, 103 그리고 184이 효소활성에 미치는 영향을 확인하기 위하여 본 실험에서는 site-directed mutagenesis 방법을 이용하여 이들 아미노산을 변형하여 효소활성을 조사하였다. 그 결과, 대조군에 비하여 활성이 현저하게 감소하는 것을 확인할 수 있었다. 또한 기질이 결합하는 binding site에 관여하는 아미노산을 확인하기 위해. 이미 구조가 알려 져 있는 Achaeoglobus fulgidus, Alicyclobacillus Acidocaldarius 그리고 Metagenomic Library에서 유래한 carboxylesterase 아미노산 서열을 비교한 결과, 기질의 binding과 관계있는 oxyanion hole의 아미노산이 보존되어 있음을 확인할 수 있었고 그 위치가 Gly 80, Gly 81 그리고 Ala 152 으로 예상되었다. 따라서 oxyanion hole이라고 예상되는 Gly 80, Gly 81 그리고 Ala 152 을 마찬가지로 site-directed mutagenesis 방법을 이용하여 mutation시켜 대조군과 비교하였을 때 활성이 감소하는 것을 확인할 수 있었다. 따라서 효소의 활성에 관여하는 catalytic triad와 binding site에 관여하는 아미노산을 확인할 수 있었다. 또한 swiss-modeling program (http://swissmodel.expasy.org/)를 통하여 metagenomic library에서 유래한 carboxylesterase의 구조를 바탕으로, S. solfataricus의 3차 구조를 예상하여, 각 아미노산이 효소의 3차 구조에서 어떠한 역할을 하는지 예상 해보았다. The carboxylesterase (E.C 3.1.1.1), a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The gene of Sulfolobus solfataricus P1 carboxylesterase was identified and over-expressed in E. coli (BBA, 2006). The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site. In this study was identified the binding sites and catalytic sites of the enzyme. In order to identify the amino acids related to substrate binding, the amino acid sequence of the enzyme was compared with that of carboxylesterase from Achaeoglobus fulgidus, of which structure has already known. (Mol.Biol, 2001). The expected amino acids related to substrate binding of the enzyme were identified by enzyme assay after site-directed mutagenesis. The amino acids related to catalytic site was also identified by the same manner as above. In addition, in previous study we observed that cysteines was also associated with the catalytic mechanism of the enzyme from the result of cysteine modification by HgCl₂and p-chloromercuribenzoate. Hence, all four cysteines in the enzyme were examined by site-directed mutagenesis. The obtained results were as follows: The amino acids related to substrate binding were Gly 80, Gly 81, and Ala 152 known as oxyanion hole. In addition, the enzyme contains a catalytic triad composed of Ser 151, Asp 244, and His 274, and also Cys 94, Cys 101, Cys 103 and Cys 184 affects the enzyme activity.

Studies on MMP inhibition mechanism of endostatin and cell survival signaling pathway induced by TIMP-1 : Endostain의 MMP 저해 기작 및 TIMP-1에 의한 세포 사멸 억제 신호 전달 과정에 관한 연구

이서진 The Graduate School, Yonsei University 2003 국내박사

Matrix metalloproteinase(MMP)들은 신혈관 생성과 종양의 전이에 필요한 세포외 기질(extracellular matrix)을 선택적으로 분해한다. Tissue inhibitor of metalloproteinases(TIMP)를 비롯한 MMP 저해 단백질은 MMP의 활성을 조절함으로써 세포외 기질 항상성 유지에 중요한 역할을 하는 것이 잘 알려져 있지만, 이들이 MMP를 저해하는 기작 및 MMP 저해 외의 생리적 역할들에 대한 보고는 미진한 실정이다. 그러므로 본 연구에서는 endostatin의 MMP 저해 기작과 TIMP-1에 의한 세포 성장과 사멸 조절에 대하여 분석하였다. 선행연구에서 endostatin이 MMP-2의 활성화와 촉매적 활성을 저해함으로써 혈관내피세포와 종양세포의 전이를 감소시킨다는 것이 알려진 바 있다. 이런 작용 기작을 이해하기 위해, surface plasmon resonance을 이용하여, proMMP-2의 기능적 부위를 고정화된 endostatin에 결합시켜서 MMP-2의 어느 부위에 endostatin이 결합하는지 결정하였다. ProMMP-2와 hinge와 hemopexin-like(HP) 부위를 포함하고 있지 않은 proMMP-2(proMMP-2△HP)는 고정화된 endostatin에 거의 결합하지 않았다. HP 부위만으로는 endostatin에 전혀 결합하지 않았지만, 활성화된 MMP-2와 MMP-2△HP는 endostatin에 유사한 정도로 결합하였다. 또한 MMP의 활성 부위에 결합하여 MMP를 저해하는 actinonin을 미리 MMP-2 및 MMP-2△HP에 incubation시에는 endostatin에 결합하는 것을 완전히 저해했다. 이러한 결과들은 endostatin이 proMMP-2와 HP 부위에는 결합하지는 않지만, MMP-2의 촉매적 활성 부위에 결합함으로써 MMP-2의 활성을 저해하는 것을 제시하고 있다. TIMP-1은 MMP를 저해하는 기능과는 별도로 세포 성장과 사멸을 조절하는 기능이 알려져 있다. 그 작용 기작을 알기 위해, 유방암 세포주인 T-47D에 TIMP-1에 의해 유도되는 세포 성장과 사멸에 대한 효과를 조사하였다. T-47D에 TIMP-1 처리 시 세포 성장에는 변화가 없었으나, 혈청 제거로 인해 유도된 세포사멸은 강하게 저해되었다. TIMP-1은 Akt와 ERK1/2의 인산화를 촉진시켰고, 이러한 Akt와 ERK1/2의 인산화와 TIMP-1에 의해 유도된 세포 사멸 저해 효과는 pertussis toxin과 Src family tyrosine kinase, protein tyrosine kinase, phosphophatidylinositol-3 kinase(PI3 kinase)의 특이적인 저해제에 의해 억제되었다. 또한, TIMP-1은 c-Src과 PI3 kinase의 kinase 활성을 증가시켰고, 이러한 활성의 증가는 pertussis toxin에 의해 저해 되었다. ERK1/2 활성화 저해는 TIMP-1에 의해 유도된 세포 사멸 저해를 약하게 감소시키는 효과를 보였다. 이러한 결과로 유방암 세포주 T-47D에서 pertussis toxin-sensitive G protein, c-Src, PI3kinase 및 Akt로 이어지는 활성화 경로를 통해 TIMP-1이 세포사멸을 저해한다는 것을 규명하였다. 지금까지의 본 연구 결과들은 종양의 형성, 성장 및 진행과정에서 endostatin과 TIMP-1의 새로운 생리적 역할과 작용 기작을 규명하고 있다. 따라서 이러한 결과들은 MMP 저해 단백질들의 다양한 생물학적인 기능을 이해하고, 이들을 MMP 관련 질병의 치료제로서 적용하는데 기여할 것이다. Matrix metalloproteinases (MMPs) mediate selective proteolytic degradation of the extracellular matrix that is required for angiogenesis and tumor metastasis. Tissue inhibitors of metalloproteinases (TIMPs) and other MMP inhibitory proteins play a critical role in the homeostasis of ECM by regulating the activity of MMPs. However, many physiological roles of MMP inhibitory proteins including TIMPs are not clearly understood. Here, I performed studies on MMP inhibition mechanism of endostatin and cell survival modulation mediated by TIMP-1 independent of its MMP inhibitory activity. It was previously reported that endostatin inhibits endothelial and tumor cellular invasion by blocking activation and catalytic activity of MMP-2. To determine which domain of MMP-2 interacts with endostatin, functional domains of MMP-2 were allowed to bind to the immobilized endostatin and examined by surface plasmon resonance. ProMMP-2 and proMMP-2△HP lacking the hinge and hemopexin-like (HP)domains bound little to the immobilized endostatin. The active MMP-2 and MMP-2△HP, but not the HP domain of MMP-2, bound to endostatin at similar levels. In addition, preincubation of MMP-2 and MMP-2△HP with the MMP inhibitor actinonin, which binds to the active site of MMP-2, abolished their binding to endostatin. These results indicate that endostatin binds neither the latent proMMP-2 nor the HP domain but to the catalytic domain of MMP-2. In addition to inhibiting MMPs, TIMP-1 is involved in the regulation of cell growth and survival. To determine its mechanism of action, I investigated effects of TIMP-1 on cell proliferation and survival and signaling pathways induced by TIMP-1 in the human breast carcinoma T-47D cell line. Treatment of T-47D cells with TIMP-1 strongly inhibited apoptosis induced by serum deprivation, but did not affect cell proliferation. TIMP-1 induced phosphorylation of Akt and extracellular signalregulated protein kinases (ERKs), but pertussis toxin and specific inhibitors of Src family tyrosine kinases, protein tyrosine kinases, and phosphophatidylinositol-3 kinase (PI3 kinase) blocked the ability of TIMP-1 to activate Akt and ERKs as well as the anti-apoptotic effect of TIMP-1. I found that TIMP-1 enhanced the kinase activities of c-Src and PI3 kinase and that this enhancement was inhibited by pertussis toxin. Inhibition of ERK activation, however, resulted in a slight decrease of the TIMP-1-induced anti-apoptotic effect. These results demonstrate that the ability of TIMP-1 to inhibit apoptosis in T-47D cells is mediated by the sequential activation of pertussis toxin-sensitive G protein, c-Src, PI3 kinase, and Akt. Taken together, these findings unveil novel physiological roles and action mechanisms of endostatin and TIMP-1 in tumorigenesis, tumor growth and progression. Therefore, these results would contribute to the understanding of biological functions of MMP inhibitory proteins and allow their applications as potential therapeutic agents in MMP-related diseases.

Synthesis of silica coated ZnO nano particles and the characteristics

김유준 성균관대학교 일반대학원 2013 국내석사

ZnO는 광촉매성을 가진 반도체 물질로서 독특한 성질로 인한 많은 응용 가능성 때문에 주목받고 있는 물질이다. 본 연구는 ZnO의 다양한 성질 중에서도 TiO2와 함께 대표적인 무기계 자외선 차단제로서의 성질을 resin에 적용하여 자외선 차단기능을 가진 기능성 resin을 만들기 위한 연구이다. 자외선을 광범위하게 차단하기 위해서는 ZnO와 TiO2의 혼합 사용이 필수적이나 이 두 물질을 그대로 사용하기엔 이들이 가진 광촉매성이 문제가 된다. 공통적인 문제로, 광촉매성으로 인해 자외선을 쬐었을 시 resin이나 resin에 함께 포함될 dye와 같은 유기물이 ZnO나 TiO2에 의해서 분해돼버릴 수 있다는 것이 있다. 또한 ZnO의 경우 화학적 안정성을 가지는 TiO2와는 다르게 대부분의 산에 용해돼버리는 성질이 있기 때문에 COOH기와 같은 acid기를 가지는 resin과 혼합 가공을 하게 되면 가공 과정중에 ZnO가 모두 용해되 버리는 문제가 있다. TiO2입자의 표면을 silica로 코팅을 함으로써 광촉매성을 억제할 수 있다는 선행 연구가 존재하기 때문에 ZnO의 표면을 silica로 코팅을 해보고 광촉매성의 억제 뿐만 아니라 COOH와 같은 산을 포함하는 medium에서 ZnO의 용해를 막아줄 수 있는지 확인을 하는 연구를 진행하였다. Silica coating은 sol-gel법을 이용했으며 silica coating의 확인은 FT-IR, HRTEM, EDS분석을 통해서 확인했으며 silica coated ZnO와 bare ZnO입자들을 acid solution 및 acid resin에 혼합을 해보고 silica coating여부에 따른 용해 정도를 비교해보았다. ZnO의 용해 정도 비교를 위한 분석은 UV-vis spectroscopy, FESEM, EDS를 사용해서 진행하였다. 또한 silica coated ZnO와 bare ZnO에 UV lamp를 통해 UV를 조사해 주면서 photoluminescence intensity와 methylene blue의 분해 정도를 비교해봄으로써 광촉매성의 억제가 가능한지 확인을 해 보았다. 그 결과 silica coating으로 인한 ZnO용해 보호 효과가 acid solution에서는 미미했으나 resin에서는 bare ZnO에 비해 silica coating으로 인한 용해 정도가 확연히 감소했음을 확인해볼 수 있었다. UV light를 조사해주면서 광촉매성의 억제를 비교해본 실험에서도 silca coating으로 인한 광촉매성의 억제를 확인할 수 있었다. 이로써 resin에 자외선 차단성을 부여하는데 발생할 수 있는 문제점을 silica coating을 통해 개선할 수 있음을 확인할 수 있었다. ZnO is one of the most common UV screening agent with TiO2. I chose ZnO and TiO2 nano particles for UV screenig resin to screen both UV-A and UV-B region effectively. But in melt blending process with acidic resin most of ZnO nanoparticles were dissolved and couldn't screen UV light. And if they were alive in resin, they would degrade resin by photo catalytic activity when exposed by UV light . So in this study, I synthesized silica coated ZnO nano particles and analyzed them if the silica coating can protect ZnO from being dissolved by acid groups and can reduce photo catalytic activity to prevent resin from being degraded by photo catalytic activity of ZnO nano particles. Silica coating was done by sol-gel process. I confirmed silica coating by FT-IR, HRTEM, EDS analysis. Silica coated ZnO nano particles were mixed with acid solution and acid resin. By UV-vis spectroscopy, FESEM and EDS analysis, I found that protective effec of silica layer is very weak in acid solution but is very effective in acid resin. Silica coated ZnO nano partilces were also measured by their photo luminescence intensity and degree of degrading methylene blue solution. With these experiments I found that silica coating can prevent ZnO from being degraded by acid group in resin and reduce photo catalytic activity.

Design and synthesis of polymer-based nanocomposites for bio, sensory and catalytic applications

Song, Sungu Sungkyunkwan university 2021 국내박사

In the modern industry, the increasing demands of higher mechanical, chemical properties and novel characteristics of materials such as bio-compatibility, green synthesis are hard to satisfy with monolithic materials. For this reason, many researchers have focused on the polymer matrix nanocomposites (PNC) due to the synergistic effects of composites inducing enhanced physical and chemical properties and adapting novel characteristics for the applications such as catalyst, bio-medicine, and sensors easily. However, PNC still has limitations in the precise size and morphology control of nanoparticles and polymers, destabilization on disperses, the uneasy orientation of the dispersed phase, and agglomeration problems. Herein, I proposed novel molecules, methods, and mechanisms to develop the novel nanocomposites based on polymers, supramolecules, carbon-nanotubes for bio-, sensory and catalytic applications. I proposed imidazoliums with thiophene groups coordinated with a gold precursor, which can be converted to N-heterocyclic carbene (NHC) polymers with gold nanoparticles (AuNPs) with disproportionation and in-situ polymerization in chapter 2. In chapter 3, a binaphthyl group was introduced for precise control of the size and shape of PNC by solvents types and molecular weights of polymers. I also proposed supramolecular nanocomposite structures using cucurbit[7]uril in chapter 4 to synthesize the various metal nanoparticles (Au, Ag, Pt, Pd, Cu, and Fe) but adopt bio-compatible properties with green synthesis. In chapter 5, I proposed a solid-state synthesis method to synthesize size-controlled dextran/metal nanoparticle PNC to solve the problems of large-scale synthesis, green synthesis, and nanoparticle storage, and test anti-cancer activity. Finally, in chapter 6, I proposed single-walled carbon nanotube-based nanocomposites aligned with a the dielectrophoresis method in portable and selective chemical warfare agent sensors. 고분자 나노복합체는 고분자에 나노사이즈의 분리된 상의 물질이 복합된 형태의 물질로서, 기존 물질의 물리적, 화학적, 전기적 성질을 향상시키는 특성과 더불어, 목표하는 복합체의 물성뿐 아니라, 바이오적합성, 환경친화적인 성질등을 고분자 구조의 설계를 통해서 쉽게 달성할 수 있는 물질로서 큰 관심을 가지고 연구가 되고 있다. 본 연구에서는 고분자 나노복합체는 분류 중 고분자 기반 금속 나노입자의 합성과 응용, 초분자 기반 금속 나노입자의 합성과 응용, 탄소나노튜브 기반 고분자 복합체를 이용한 화학 센서로의 응용에 대하여 연구하였다. 우선, 이미다졸리윰 기반 물질에 금속 전구체가 결합된 형태의 단분자에서, 싸이오펜 그룹을 도입하여 단분자내에서 불균등화반응에 의해 생성된 금나노입자가 이미다졸리윰 물질에 단단히 결합하여 금속나노입자의 위치를 특정할수 있음과 동시에, 전기적 특성을 보이는 싸이오펜 그룹이 금나노입자에 의해 고분자화 반응이 진행되어서, 2TNHC-AuCl 단분자 하나로 고분자 금 나노입자 복합체를 형성하는데 성공하였다. 이에 이에서 바이나프틸 단분자 구조를 도입하여, 금 전구체를 이용해 금속나노입자의 합성과 고분자 합성을 동시에 진행하였으며, 용매에 따른 나노복합체의 크기 조절의 메커니즘 연구를 진행하였다. 이에, 보다 구체적인 복합체의 성질을 도입하고 응용하기 위하여 Cucurbit[7]uril 초분자를 도입하여 NaOH를 통하여 다양한 금속의 나노입자를 합성하고, 생체친화적인 성질을 보이는 CB7의 성질이 도입되었음을 확인하였고 4-나이트로페놀의 촉매 작용 및 항암성질에 응용하였다. 이어서, 초분자가 아닌 덱스트란을 고분자 이용하여 덱스트란의 분자량, 비율등에 의해 고분자 나노복합체의 크기 및 모양이 조절되는 것을 확인하였으며, 고체합성 반응을 도입하여 대량합성 및 나노복합체의 보관에 있어서 높은 안정성을 보이는 물질을 합성하였다. 또한, 보다 다양한 분야에의 응용을 위하여 탄소나노튜브를 도입하여, 유전영동을 통해 고정된 탄소나노튜브에 공유결합과 비공유결합 형태의 탄소나노튜브 기반 나노복합체를 형성하여 정전용량센서에 응용하여 화학작용제를 탐지할 수 있는 센서를 개발하였으며, 유사작용제 및 실작용제를 탐지하는데 성공하였으며, PCA 분석을 통하여 선택성을 보이는 센서를 개발하였다.