단백질 발현 및 정제 조건 탐색

원영한 충북대학교 2014 국내석사

Escherichia coli is one of the most widely used hosts for the production of heterologous proteins because of its ability to grow rapidly and at high density on inexpensive substrates, its well-characterized genetics and the availability of an increasingly large number of cloning vectors and mutant host strains. But all proteins are not soluble in E.coli. The protein structural study through the crystallization of human-derived protein has some difficulty with over expression of huge amount of human-derived protein in E.coli. In order to overcome the difficulties affecting protein over expression, the variety changes of expression condition, such as temperature, media type, IPTG concentration, heat shock, cold shock, were given systematically for checking the solubilization of the non-soluble proteins to the target protein expression batch. By this systematic method, some proteins such as 14-3-3, Exportin 6, Topoisomerase V, SIRT1(241-356), SIRT2(34-356), SIRT2(50-356) could be expressed in a large amount which is sufficient for the crystallization of the protein although the expression conditions of each protein was different repectively. These results suggest a hint for the overexpression of the non-soluble protein.

2-크로만카복실산 N-(monosubstituted) 페닐아마이드 유도체 분자설계 및 합성

원선우 충북대학교 2008 국내석사

Nuclear factor (NF)-kB, an inducible transcription factor, regulates the immune response and plays critical roles in the pathogenesis of chronic inflammatory diseases and a variety of human cancers. It has been suggested that NF-kB function inhibitors may be useful as both anti-inflammatory agents and antitumor agents. During the search for a novel compound that can inhibit NF-kB activation, 6-hydroxy-7-methoxy-2-chromancarboxylic acid phenyl amide (KL-1156) was identified as a good inhibitor of NF-kB activation. In the present study, we report the synthesis and inhibitory effect of 2-chromancarboxylic acid N-(monosubstituted) phenylamide derivatives. 6-Hydroxy and 7-methoxy substituents of compound KL-1156 were removed for SAR(structure activity relationship). In methyl substituted series, 2-chromancarboxamide for methyl substituent was introduced at 6 or 7-position of the target compounds. Substituents on N-phenyl ring of the target compounds were selected considering their electronic and hydrophobic character. Antioxidant activities of the target compounds were evaluated by 1,1- diphenyl-2-picrylhydrazyl (DPPH) scavenging activity and inhibition of lipid peroxidation. 2-Chromancarboxylic acid phenylamide derivatives hardly expressed antioxidant activity. Their NF-kB inhibitory activities were evaluated on lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7. The NF-kB inhibitory activities of the compounds were compared with that of KL-1156 (IC50 : 43.9 mM). The target compounds contained various substituents (H, Cl, OMe, CH3, CF3, and NO2) on phenyl ring. The positional effects of the substituents were also explored by examining the compounds with substituents at various position (2-; 3-; 4-; ). Consequently, fiftyseven 2-chromancarboxylic acid N-(mono- substituted)phenylamide derivatives were synthesized, their SAR was examined. Best activation have been showed by 2-chromancarboxylic acid 4-chlorophenylamide (IC50 : 18.2 mM). Eight compounds exhibited more potent activity than KL-1156. The positional effects of substituents on inhibitory activities are contradictory. Direct correlation was not observed based on the electronic and hydrophobic character of the substituents.

임포틴 베타와 스네일 징크핑거 복합체의 구조연구

최새해 충북대학교 대학원 2013 국내박사

During embryonic development in vertebrate, a morphological change is an important process. Epithelial Mesenchymal Transition (EMT) is a kind of cytogenesis that non-motile epithelial cell change their morphology to motile, fibroblast-like mesenchymal cell. This process promotes gastrulation for mesoderm formation in embryogenesis, and organizes neural crest and heart cushions in cyto-differentiation. EMT is a normal development process, also a fundamental of carcinoma metastasis. Cancer metastasis occurs with migration of invasive epidermoid carcinoma cell by blood and other factors, which is more dangerous than primary tumors. E-cadherin is a transmembrane protein for cell adhesion along the lateral surface of epithelial cell, which decides a feature of cell phenotype. As E-cadherin loss the function, cell adhesion turns loose. This process is controlled by Snail family. Snail1 regulates the expression of E-cadherin protein as transcription repressor by inhibiting transcription of the gene CDH1. Snail is a zinc finger protein, containing a SNAG (Snail/Gli) domain to recruit histone deacetylase family in the N-terminal region, and four zinc finger domains for binding with DNA and protein in the C - terminal region. Snail zinc finger domains consist of three C2H2 type and one C2HC type zinc finger. To repress the expression of E-cadherin, Snail is translocated into nucleus by carrier proteins. Importin β, a nuclear import carrier, recognizes NLS of cargo protein, or binds to IBB domain of importin α which interacts with a cargo protein. Importin β is composed of 19 of HEAT repeats, which structure was determined with the binding protein such as importin α, SREBP-2, PTHrP, RanGTP, FG repeats. Recent study revealed that Snail zinc finger is recognized by importin β, importin 7, transportin while importin α inhibits the nuclear translocation of Snail1. In this study, we determined X-ray structure of importin β/Snail1 zinc finger domain complex to understand the selective nuclear import mechanism of Snail family proteins. Importin β and Snail zinc finger proteins were expressed in E.coli respectively, and co-purified by mixing each expressed cells. A complex crystal shows 2.5 Å resolution and belonged to the space group, C2 with unit-cell parameter a = 228.2, b = 77.5,c = 72.0, α = 90.0, b = 100.9, γ = 90.0. The structure showed that fourteen residues located evenly on Snail four zinc fingers are necessary to bind importin β. After site directed mutagenesis of Snail1 zinc finger with structural information, binding affinity analysis and nuclear import assay were executed. The result showed that all four zinc fingers are important to stable interaction with importin β and nuclear import. Also, the length of C-terminal tail of zinc finger is a key factor for selectivity of importin β from the result of size exclusion chromatography and nuclear transport assay with Snail1 and WT1(+) zinc finger C-terminal GFP mutants. 척추동물의 배발생 과정에서 세포의 형태적 변화는 중요한 과정이다. 상피세포와 간엽세포는 가장 기본적인 세포의 형태로, Epithelial- Mesenchymal Transition (EMT)는 부착형의 상피세포가 이동성을 갖는 간엽세포로 분화하는 세포형성성의 한가지 형태이다. 이 기작은 배발생 과정에서 세포의 만입을 유도해 중배엽을 형성하고, 세포분화 과정에서 신경능의 발생, 심장내융기의 형성 등을 유도한다. EMT는 정상적인 발생의 과정이지만, 상피세포 암의 전이과정이기도 하다. 전이는 EMT에 의해 기질로 침습하는 성질을 갖게 된 상피세포 암이 혈류와 다른 요소에 의해 이동되어 발생하는데, 원발 종양보다 더 위험하다. EMT의 과정에서 E-cadherin은 세포 경계면에 위치하여 세포간 부착을 유지하는 단백질로 세포의 특성을 결정하는 중요한 단백질이다. EMT로 E-cadherin이 소실되면 세포간 부착이 느슨해 지는데, E-cadherin은 대표적으로 Snail family에 의해 조절 받는다. Snail은 CDH1 유전자의 전사를 억제함으로써 E-cadherin 단백질의 발현을 조절하는 전사억제 단백질이다. Snail은 N-말단에 Histone deacetylase 단백질과 결합하는 SNAG 도메인과 C-말단에 DNA, 단백질과 결합하는 zinc finger 도메인으로 구성되어 있다. Snail의 zinc finger 도메인은 3개의 C2H2 type과 1개의 C2HC type의 zinc finger motif를 갖는다. Snail이 E-cadherin의 발현을 억제하기 위해서는, 핵 수송 단백질에 의해 핵 내로 이동되어야 한다. Importin β는 핵으로 단백질을 운반하는 수송단백질로 cargo 단백질의 NLS를 인식하거나, 혹은 cargo 단백질과 결합한 importin α 의 IBB 도메인과 결합하여 핵 내 수송을 한다. Importin β는 19개의 HEAT repeat 구조로 된 단백질로 importin α, SREBP-2, PTHrP, RanGTP, FG repeat 등의 단백질과 함께 복합체 구조가 밝혀져 있다. 최근 Snail의 zinc finger 도메인이 NLS로써, importin β, importin 7, transportin과 같은 몇몇 importin family에 의해 인식됨이 밝혀졌고, 또한 importin α에 의해 핵내 수송이 저지됨이 알려졌다. 본 연구는 EMT와 같은 방식의 암전이 조절기작과, 더불어 다양한 ZF 단백질의 핵 수송 선택 기작을 알기위해 importin β/Snail1 ZF complex의 X-ray 구조를 규명하였다. Importin β와 Snail zinc finger 복합 단백질은 각각 대장균에서 발현하였으며, 함께 섞어서 정제하였다. 단백질 결정은 2.5 Å의 분해능을 보였으며, space group은 C2, unit-cell parameter는 a = 228.2, b = 77.5, c = 72.0, α = 90.0, β = 100.9, γ = 90.0 이다. 구조 해석 결과, Snail은 4개의 zinc finger 도메인 안에 고르게 분포하는 14개의 아미노산이 importin β와 중요한 결합을 이루고 있다. 이 결과를 바탕으로 돌연변이 실험과 핵 수송실험을 한 결과, 4개의 zinc finger가 모두 importin β와의 결합과 핵수송에 중요하다는 것을 확인하였다. 또한, importin β와 직접적인 결합을 하는 단백질 Snail1과 WT1(+)의 zinc finger 도메인 C-말단에 GFP를 붙인 돌연변이 실험과 핵 수송실험 결과, C-말단의 길이가 zinc finger 단백질의 importin β에 의한 직접적인 수송을 선택하는데 중요한 역할을 하는 것을 알 수 있었다.

GP130-mediated aberrant mitophagy in sorafenib- resistant HCC and development of the TGF-βRI inhibitor compound 10q for colorectal cancer

HWAN MA 충북대학교 일반대학원 2025 국내박사

큰꽃삽주 뿌리의 성분 및 항헬리코박터 파일로리 활성

정소영 충북대학교 2024 국내석사

Atractylodes macrocephala is a perennial herb that widely distributed in East Asia. The roots of this plant contain sesquiterpenes and polyacetylenes as major constituents, and traditionally used to stimulate appetite and treat diarrhea. It is also reported to possess anti-inflammatory and antibacterial activities. Helicobacter pylori exists in the stomach and causes indigestion and gastritis. In the screening of anti-H.pyrori activity, the fractions of A. macrocephala roots showed strong inhibitory activity. While antibacterial activity against Staphylococcus aureus and Escherichia coli has been studied, there have been no reports on the anti-H.pylori activity. Therefore, this study aims to identify the chemical constituents of A. macrocephala and evaluate its anti-H.pylori activity. Purification of n-hexane and CH2Cl2 fractions which showed anti-H.pylori activity using various chromatographic techniques yielded 21 compounds. The structures of isolated compounds were identified as 10 sesquiterpenes, 4 polyacetylenes, 1 sterol, 4 phenols, and 2 indoles based on spectroscopic methods. Among the isolated compounds, compound 6 were first reported from this plant. Measurement of anti-H.pylori activity of isolated compounds showed that two sesquiterpenes, atracylenolide Ⅲ (2) and atracylenolide I (7) showed strong inhibitory effects with MIC50 values of 13.6 and 90.1 µM, respectively. Chromatographic analysis of the total extract revealed that compound 2 is major component of A. macrocephala roots and contained 0.13 mg/g sample. Conclusively, 21 compounds were purified and identified from the roots of A. macrocephala. Sesquiterpenes are major components of A. macrocephala roots and its major sesquiterpene, atracylenolideⅢ (2) showed strong anti-H.pylori activity. Therefore, the roots of A. macrocephala might be beneficial in the prevention and treatment of H. pyrori infection Keywords : Atractylodes macrocephala, sesquiterpenes, polyacetylenes, atracylenolide Ⅲ, anti-H.pylori activity. * A thesis for the degree of Master in August 2024.

다발성 경화증 동물 모델에서 중간엽줄기세포와 미코페놀레이트모페틸의 병용치료 효과

이수정 충북대학교 2024 국내석사

Multiple sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system. Immunosuppressants, such as mycophenolate mofetil (MMF), have a reported relative safety profile as treatments of MS. Mesenchymal stem cells (MSCs), which have immunomodulatory and regenerative properties, have also been proposed as a potential cell therapy for MS. MSCs have been shown to inhibit the activation and proliferation of immune cells, including T and B cells, which play a key role in the pathogenesis of MS, and have been shown to promote the regeneration of damaged nerve cells. In this study, we examined whether combined administration of MMF and MSCs in Experimental autoimmune encephalomyelitis (EAE) mice, a widely used animal model * A thesis for the degree of Master in August 2024. for MS research, would be more effective than each alone. Our data showed that compared to each therapy, the combination therapy was more effective in reducing the disease progression in EAE mice and in reducing the cytokine expression in the spinal cord, spleen, and lymph nodes. In summary, our results indicate that combination therapy of MMF and MSCs might be effective in ameliorating therapy of MMF and MSCs might be effective in ameliorating the symptoms in EAE mice, which provides information of how to treat MS patients. Key words: Multiple sclerosis, Mesenchymal stem cell, Mycophenolate mofetil, Experimental autoimmune encephalomyelitis mice

PLK1 결정에 결합하는 조각 화합물의 탐색

최진광 충북대학교 2024 국내석사

Polo Like Kinase1(PLK1) plays a critical role in various cellular processes during cytokinesis, including centrosome maturation, spindle assembly, and microtubule-dynamics attachment. In cancer cells, which undergo abnormal division, the activity of PLK1 is significantly higher than in normal cells. Therefore, research is needed to find inhibitors of PLK1 PBD to induce growth delay and cell death in cancer cells. To discover inhibitors of the target protein, the Fragment-based lead discovery(FBLD) technique, which maximizes efficiency compared to HTS techniques, was employed. FBLD screening, which utilizes small fragment compounds with a high binding potential, offers the advantage of sampling a broader chemical space. The three-dimensional structure of the target protein was determined using X-ray crystallography and observed using PanDDA. This study aims to discover fragment compounds that bind to PLK1 PBD. Human PLK1 (371-594) was crystallized under 12.5% PEG 3350 conditions. Subsequently, six fragment compounds were discovered using a Fragment Library, with four of them binding to the active site. This provides a valuable opportunity for discovering lead drug compounds through techniques such as fragment linking, growing, and merging using FBLD.

폐암세포에서 (E)-2,4-bis(p-hydroxyphenyl)2-butenal 의 항암효과

SARANYA, KOLLIPARA PUSHPA 충북대학교 대학원 2013 국내석사

분자 네트워킹 기법을 이용한 저백피의 일산화질소 생성 저해 성분 분리

안범균 충북대학교 2024 국내석사

Ailanthus altissima, which belongs to the family Simaroubaceae, has been traditionally used to treat various ailments such as bleeding, epilepsy, and asthma in Korea, China, and India. Previous phytochemical studies on the bark of A. altissima have reported the presence of various constituents, including prenylated coumarins, quassinoids, and triterpenoids. Meanwhile, prenylation of structures, like coumarins, flavonoids, and diarylheptanoids was reported with various biological activities, such as anti-inflammatory, anti-HIV, and anti-tumor activities. Herein, molecular networking (MN) was used for the targeted isolation of prenylated coumarins as the dereplication strategy. The MeOH extract of A. altissima was partitioned with n-hexane, CH2Cl2, EtOAc, and n-BuOH. All fractions were analyzed by UHPLC-ESI-FT-MS, and the MS/MS data of the CH2Cl2 fraction that exhibited significant inhibitory effects on NO production (IC50 = 28.6 µg/mL) were organized using the GNPS (Global Natural Products Social molecular networking) web platform to construct the MN. In the MN, a potential prenylated coumarin cluster was found using in silico tool NAP (Network Annotation Propagation) and target-isolation of the cluster was performed by various chromatographic techniques, such as MPLC and preparative HPLC. As a result, 14 compounds, including five prenylated coumarins (1-5), were obtained through MN-guided isolation from the bark of A. altissima. The chemical structures of the isolated compounds were elucidated using spectroscopic and spectrometric analysis such as 1D NMR, 2D NMR, and HR-ESI-FT-MS. All compounds were evaluated for their inhibitory effects against NO production. Compounds 1-3 exhibited significant inhibitory effects with IC50 values of 11.8, 36.1 and 27.9 μM, respectively. Conclusively, the prenylated coumarins that inhibited NO production were isolated through dereplication strategy using MN from the bark of A. altissima. It suggests that various bioactive compounds can be target isolated in another natural products without time-consuming using MN-guided isolation. *A thesis for the degree of Master in August 2024.

다발성 경화증 동물 모델에서 뇌척수내 주입된 중간엽 줄기세포의 치료 효과

박근오 충북대학교 2024 국내석사

Multiple sclerosis (MS) is an autoimmune inflammatory disorder of the central nervous system. Mesenchymal stem cells (MSCs) play a crucial role in suppressing the activation and proliferation of immune cells, including T and B cells, as well as astrocytes and microglia present in the central nervous system (CNS). MSCs also promote the regeneration of damaged nerve cells. In this study, I investigated the injection routes to ameliorate MS symptoms in experimental autoimmune encephalomyelitis (EAE) mice, a widely used animal model to study MS. Intrathecal (IT) injection of MSCs alleviated disease symptoms and reduced expression of IFN-γ and IL-6. IT injection of MSCs also reduced cytokine expression * A thesis for the degree of Master in February 2024. in the spleen and lymph nodes. However, intravenous infusions of MSCs did not improve disease progression. I found that these outcomes might be due to the distribution differences of MSCs. In IT injection, more MSCs are located in the spinal cord and brain than in intravenous (IV) injection. In summary, the present a data show that local injection of MSCs into the spinal cord is necessary to ameliorate MS symptoms, which provides information on how to inject MSCs for the treatment of EAE. Key words: Multiple sclerosis, Mesenchymal stem cell, Experimental autoimmune encephalomyelitis, Intrathecal injection