미백물질 탐색을 위한 HTS System으로서의 Protein Chip의 적용가능성에 대한 연구 = Development of Protein Chip for HTS System of Depigmenting Agent Screening

다국어 초록 (Multilingual Abstract)

MITF (Microphthalmia-associated transcription factor) plays a key role in the pigmenting process in the skin. Binding of MITF on its DNA binding domain, E-box of the corresponding promoter, initiates the production of melanin, a key determining factor of skin color. A protein chip was developed to screen the depigmenting agents. MITF was produced as a fusion protein of maltose binding protein (MBP) and was immobilized on a cyclodextrin-coated glass plate. Binding of Cy-3 labeled 8-mer E-box DNA was monitored by fluorescence. Kinetics of DNA binding with MITF showed Langumuir isotherm and its kinetic constants were determined. Based on the computer-simulated structure, 27 chemicals were selected and were investigated as a MITF-DNA binding inhibitor. Reasonable correlation of melanin synthesis inhibition was observed between the chip-based estimation and cell-based melanin assay. This result showed that MITF chip can be used as a HTS screening tool of depigmenting agents.

국문 초록 (Abstract)

미백물질의 high-throughput screening (HTS) system으로서 protein chip의 적용가능성을 확인하고자 이 실험을 수행하였다. 피부색을 결정 짓는 melanogenesis의 주요 조절 인자인 Microphthalmia transcription factor (MITF)와 MITF의 주 결합 DNA인 tyrosinase promoter의 결합 (protein-DNA결합)을 protein chip에 적용하여 depigmenting agent 선별에 응용하였다. Maltose Binding Protein(MBP)과 fusion하여 생산(MBPMITF)된 MBP-MITF를 cyclodextrin으로 코팅된 유리기판에 고정시키는 방법을 이용하여 protein chip을 제작하고, tyrosinase promoter 내 MITF와의 특이적인 결합 부위인 E-box (CATGTG)를 기준으로 제작한 22-mer의 oligonucleotide를 Cy3로 표지 하여 형광으로 그 결합 정도를 측정하였다. MITF와 DNA의 결합 반응 정도는 Langumuir isotherm으로 보이고 그 반응 상수를 구하였다.
MITF와 tyrosinase promoter의 결합 저해 인자로써는, 현재까지 알려진 MITF와 tyrosinase의 결합 구조를 토대로 Chemical DB를 통해 MITF 내 결합부위의 구조적 특징을 확인 하고 결합 저해 가능성이 있는 27개의 후보물질을 virtual screening을 통해 선별·합성하였다. 이 경쟁자 물질들을 protein chip에 적용하여 효과를 보이는 후보물질을 선정하고, cell test를 통해 최종 3개의 물질을 선정 하여, 그 저해 효과를 확인하고 수치화 하였으며, Electrophoretic Mobility Shift Assay를 통해 sample 처리 전후의 단백질과 DNA의 결합 정도 변화를 확인 하였다.

목차 (Table of Contents)

• 1. INTRODUCTION = 1
• 1.1. Process of Skin Pigmentation = 1
• 1.2. Microphthalmia-associated transcription factor = 3
• 1.3. The E-box (CATGTG) in the Tyrosinase = 7
• 1.4. Protein Microarrays = 8
• 2. OBJECTIVES = 10
• 3. MATERIALS AND METHODS = 11
• 3.1. Protein Chip = 11
• 3.1.1. Production of MBP Fusion MITF = 11
• 3.1.2. Design of Oligo-DNA (E-Box: CATGTG) = 13
• 3.1.3. Manufacture of MITF Target Compound (MITF Mimic) = 14
• 3.1.4. Slide Glass Modification = 17
• 3.1.5. Protein Chip Construction = 19
• 3.1.6. Analysis of Protein Chip Data = 21
• 3.2. Animal Cell Test = 22
• 3.2.1. Cell Culture = 22
• 3.2.2. Assay of Melanin Content = 23
• 3.2.3 Determination of Cell Proliferation = 24
• 3.3. Eelectrophoretic Mobility Shift Assay = 25
• 3.3.1. Nuclear Extract = 25
• 3.3.2. Design of Oligonucleotide (E-Box: CATGTG) = 26
• 3.3.3. EMSA Assay = 26
• 4. RESULTS = 27
• 4.1. Protein Chip = 27
• 4.1.1. Optimization of MITF-DNA Binding Concentration = 27
• 4.1.2. Inhibitory effect of MITF Target Compounds (MITF mimics) on MITF-DNA Binding = 31
• 4.2. Animal Cell Test = 35
• 4.2.1. Effect of MITF Target Compounds (MITF mimics) on Melanin Content and Cell Proliferation in B16F10 cells = 35
• 4.2.2. Selection of the Effective Candidates = 37
• 4.3. Eelectrophoretic Mobility Shift Assay = 38
• 4.3.1. Optimization of Nuclear Extract Concentration in EMSA = 38
• 4.3.2. MITF Target Compound (compound #18) Inhibition Effect on MITF-DNA Binding = 42
• 5. DISCUSSION = 44
• 6. REFERNCES = 46
• 7. ACKNOWLEDGMENT = 51