인간 TEAD1 YAP 결합 도메인의 구조 규명 연구 및 초파리 DARP의 구조 기반 탈인산화 효소 활성 연구

다국어 초록 (Multilingual Abstract)

TEAD family proteins are downstream transcription factors in Hippo signaling that plays a critical role in embryo development, organogenesis, and tissue homeostasis. TEAD activity is regulated by interacting with transcriptional co-activators like YAP that cannot bind DNA directly and is known to be implicated in oncogenesis of various tumors. Therefore interference of the YAP-TEAD binding is presumed to be a putative anticancer strategy. In particular, lots of studies have shown that up-regulated gene expression by TEAD1 causes cancers of the stomach, renal cell, ovaries and prostate. However to date, the apo form structure of YAP-binding domain (YBD) of human TEAD1 was not elucidated. Herein, I reports the crystal structure of YBD of human TEAD1 in the apo form with a high resolution of 1.7 Å. While the YBD of TEAD1 looks like a dimeric form in an asymmetric unit, it exists as a monomeric form, which was confirmed using the Protein Interfaces, Surfaces, and Assemblies server and size exclusion chromatography-multiangle light scattering analysis. I also compared and analyzed TEAD1 YBD via structural alignment with the TEAD1-YAP complex and other human TEAD YBDs (TEAD 2-4).

다국어 초록 (Multilingual Abstract)

Protein phosphorylation and dephosphorylation are reversibly regulated by protein kinase and phosphatase and are important mechanisms that regulate signal transduction within cells. The target residues of protein dephosphorylation, which are present in many organisms, are mainly serine, threonine and tyrosine. However, recent studies have revealed dephosphorylation targets such as aspartate, histidine, cysteine and arginine. Recently, arginine phosphatase, YwlE, is found with the structure in the bacteria, Bacillus subtilis. This enzyme is known to be a protein that is involved in stress response and plays an important role in regulating protein homeostasis in bacteria. The discovery of arginine phosphatase played a role in the characterization of arginine phosphatase. This finding could lead to the discovery of the first eukaryotic arginine phosphatase, the Drosophila DARP protein. In this study, I was able to determine the crystal structure of Drosophila DARP protein using diffraction data with good resolution of 2.2 Å. I also compared DARP protein with YwlE and representative LMW-PTP structures and found the important feature for arginine phosphatase. Based on the characteristics of the arginine phosphatase found from the structure comparison, I measured the phosphatase activity.

목차 (Table of Contents)

• Part 1 : 인간 TEAD1 YAP 결합 도메인의 구조 규명 1
• Ⅰ. 서론 1
• Ⅱ. 재료 및 실험방법 8
• 1. 유전자 재조합 클로닝 8
• 2. 인간 TEAD1 YBD 단백질의 발현과 정제 10
• 2.1. 단백질의 발현 확인 10
• 2.2. 세포 배양과 용해 11
• 2.3. Ni-NTA 크로마토그래피 11
• 2.4. 투석 (Dialysis) 과 트롬빈 (thrombin) digestion 12
• 2.5. 이온 교환 크로마토그래피 13
• 2.6. Size exclusion 크로마토그래피 13
• 3. 인간 TEAD1 YBD 단백질의 결정화 14
• 4. 결정의 X-선 회절 실험 14
• 5. 데이터 분석 15
• 6. Size exclusion chromatography-multiangle light scattering (SEC-MALS) 16
• Ⅲ. 결과 및 고찰 17
• 1. 인간 TEAD1 YBD 단백질의 발현 및 정제 17
• 2. 단백질 결정화와 데이터 수집 및 분석 24
• 3. 인간 TEAD1 YBD 단백질의 SEC-MALS 29
• 4. 인간 TEAD1 YBD 단백질의 구조 33
• 5. 인간 TEAD1 YBD 단백질 간의 구조 비교 35
• 6. 인간 TEAD family 구성 단백질 간의 구조 비교 41
• Ⅳ. 결론 45
• Part 2 : DARP의 구조 규명 및 탈인산화 기능 연구 48
• Ⅰ. 서론 48
• Ⅱ. 재료 및 실험방법 56
• 1. 유전자 재조합 클로닝 56
• 2. 초파리 DARP 단백질의 발현과 정제 58
• 2.1. 단백질의 발현 확인 및 세포 배양과 용해 58
• 2.2. 단백질의 정제 58
• 3. 초파리 DARP 단백질의 결정화 59
• 4. 결정의 X-선 회절 실험 59
• 5. 데이터 분석 60
• 6. 탈인산화 효소 활성 분석 60
• Ⅲ. 결과 및 고찰 62
• 1. 구조 규명을 위한 초파리 DARP 단백질의 발현 및 정제 62
• 2. 단백질 결정화와 데이터 수집 및 분석 68
• 3. 초파리 DARP 단백질의 구조 73
• 4. 초파리 DARP 단백질과 다른 LMW-PTP의 구조 비교 76
• 5. 탈인산화 효소 활성 연구를 위한 돌연변이 도입 84
• 6. 초파리 DARP 단백질의 탈인산화 효소 활성 연구 95
• Ⅳ. 결론 101
• 참고 문헌 105
• Abstract 110