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系統識別號 U0026-2607201421392700
論文名稱(中文) 馬來腹蛇蛇毒蛋白其RGD loop、 linker區域與C端突變蛋白的結構與辨識整合蛋白活性的關聯性研究
論文名稱(英文) Structure-activity relationships of the RGD loop, linker region, and C-terminus of Rhodostomin mutants in the recognition of integrins
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 102
學期 2
出版年 103
研究生(中文) 張耀宗
研究生(英文) Yao-Tsung Chang
學號 S58961581
學位類別 博士
語文別 英文
論文頁數 148頁
口試委員 指導教授-莊偉哲
口試委員-陳金榜
口試委員-蘇士哲
口試委員-王淑鶯
口試委員-鄭宏祺
口試委員-鄭文義
中文關鍵字 整合蛋白  去整合蛋白  馬來腹蛇蛇毒蛋白  連結區域  C端  突變蛋白  細胞黏著競爭實驗  核磁共振圖譜  X射線結晶學  分子嵌合方法 
英文關鍵字 integrin  disintegrin  rhodostomin  RGD loop  C-terminal region  linker region  cell adhesion assay  NMR  X-ray crystallography  HADDOCK Docking 
學科別分類
中文摘要 整合蛋白(integrin)是一群由α及β次單元體所形成的異構穿膜雙體蛋白,它們表現在細胞的表面上並賦予細胞黏著能力與媒介細胞與細胞或細胞與細胞間質的交互作用。由於整合蛋白的異常與許多疾病的發生有關,包括腫瘤新生、癌細胞轉移、骨質疏鬆症及凝血功能不全症,突顯了整合蛋白作為藥物設計的目標。去整合蛋白則是一群在蛇毒液所發現的強效整合蛋白抑制劑。馬來蝮蛇蛇毒蛋白(Rhodostomin, Rho)屬於去整合蛋白中的一員,具有48PRGDMP辨識序列、連接區域為39SRAGKICRI序列功能區與C端為65PRYH序列並形成6對雙硫鍵。在本篇研究中,我使用馬來蝮蛇蛇毒蛋白當作骨架來研究去整合蛋白與整合蛋白間的辨識機制。藉由運用細胞黏著競爭實驗、核磁共振圖譜(NMR)、X射線結晶學與分子嵌合方法,來探討以下4個關於去整合蛋白與整合蛋白之間結構與功能性相關性的研究主題: (1)含有48ARGDWN序列馬來蝮蛇蛇毒蛋白與其C端區域能互相協同並調控與整合蛋白αIIbβ3之間的辨識。為了瞭解RGD loop與C端區域在去整合蛋白所扮演的角色,我們利用酵母菌產製了2種RGD序列(48PRGDMP和48ARGDWN)搭配5種C端區域序列(65P、65PR、65PRYH、65PRNGLYG和65PRNPWNG)組合的馬來蝮蛇蛇毒突變蛋白。C端區域對於整合蛋白的影響程度為αIIbβ3 > αVβ3 > α5β1。48ARGDWN-65PRNPWNG突變蛋白具有最高辨識整合蛋白αIIbβ3的選擇性;然而,48PRGDMP-65PRNPWNG突變蛋白則不具有任何整合蛋白的選擇性。NMR結構分析48ARGDWN-65PRYH、48ARGDWN-65PRNGLYG與48ARGDWN-65PRNPWNG突變蛋白證實了他們的C端區域與RGD loop進行交互作用,尤其是他們的W52胺基酸分別與65PRYH上的H68胺基酸、65PRNGLYG上的L69胺基酸與65PRNPWNG上的N70胺基酸進行交互作用。在48ARGDWN-65PRNPWNG與整合蛋白αIIbβ3電腦嵌合分析中也發現胺基酸N70能與αIIb上的D159胺基酸形成氫鍵,胺基酸W69能與β3上的K125胺基酸形成cation-pi作用力。我們的結果說明了去整合蛋白的RGD loop與C端區域能夠進行協同作用進而影響其結構與對整合蛋白的辨識; (2) 48ARGDDP突變蛋白對於整合蛋白αVβ3之間的選擇性抑制機制。為了瞭解胺基酸X在ARGDX序列中扮演的角色,我們產製了一系列的48ARGD52XP突變蛋白。X52胺基酸對於整合蛋白的影響程度為α5β1 (86-fold) > αIIbβ3 (41-fold) > αVβ3 (14-fold)。其中48ARGDDP突變蛋白能專一性辨識整合蛋白αVβ3,其抑制整合蛋白αVβ3、αIIbβ3、αVβ5、αVβ6與α5β1的IC50數值分別為45.3、5117.2、6886、14980.3與5044.5 nM。X-ray結構分析證明了Arg49與Asp52胺基酸之間的Cα原子距離為5.9 Å,與目前已知能專一性抑制整合蛋白αVβ3的結構需求條件一致。M52D突變也導致負電區域出現,推測與下降抑制整合蛋白αIIbβ3、αVβ5、αVβ6與α5β1活性相關。而在體外試驗也證明48ARGDDP突變蛋白仍保有抑制內皮細胞的爬行與血管生成作用,說明了此突變蛋白應用於抑制血管增生的潛力; (3) 48ARGDPP突變蛋白喪失與整合蛋白之間辨識的機制。48ARGDPP是個沒有活性的突變蛋白,其抑制整合蛋白αVβ3、αIIbβ3、αVβ5、αVβ6與α5β1的IC50數值分別為41260、64665、35247、15055.3與62460 nM。X-ray結構分析發現48ARGDPP突變蛋白具有兩種構型與P52突變導致了RGD區域結構的改變。位置52的Met突變成Pro導致構型A上的D51-P52之間的胜肽鍵與構型B上的P52-P53之間的胜肽鍵產生反式構型。而在胺基酸R49與P52兩個Cα之間的距離增加成8.0 Å,說明了RGDX功能域(motif)的β轉折結構(turn)的破壞。接著,藉由拉氏圖(Ramachandran plot)分析發現胺基酸D51已經轉變成延展的構型,並導致RGDX功能域的失活。我們的結果也同樣證實了RGDX胜肽上的β轉折結構的重要性; (4) 39KKARTICAR-48GRGDNP-65PRYH (KG) 與39KKARTICAR-48GRGDNP-65PGLYG (KG-P)突變蛋白下降對於抑制整合蛋白αIIbβ3活性的機制。KG與KG-P突變蛋白相較於Rho而言,大大的降低了對於抑制血小板凝集的活性,分別為56與384倍的下降。X-ray結構分析同樣發現他們的C端區域會與RGD loop進行交互作用,例如在KG突變蛋白上可以發現R56胺基酸與Y67胺基酸形成氫鍵; 在KG-P突變蛋白上可以發現D55胺基酸與L67與Y68胺基酸形成氫鍵,R49與N52胺基酸與Y68胺基酸形成氫鍵。它們具有較窄的RGD loop,表面則是呈現不同的電荷分布。從蛋白交互作用嵌合程式分析中我們則發現了此由氨基酸Arg46與Arg66所形成的正電區塊會與整合蛋白αIIb上insert-3區域的D159胺基酸形成鹽橋,在其他α次單元上則具有較短的insert-3而無法曝露出來與正電區塊作用。我們回去觀察野生型Rho的表面電荷分布,發現氨基酸Arg46與Arg66在立體結構上是聚集且形成正電區塊的,可是相較於KG與KG-P則發現正電區塊由於突變的關係而逐漸消失。我們的結果說明了去整合蛋白的氨基酸Arg46與Arg66所形成的正電區塊對於辨識整合蛋白αIIb上insert-3區域的負電區塊扮演重要的角色。我們的結果說明了去整合蛋白的RGD loop、連接區域與C端區域能夠進行協同作用進而影響其結構與對整合蛋白的辨識。總結來說,藉由解答去整合蛋白與整合蛋白結構與功能之間的相關性,加速連結了我們的基礎研究到臨床藥物的開發。
英文摘要 Integrins are αβ heterodimeric receptors that mediate cell-cell and cell-extracellular matrix interactions. Because integrins are involved in tumor progression, thrombosis, and osteoporosis, they are important therapeutic targets. Disintegrins are a family of potent integrin inhibitors that found in snake venoms. Rhodostomin (Rho) is a disintegrin containing a 48PRGDMP motif, a 39SRAGKICRI linker region, and a 65PRYH C-terminus with six disulfide bonds. In this dissertation, I used Rho as protein scaffold to study the interactions between integrins and disintegrins. Using cell adhesion assay, nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and molecular docking, four structure-activity relationships between integrins and Rho mutants were identified: (1) the 48ARGDWN motif and C-terminus of Rho mutants acted synergistically and regulated the recognition of integrin αIIbβ3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing 65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αVβ3 > α5β1. The 48ARGDWN-65PRNPWNG protein was the most selective integrin αIIbβ3 mutant; however, the 48PRGDMP-65PRNPWNG mutant did not exhibit any integrin selectivity. NMR structural analyses of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN-65PRNPWNG mutant into integrin αIIbβ3 indicated that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-pi interaction with the β3 K125 residue. Our results demonstrated that the RGD loop and C-terminus of disintegrins acted in a synergistic manner, resulting in their functional and structural differences in integrin binding; (2) Rho 48ARGDDP mutant selectively inhibited integrin αVβ3. To study the role of the C-terminal residue adjacent to the ARGD motif, we expressed Rho 48ARGD52XP mutants. The effect of the 52 residue position on their integrin binding activities was α5β1 (86-fold) > αIIbβ3 (41-fold) > αVβ3 (14-fold). The 48ARGDDP mutant was integrin αVβ3-specific mutant and inhibited integrins αVβ3, αIIbβ3, αVβ5, αVβ6, and α5β1 with the IC50 values of 45.3, 5117.2, 6886, 14980, and 5117.2 nM. X-ray structural analysis showed that the distance between the α carbons of Arg49 and Asp52 was 5.9 Å that is consistent with the structural requirement for integrin αVβ3-specific antagonist. The Met to Asp mutation caused a negative surface charge, which might be related to the lower activities toward integrins αIIbβ3, αVβ5, αVβ6, and α5β1. In vitro study showed that Rho 48ARGDDP mutant inhibited HUVEC migration and tube formation in a dose-dependent manner, suggesting its potential use as an anti-angiogenic agent; (3) the M to P mutation of the C-terminal residue adjacent to the ARGD motif abolished its binding to integrins. The 48ARGDPP mutant was an inactive integrin antagonist, which inhibited integrins αVβ3, α5β1, αIIbβ3, αVβ5, and αVβ6 with the IC50 values of 41260, 62460, 64665, 35247, and 15055.3 nM. X-ray structure analysis showed that Rho 48ARGDPP mutant has two conformations, and the P52 residue caused conformational change of the RGD motif. Met to Pro mutation in residue 52 caused the cis formation of D51-P52 peptide bond in conformer A, and that of P52-P53 in conformer B. The distance between the α carbons of Arg49 and Pro52 was increased up to 8.0 Å, indicating the disruption of turn conformation in the RGDX motif caused by the P52 residue. According to Ramachandran plot analysis, the P52 mutation modulated the D51 residue into an extended conformation and resulted in the loss of function of the RGDX motif. Our results demonstrated that the importance of the turn conformation in the RGDX motif of integrin ligands for integrin recognition; and (4) Rho 39KKARTICAR-48GRGDNP-65PRYH (KG) and 39KKARTICAR-48GRGDNP-65PGLYG (KG-P) mutants exhibited lower αIIbβ3 integrin inhibitory activity. The inhibitory activities of platelet aggregation by KG and KG-P mutants were 56 and 384 times lower than that by Rho. X-ray structural analyses of KG and KG-P mutants showed that their C-terminal regions interacted with the RGD loop: the R56 residue interacted with the Y67 residue in KG mutant, and the D55 residue interacted with the L67 and Y68 residues as well as the R49 and N52 residues interacted with Y68 residue in KG-P mutant. They had relatively narrower RGD loop and different electrostatic surface in comparison with those of Rho. The docking experiments showed that the positive charge patch formed by the R46 and R66 residues of Rho had salt bridge interactions with the negative charge D159 on the insert-3 region of αIIb subunit. KG and KG-P mutants did not have the positive charge patch due to the lack of the R46 residue in KG mutant, and the lack of the R46 and R66 residues in KG-P mutant. These results suggested that this positive charge patch may be important for the interaction of integrin αIIbβ3 with disintegrins. Our results demonstrated that the RGD loop, the linker region, and C-terminus of disintegrins acted in a synergistic manner, resulting in their functional and structural differences in integrin binding.
論文目次 CHINESE ABSTRACT I
ABSTRACT IV
ACKNOWLEDGMENT VII
TABLE OF CONTENTS VIII
LIST OF TABLES XII
LIST OF FIGURES XIV
ABBREVIATION XVI
CHAPTER 1 INTRODUCTION 1
1.1 BACKGROUND 1
1.2 INTEGRINS 2
1.2.1 Overview of integrin 2
1.2.2 Integrin and ECM 2
1.2.3 Three-dimensional structures of RGD-binding integrins 3
1.2.4 Distribution and biological function of RGD-binding integrins 4
1.2.5 The roles of RGD-binding integrins in diseases and cancers 4
1.2.6 Strategies to therapeutically target RGD-binding integrins 5
1.2.6.1 Selective inhibitor 5
1.2.6.2 Receptor antagonists that do not induce conformational changes 6
1.2.6.3 Activation state-specific inhibitors 6
1.3 DISINTEGRINS 7
1.3.1 Overview of disintegrins 7
1.3.2 Functional regions of disintegrin 8
1.3.2.1 RGD loop 8
1.3.2.2 C-terminal region 8
1.3.2.3 Linker region 9
1.3.3 Biomedical applications of disintegrins 9
1.4 RHODOSTOMIN (RHO) 10
1.4.1 Overview of Rho 10
1.4.2 3D Structure of Rho 10
1.5 THE PICHIA PASTORIS EXPRESSION SYSTEM 11
1.6 CHALLENGES IN THE DEVELOPMENT OF INTEGRIN-SELECTIVE RHO MUTANT 11
1.6.1 No disintegrin-integrin complex is currently available 11
1.6.2 Limited structure–activity relationship studies on disintegrin and integrin 11
1.7 THE USE OF CELL-BASED ASSAY IN PROTEIN DRUG DESIGN 12
1.7.1 Cell adhesion assay 12
1.8 THE USE OF STRUCTURE-BASED TECHNIQUES IN PROTEIN DRUG DESIGN 12
1.8.1 Protein structure determination 12
1.8.1.1 Nuclear magnetic resonance (NMR) spectroscopy 12
1.8.1.2 X-ray crystallography 13
1.8.2 Investigation of protein-protein interaction 14
1.8.2.1 Molecular docking (HADDOCK) 14
1.9 RESEARCH MOTIVATION AND SPECIFIC AIMS 14
1.9.1 Functional and structural characterization of Rho 48ARGDWN-C terminal mutants involved in integrins recognition 14
1.9.2 Functional and structural characterization of Rho 48ARGDXP mutants involved in integrins recognition 15
1.9.3 Functional and structural characterization of Rho KG and KG-P mutants involved in integrins recognition 16
CHAPTER 2 MATERIALS AND METHODS 18
2.1 CONSTRUCTION, EXPRESSION, AND PURIFICATION OF RHO AND ITS MUTANTS 18
2.1.1 Construction of Rho mutants with and without his-tag fusion protein 18
2.1.2 Expression of labelled and unlabelled proteins 19
2.1.3 Purification of his-tag fusion and tag-free proteins 19
2.2 MASS SPECTROMETRIC MEASUREMENT 20
2.3 PLATELET AGGREGATION INHIBITION ASSAY 20
2.4 CELL CULTURE 21
2.5 PURIFICATION OF FIBRONECTIN 22
2.6 PURIFICATION OF VITRONECTIN 22
2.7 CDNA CLONING AND CONSTRUCTION OF INTEGRIN ΑVΒ3 AND ITS MUTANTS- EXPRESSION VECTORS 23
2.8 K562 CELL TRANSFECTION AND FACS SORTING 23
2.9 FLOW CYTOMETRY ANALYSIS 24
2.10 CELL ADHESION COMPETITION ASSAY 24
2.11 NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY 26
2.11.1 NMR spectroscopy 26
2.11.2 NMR structure determination 26
2.12 PROTEIN CRYSTALLOGRAPHY 27
2.12.1 Crystallization of Rho 48ARGDDP, 48ARGDPP, KG, and KG-P mutants 28
2.12.2 Diffraction data collection, structure determination and refinement 28
2.13 MOLECULAR DOCKING 29
2.13.1 Molecular docking of Rho mutants into integrin αVβ3 29
2.13.2 Molecular docking of Rho mutants into integrin αIIbβ3 30
2.13.3 Molecular docking of Rho mutants into integrin α5β1 30
2.14 FBS-INDUCED HUVEC TRANSWELL MIGRATION ASSAY 31
2.15 FBS- AND BFGF-INDUCED HUVEC TUBE FORMATION ASSAY 32
2.16 EXPRESSION OF LIBS EPITOPE 32
2.17 PROTEIN DATA BANK ACCESSION NUMBER AND NUCLEAR MAGNETIC RESONANCE ASSIGNMENT 33
CHAPTER 3 RESULTS 34
3.1 EXPRESSION, PURIFICATION, AND MASS CHARACTERIZATION OF RHO MUTANTS 34
3.2 THE ANALYSIS OF INTEGRIN EXPRESSION IN CELL LINES USING FLOW CYTOMETRY 34
3.3 THE INHIBITION OF PLATELET AGGREGATION AND CELL ADHESION BY RHO MUTANTS 35
3.3.1 The inhibition of platelet aggregation and cell adhesion by Rho 48ARGDWN-C terminal mutants 35
3.3.1.1 The inhibition of platelet aggregation by Rho 48ARGDWN-C terminal mutants 35
3.3.1.2 The inhibition of cell adhesion by Rho 48ARGDWN-C terminal mutants 36
3.3.2 The inhibition of platelet aggregation and cell adhesion by Rho 48ARGDXP mutants 37
3.3.2.1 The inhibition of platelet aggregation by Rho 48ARGDXP mutants 38
3.3.2.2 The inhibition of cell adhesion by Rho 48ARGDXP mutants 38
3.3.3 The inhibition of platelet aggregation and cell adhesion by Rho KG and KG-P mutants 41
3.3.3.1 The inhibition of platelet aggregation by Rho KG and KG-P mutants 41
3.3.3.2 The inhibition of cell adhesion by Rho KG and KG-P mutants 42
3.4 STRUCTURAL, DYNAMIC, AND DOCKING ANALYSES OF RHO 48ARGDWN MUTANTS 43
3.4.1 NMR structures determination of Rho 48ARGDWN-65PRYH, -65PRNGLYG, and -65PRNPWNG mutants 43
3.4.2 Structural differences among Rho 48ARGDWN-65PRYH, -65PRNGLYG, and -65PRNPWNG mutants 44
3.4.3 Dynamics differences among Rho 48ARGDWN-65PRYH, -65PRNGLYG, and -65PRNPWNG mutants 45
3.4.4 Interaction differences in the docking models of integrin αIIbβ3-Rho 48ARGDWN-65P, -65PRNGLYG, and -65PRNPWNG mutants complexes 46
3.5 STRUCTURAL ANALYSIS AND BIOLOGICAL FUNCTION EVALUATION OF RHO 48ARGDDP MUTANT 47
3.5.1 Crystal structure of Rho 48ARGDDP mutant 47
3.5.2 Structural difference between Rho and its 48ARGDDP mutant 48
3.5.3 Interaction differences in the docking models of Rho and integrins complexes: implication for RGDX-integrins interactions 49
3.5.4 Mutagenesis study of the interactions between Rhodostomin and integrin alphaVbeta3 50
3.5.4.1 The adhesion of transfected K562 cells on vitronectin was integrin alphaVbeta3-specific 50
3.5.4.2 Effects of Rho and its 48ARGDXP mutants on the inhibition of K562-alphaVbeta3 cell adhesion 51
3.5.5 The inhibition of FBS-induced HUVEC transwell migration by Rho and its 48ARGDDP mutant 52
3.5.6 The inhibition of HUVEC tube formation by Rho and its 48ARGDDP mutant 52
3.6 STRUCTURAL STUDY OF THE RHODOSTOMIN 48ARGDPP MUTANT 53
3.6.1 Crystal structure of Rho 48ARGDPP mutant 53
3.6.2 Differential NMR spectroscopy between Rho 48ARGDMP and 48ARGDPP mutant 54
3.6.3 Structural difference between Rho and its 48ARGDPP mutant 54
3.7 STRUCTURAL AND DOCKING ANALYSIS OF RHODOSTOMIN KG AND KG-P MUTANTS 55
3.7.1 Crystal structure of Rho KG and KG-P mutants 55
3.7.2 Structural difference between Rho, its KG, and KG-P mutants 57
3.7.3 Interaction differences in the docking models of integrin alphaIIbbeta3- Rho and its KG-P mutant complexes 58
3.7.4 Interaction differences in the docking models of integrins-Rho complexes 59
3.8 EFFECTS OF RHO AND ITS 48ARGDWN MUTANTS ON THE EXPRESSION OF LIBS EPITOPES ON BETA3 SUBUNIT OF ALPHAIIBBETA3 AND ALPHAVBETA3 INTEGRIN 59
3.9 EFFECTS OF RHO AND ITS MUTANTS ON INHIBITING MN2+-ACTIVATED AND RESTING CHO-ALPHAVBETA3 CELL 61
CHAPTER 4 DISCUSSION 62
4.1 THE SYNERGISTIC EFFECT BETWEEN THE 48ARGDWN MOTIF AND C-TERMINUS OF RHO MUTANTS IN RECOGNIZING INTEGRIN ALPHAIIBBETA3 62
4.2 THE STRUCTURE-ACTIVITY RELATIONSHIPS OF THE SELECTIVE INTEGRIN ALPHAVBETA3 TARGETING ACTIVITY OF RHODOSTOMIN 48ARGDDP MUTANT 65
4.3 THE STRUCTURE-ACTIVITY RELATIONSHIP STUDY OF THE LOSS OF INTEGRINS INHIBITORY ACTIVITY OF RHODOSTOMIN 48ARGDPP MUTANT 67
4.4 THE STRUCTURE-ACTIVITY RELATIONSHIP STUDY OF THE LOWER INTEGRIN ALPHAIIBBETA3 INHIBITORY ACTIVITY OF RHODOSTOMIN KG AND KG-P MUTANTS 68
CHAPTER 5 CONCLUSIONS 71
REFERENCES 75
TABLES 86
FIGURES 107
PUBLICATIONS 134
APPENDIX 135


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