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系統識別號 U0026-0808201216240000
論文名稱(中文) 利用Echistatin 和 Rhodostomin的RGD 序列及C 端區域來探討其在整合蛋白辨識上的角色
論文名稱(英文) The role of the RGD motif and C-terminus of Echistatin and Rhodostomin in 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 100
學期 2
出版年 101
研究生(中文) 陳怡均
研究生(英文) Yi-Chun Chen
學號 s58921141
學位類別 博士
語文別 英文
論文頁數 117頁
口試委員 指導教授-莊偉哲
召集委員-鄭文義
口試委員-周三和
口試委員-陳金榜
口試委員-王淑鶯
口試委員-羅玉枝
中文關鍵字 C端  去整合蛋白  雙硫鍵  畢赤酵母  結構  動力學  受質誘導結合位  爬移 
英文關鍵字 C-terminus  disintegrin  disulfide bond  Pichia pastoris  structure  dynamic  LIBS  migration 
學科別分類
中文摘要 整合蛋白(Integrin)是由α、β異質雙聚體所組成的細胞表面接受體,其與細胞的增生,爬移及存活相關。由於它們促成許多常見疾病像是血栓,免疫系統失調,癌症及骨質疏鬆等的啟動與進行,因此整合蛋白被視為許多疾病的治療標的。由蛇毒分離而來的去整合蛋白(Disintegrin)是整合蛋白的天然拮抗劑。根據蛇毒蛋白的大小及雙硫鍵的數目,其可被分類成短、中、長鏈及雙聚體等四類。為瞭解短鏈及中鏈去整合蛋白在結構、動力學與功能上的相關性,我們利用酵母菌(Pichia pastoris)系統,表現了具有49個氨基酸及4對雙硫鍵的短鏈去整合蛋白Echistatin (Ech),和具有68個氨基酸及6對雙硫鍵的中鏈去整合蛋白rhodostomin (Rho)及與其彼此C端相同的嵌合體 (chimera)突變株。我們成功得到了重組Ech 及其突變株每升菌液2-7 mg的產量,並確定其具有抑制血小板凝集的活性 (IC50=210.5 nM)及正確的構型。取代Ech的RGD loop序列 (ARGDDM)及C端序列 (HKGPAT)成為Rho的RGD loop序列 (PRGDMP)及C端序列 (YH)引起其對αIIbβ3,α5β1及αvβ3整合蛋白的活性較Rho下降了7.3-,4.5-及2.0-倍。而取代Rho的RGD loop序列 (PRGDMP)及C端序列 (YH)成為Ech的RGD loop序列 (ARGDDM)及C端序列 (HKGPAT)引起其對αIIbβ3,α5β1及αvβ3整合蛋白的活性較Ech下降了14.2-,3.1-及6.7-倍。此結果說明,不同骨架蛋白會影響其對整合蛋白的活性。在動力學的分析中,可見Ech和Rho-ech在相同ARGDDM motif 的動力學特質相似,然而Rho-ech C端的HKGPAT序列卻明顯較Ech變動性大。由Ech C端突變株的血小板凝集試驗中,顯示C端的長度及K45殘基對於其和αIIbβ3的作用是重要的。Ech及其突變株亦可誘導αIIbβ3及αvβ3整合蛋白上受質誘導結合位(LIBS;ligand induce binding site)之抗原決定位(epitope)的表現。而此能力與其在細胞黏著試驗中的活性相關。比較Ech及其C端截切的突變株可見C端對誘導LIBS epitope的表現是必須的。從NMR的分析中,顯示不同骨架蛋白會影響其RGD loop上D侧鏈的方向,同時亦影響RGD motif和C端的動力學特性。在對接的結構中,亦證實了不同骨架蛋白會影響其C端的方向,導致其對整合蛋白的作用活性不同。此外,我們發現重組Ech具有抑制黑色素細胞癌(A375)爬移的能力。其抑制黑色素細胞癌及神經膠質瘤(U373 MG)遷移的IC50可達1.5及4.9 nM。而對胰臟癌(Panc-1)的IC50卻達123.9 nM。Ech對不同癌細胞的抑制效果不同,可能源自於癌細胞所表現的整合蛋白種類及表現量不同。總而言之,此研究指出雙硫鍵型式對去整合蛋白的RGD區及C端構型是重要的,因而造成其在功能、結構與動力學上的差異。此發現,可提供以去整合蛋白為骨架蛋白來做為抗腫瘤藥物設計的基礎。
英文摘要 Integrins are α/β heterodimeric cell surface receptor and are involved in cell proliferation, migration and cell survival. Because they contribute to the initiation and progression of many common diseases, such as thrombosis, immune system disorders, cancer, and osteoporosis, they are attractive therapeutic targets for many diseases. Disintegrins are potent integrin inhibitors found from snake venoms. According to the size and the number of disulfide bonds, they can be classified into small, medium, long, and dimeric disintegrins. To study structure, dynamics, and function relationships of short and medium disintegrins, we expressed echistatin (Ech), rhodostomin (Rho), their corresponding and C-terminal mutants in Pichia pastoris. Ech, a 49-residue short disintegrin with four disulfide bonds, and Rho, a 68-residue medium disintegrin with six disulfide bonds, are RGD-containing proteins. We successfully expressed recombinant Ech and its mutants and purified them to homogeneity with the yields of 2-7 mg/L. Recombinant Ech inhibited platelet aggregation with the IC50 value of 210.5 nM. Functional and structural analyses showed that Ech produced in P. pastoris retained its function and native fold. The replacement of Ech’s RGD loop (ARGDDM) and C-terminal (HKGPAT) sequences with Rho’s (PRGDMP) and (YH) sequences caused 7.3-, 4.5-, and 2.0-folds less active than those of Rho in inhibiting integrins αIIbβ3, α5β1, and αvβ3. The replacement of Rho’s RGD loop (PRGDMP) and C-terminal (YH) sequence with Ech’s (ARGDDM) and (HKGPAT) sequences caused 14.2-, 3.1-, and 6.7-folds less active than those of Ech in inhibiting integrins αIIbβ3, α5β1, and αvβ3. These results suggest that disintegrins scaffolds are sensitive for the activities of integrins. Backbone dynamics analysis showed that Ech and Rho with an ARGDDM motif exhibited similar dynamical properties for the RGD motif; however, C-terminus of Rho with a HKGPAT sequence was more flexible than that of Ech. Platelet aggregation analysis of Ech C-terminal mutants showed that the length of C-terminus and the K45 residue are important for interacting with integrin αIIbβ3. Ech and its C-terminal mutants can also induce ligand-induced binding site (LIBS) on integrins αIIbβ3 and αvβ3, and their activity is correlated with their inhibition activity of cell adhesion. The comparison of Ech with its C-terminal deletion mutants also showed that the length of C-terminus is important for LIBS induction. NMR analysis showed that disintegrin scaffolds affect the orientation of the D residue and dynamical properties of the RGD motif and C-terminus. Docking structure also confirmed that disintegrin scaffolds affect the orientation of C-terminus, resulting in their differences in interacting with integrins. These results indicate that disulfide patterns of disintegrins are important for the conformation of their C-terminus, resulting in their functional, structural, and dynamic differences. In addition, we found that recombinant Ech can inhibit the migration of human A375 melanoma cell. Ech can inhibit the migration of human melanoma A375 and glioblastoma U373 MG cells with the IC50 values of 1.5 and 4.9 nM. In contrast, it inhibited human pancreatic tumor cell Panc-1 with the IC50 value of 123.9 nM. The differences in its activity may be due to expression levels of integrins in cancer cells. In conclusion, our study indicates that disulfide patterns of disintegrins are important for the conformation of their RGD motif and C-terminus, resulting in their functional, structural, and dynamic differences. These findings may serve as the basis for the design of anti-tumor drugs using disintegrin scaffolds.
論文目次 TABLE OF CONTENTS
CHINESE ABSTRACT I
ABSTRACT III
ACKNOWLEDGMENT V
TABLE OF CONTENTS VI
LIST OF TABLES VIII
LIST OF FIGURES IX
ABBRIEVATION XI
CHAPTER 1 INTRODUCTION 1
1.1 Rationale of this dissertation 1
1.2 Integrins 2
1.3 Disintegrins 5
1.4 Rhodostomin (Rho) 6
1.5 Echistatin (Ech) 8
1.6 Pichia pastrosis expression system 9
1.7 NMR spectroscopy in protein structure and dynamics 10
1.8 Research motivation and specific aims 11
CHAPTER 2 MATERIALS AND METHODS 13
2.1. Expression of Ech and its mutants in P. pastoris and purification 13
2.2. Fibronectin purification 14
2.3. Mass spectrometric measurements 15
2.4. NMR spectroscopy 15
2.5. NMR assignment 16
2.6. Platelet aggregation assay 16
2.7. Cell adhesion assay 16
2.8. Expression of LIBS epitope 18
2.9. Cell migration assay 18
2.10. Structure determination 19
2.11. Backbone dynamics analysis 20
2.12. Molecular docking 21
CHAPTER 3 RESULTS 23
3.1. Expression, purification, and characterization of Ech, Rho, and their
mutants 23
3.2. NMR analysis of Ech 23
3.3. Inhibition of platelet aggregation by Ech and its mutants 24
3.4. Inhibition of cell adhesion by Ech and its truncated and alanine mutants 25
3.5. Analysis of LIBS expression by Ech and its mutants 26
3.6. Inhibition of melanoma cell migration by Ech and its mutants 26
3.7. NMR analysis of Ech and Rho-ech mutant 27
3.8. Inhibition of platelet aggregation by Ech, Rho, and their mutant 28
3.9. Inhibition of cell adhesion by Ech, Rho, and their mutants 29
3.10. Structure determination of Rho-ech 30
3.11. Structural difference between the RGD motif of Ech and Rho-ech 31
3.12. Dynamics analysis of Ech and Rho-ech mutant 31
3.13. Comparison of the dynamical properties of Ech and RGD-containing proteins 33
3.14. Structural differences between the integrin complexes of Ech and Rho-ech mutant 33
CHAPTER 4 DISCUSSION 36
CHAPTER 5 CONCLUSIONS 40
REFRENCES 42
TABLES 55
FIGURES 70
PUBLICATIONS 108
APPENDIX 110
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