臺灣博碩士論文加值系統

整合蛋白家族是一群表現在大部分細胞細胞膜上的異構穿膜雙體蛋白，並且調控著細胞與細胞之間和細胞與細胞間質間的交互作用。整合蛋白αvβ6位於表皮細胞並且會再傷口癒合、發炎、和癌症發生時會被大量誘發。整合蛋白αIIbβ3則是在止血以及血栓形成中的血小板凝集扮演重要角色。先前已有報告指出具有ARGDLXXL和KGD motif的蛋白可以分別具有專一性的結合到整合蛋白αvβ6和αIIbβ3。在本研究中，蛇毒去整合蛋白，馬來腹蛇蛇毒蛋白 (rhodostomin, Rho) 和龜殼花蛇毒蛋白 (trimucrin, Tmu) 被利用來當作骨架，以設計針對整合蛋白αvβ6和αIIbβ3的專一性拮抗劑。在設計整合蛋白αvβ6專一性拮抗劑的實驗中，我們發現48ARGDRP和48ARGDKP分別針對整合蛋白αvβ6有很好的活性，IC50值分別為24.0和36.4 nM。相反的，48ARGD(D/E)P和48ARGDLP則具有很差的活性，IC50值分別為大於5000和357.3 nM。這些結果說明了若要和整合蛋白αvβ6結合，RGD右側的胺基酸要為正電荷的胺基酸比較好，而非負電荷的胺基酸。總結目前針對整合蛋白αvβ6的拮抗蛋白活性分別為48ARGDRP 〉 48ARGDKP 〉 48ARGDWP 〉 48ARGDMP 〉 48ARGDAP 〉 48ARGDLP〉 48ARGDDP。我們也將在Helicobacter pylori發現的序列48ARGDLAAL引入馬來腹蛇蛇毒蛋白中，但發現這個突變白的活性很低，IC50值大於2000 nM。這說明了引入相同的RGD序列到不同的骨架蛋白中可能會導致不同的構型改變。此外，52RP的衍生突變蛋白比起52LA的衍生突變蛋白有更好的活性，這說明了馬來腹蛇蛇毒蛋白中若有52RP會較容易和整合蛋白αvβ6結合。從48ARGDRP和整合蛋白αvβ6結合的docking結果，可以發現與在LAP和整合蛋白αvβ6結合的結果中有不同的交互作用，這些作用力的差異或許可以做為未來拮抗劑專一性設計的一個新方向。在優化整合蛋白αIIbβ3專一性拮抗劑的實驗中，我們發現41KKKRT-50AKGDRR、 41MKKGT-50AKGDRR和41IEEGT-50AKGDRP的突變蛋白分別具有IC50值127.1、 115.6和511.2 nM。這些結果指出龜殼花蛇毒蛋白的連接區域若有較多帶正電荷的胺基酸會較容易和整合蛋白αIIbβ3結合。本項研究的結果將能提供未來針對可用於纖維化及心肌梗塞的整合蛋白αvβ6和αIIbβ3專一性拮抗劑藥物設計的基礎。

Integrins are a family of heterodimeric receptors that are expressed on the surface of most cells, where they mediate cell-cell and cell-extracellular matrix interactions. Integrin αvβ6 is epithelial-specific and strongly induced during wound healing, inflammation and carcinogenesis. Integrin αIIbβ3 plays a critical role in platelet aggregation that is essential for hemostasis and thrombosis. It was reported that proteins with ARGDLXXL and KGD motifs can selectively bind to integrins αvβ6 and αIIbβ3, respectively. In this study, we propose to use rhodostomin (Rho) and trimucrin (Tmu), snake venom disintegrins, as scaffolds to develop integrins αvβ6 or αIIbβ3-specific antagonists. In the study on the design of integrin αvβ6-specific antagonists, we found that the 48ARGDRP and 48ARGDKP mutants exhibited high affinity with the IC50 values of 24.0 and 36.4 nM. In contrast, the 48ARGD(D/E)P and 48ARGDLP mutants exhibited low affinity with the IC50 values of 〉 5000 and 357.3 nM. These results suggest that the binding of integrin αvβ6 prefers the C-terminal residue adjacent to the RGD motif with positively charged residues but not with negatively charged and hydrophobic residues. In summary, we found that the relative affinity of 48ARGDXP mutants to integrin αvβ6 were 48ARGDRP 〉 48ARGDKP 〉 48ARGDWP 〉 48ARGDMP 〉 48ARGDAP 〉 48ARGDLP〉 48ARGDDP. We also incorporated the 48ARGDLAAL amino acid sequence of CagL in Helicobacter pylori, the integrin αvβ6-binding loop, into Rho, and it exhibited low affinity with the IC50 value of about 2000 nM. These results suggested that the corporation of the RGD motif into different scaffolds may exhibit different conformations. In addition, 52RP derived mutants exhibited better activity than mutants derived from 52LA, suggesting that binding of integrins αvβ6 prefers 52RP in Rho. From the docking results of 48ARGDRP binding to integrin αvβ6, we can see the difference interactions that LAP binding to integrins αvβ6 processes, which may provide us a new perspective for future design in selectivity. In the study on the design of integrin αIIbβ3-specific antagonist, we found that 41KKKRT-50AKGDRR, 41MKKGT-50AKGDRR, and 41IEEGT-50AKGDRP mutants had the IC50 values of 127.1, 115.6, and 511.2 nM. These results indicate that the binding of integrin αIIbβ3 prefers positively charged residues in the linker region of Tmu. The results of this study will serve as the basis for the design of integrin αvβ6- or αIIbβ3-specific drugs for the treatments of fibrosis and myocardial infarction.

CHINESE ABSTRACT……………………………………………………...I
ABSTRACT………………………………………………………………...III
ACKNOWLEDGEMENT………………………………………...………..V
TABLE OF CONTENTS…………………………………………………..VI
LIST OF TABLES……………………………………………………….. ..IX
LIST OF FIGURES………………………………………….….…..………X
ABBREVIATIONS.………………………………………………………..XI
CHAPTER I INTRODUCTION……………………………1
1.1 Integrins…………………………………1
1.2 The roles of RGD-binding integrins in fibrosis………………2
1.3 TGF-β and its activation………………………………………………...3
1.4 The roles of integrin αIIbβ3 in platelet aggregation…….4
1.5 Strategies to therapeutic target RGD-binding integrins.5
1.6 Disintegrins………………………………………………..6
1.7 Rhodostomin…………………………………………7
1.8 Trimucrin……………………………………………..7
CHAPTER II RATIONALE AND SPECIFIC AIMS……………..9
CHAPTER III MATERIALS AND METHODS…………11
3.1 Construction of Rho and Tmu mutants…………..11
3.1.1 Fast cloning……………………………………….11
3.1.2 XL1-blue transformation…………………………11
3.1.3 X-33 transformation…………………………...12
3.2 Expression of Rho and Tmu proteins…………………………….13
3.2.1 Small scale protein expression of Rho and Tmu mutants..13
3.2.2 Large scale protein expression of Rho and Tmu mutants…….13
3.3 Purification of Rho and Tmu proteins………………14
3.3.1 Nickel chelating chromatography…...……………………14
3.3.2 CaptoMMC chromatography...……………………………...15
3.3.3 RP-HPLC of Rho and Tmu proteins………………………...15
3.4 Mass spectrometric measurement………………………………..16
3.5 Cell adhesion competition assay……………………………………16
3.6 Platelet aggregation assay…………………………………..17
CHAPTER IV RESULTS…………………………………….19
4.1 Expression, purification and mass characterization of Rho and Tmu mutants………..19
4.2 The effects of RGD motif of Rho on integrin αvβ6 activities………………19
4.2.1 The effects of the X site of Rho-ARGDXP on their activities in cell adhesion of integrin αvβ6………….....19
4.2.2 The effects of Rho-ARGDXX motif on their activities in cell adhesion of integrin αvβ6…………………………………......20
4.2.3 The effects of Rho-ARGDLXXL motif on their activities in cell adhesion of integrin αvβ6…………………………..21
4.3 Molecular docking result of integrin αvβ6 bound to Rho-ARGDRP mutant………….22
4.4 The effects of linker region and RGD motif of Tmu on integrin αIIbβ3 activities and platelet aggregation…………...22
CHAPTER V DISCUSSION………………………………………......…..23
5.1 The roles of the 52~55 residues of Rho in integrin αvβ6 recognition………23
5.2 Structural comparison of β1, β3 and β6 subunits and structural docking ………….24
5.3 The roles of linker region of Tmu in integrin αIIbβ3 recognition…………………..25
CHAPTER VI CONCLUSION AND FUTURE PERSPECTIVE………26
REFERENCES………………………………………..28 RECIPES……………………………………………………………………34
TABLES……………………………………………………………………..41
FIGURES…………………………………………………………………...47
APPENDIX TABLES………………………………………………………63
APPENDIX FIGURES……………………………………………………..70

Alanko, J., Mai, A., Jacquemet, G., Schauer, K., Kaukonen, R., Saari, M., Goud, B., and Ivaska, J. (2015). Integrin endosomal signalling suppresses anoikis. Nature Cell Biology 17, 1412–1421.
Arnaout, M.A., Goodman, S.L., and Xiong, J.-P. (2007). Structure and mechanics of integrin-based cell adhesion. Current Opinion in Cell Biology 19, 495–507.
Benoit, Y.D., Groulx, J.-F., Gagné, D., and Beaulieu, J.-F. (2012). RGD-Dependent Epithelial Cell-Matrix Interactions in the Human Intestinal Crypt. Journal of Signal Transduction 2012, 1–10.
Civera, M., Arosio, D., Bonato, F., Manzoni, L., Pignataro, L., Zanella, S., Gennari, C., Piarulli, U., and Belvisi, L. (2017). Investigating the Interaction of Cyclic RGD Peptidomimetics with αVβ6 Integrin by Biochemical and Molecular Docking Studies. Cancers 9, 128.
Demircioglu, F., and Hodivala-Dilke, K. (2016). αvβ3 Integrin and tumour blood vessels—learning from the past to shape the future. Current Opinion in Cell Biology 42, 121–127.
Desgrosellier, J.S., and Cheresh, D.A. (2010). Integrins in cancer: biological implications and therapeutic opportunities. Nature Reviews Cancer 10, 9–22.
Dong, X., Hudson, N.E., Lu, C., and Springer, T.A. (2014). Structural determinants of integrin β-subunit specificity for latent TGF-β. Nature Structural ＆ Molecular Biology 21, 1091–1096.
Ginsberg, M.H., Partridge, A., and Shattil, S.J. (2005). Integrin regulation. Current Opinion in Cell Biology 17, 509–516.
Gupta, S., Pfannkoch, E., and Regnier, F.E. (1983). High-performance cation-exchange chromatography of proteins. Analytical Biochemistry 128, 196–201.
Hatley, R.J.D., Macdonald, S.J.F., Slack, R.J., Le, J., Ludbrook, S.B., and Lukey, P.T. (2018). An αv-RGD Integrin Inhibitor Toolbox: Drug Discovery Insight, Challenges and Opportunities. Angewandte Chemie International Edition 57, 3298–3321.
Henderson, N.C., and Sheppard, D. (2013). Integrin-mediated regulation of TGFβ in fibrosis. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1832, 891–896.
Humphries, M.D., Stewart, R.D., and Gurney, K.N. (2006). A Physiologically Plausible Model of Action Selection and Oscillatory Activity in the Basal Ganglia. J. Neurosci. 26, 12921.
Hynes, R.O. (2002). Integrins: Bidirectional, Allosteric Signaling Machines. Cell 110, 673–687.
John, A.E., Porte, J., Jenkins, G., and Tatler, A.L. (2017). Methods for the Assessment of Active Transforming Growth Factor-β in Cells and Tissues. In Fibrosis, L. Rittié, ed. (New York, NY: Springer New York), pp. 351–365.
Kapp, T.G., Fottner, M., Maltsev, O.V., and Kessler, H. (2016). Small Cause, Great Impact: Modification of the Guanidine Group in the RGD Motif Controls Integrin Subtype Selectivity. Angewandte Chemie International Edition 55, 1540–1543.
Kotecha, A., Wang, Q., Dong, X., Ilca, S.L., Ondiviela, M., Zihe, R., Seago, J., Charleston, B., Fry, E.E., Abrescia, N.G.A., et al. (2017). Rules of engagement between αvβ6 integrin and foot-and-mouth disease virus. Nature Communications 8, 15408.
Leask, A., and Abraham, D.J. (2004). TGF-β signaling and the fibrotic response. The FASEB Journal 18, 816–827.
Levental, K.R., Yu, H., Kass, L., Lakins, J.N., Egeblad, M., Erler, J.T., Fong, S.F.T., Csiszar, K., Giaccia, A., Weninger, W., et al. (2009). Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling. Cell 139, 891–906.
Ley, K., Rivera-Nieves, J., Sandborn, W.J., and Shattil, S. (2016). Integrin-based therapeutics: biological basis, clinical use and new drugs. Nature Reviews Drug Discovery 15, 173–183.
Li, C., Wen, A., Shen, B., Lu, J., Huang, Y., and Chang, Y. (2011). FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnology 11, 92.
Liu, L., You, Z., Yu, H., Zhou, L., Zhao, H., Yan, X., Li, D., Wang, B., Zhu, L., Xu, Y., et al. (2017). Mechanotransduction-modulated fibrotic microniches reveal the contribution of angiogenesis in liver fibrosis. Nature Materials 16, 1252–1261.
Lo, S.H. (2006). Focal adhesions: What’s new inside. Developmental Biology 294, 280–291.
Luo, B.-H., and Springer, T.A. (2006). Integrin structures and conformational signaling. Current Opinion in Cell Biology 18, 579–586.
Maltsev, O.V., Marelli, U.K., Kapp, T.G., Di Leva, F.S., Di Maro, S., Nieberler, M., Reuning, U., Schwaiger, M., Novellino, E., Marinelli, L., et al. (2016). Stable Peptides Instead of Stapled Peptides: Highly Potent αvβ6-Selective Integrin Ligands. Angewandte Chemie International Edition 55, 1535–1539.
Margadant, C., and Sonnenberg, A. (2010). Integrin–TGF‐β crosstalk in fibrosis, cancer and wound healing. EMBO Rep 11, 97.
Nagae, M., Re, S., Mihara, E., Nogi, T., Sugita, Y., and Takagi, J. (2012). Crystal structure of α5β1 integrin ectodomain: Atomic details of the fibronectin receptor. J Cell Biol 197, 131.
Nieberler, M., Reuning, U., Reichart, F., Notni, J., Wester, H.-J., Schwaiger, M., Weinmüller, M., Räder, A., Steiger, K., and Kessler, H. (2017). Exploring the Role of RGD-Recognizing Integrins in Cancer. Cancers 9, 116.
Niu, G., and Chen, X. (2011). Why Integrin as a Primary Target for Imaging and Therapy. 18.
Rafii, S., Butler, J.M., and Ding, B.-S. (2016). Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325.
Reynolds, A.R., Hart, I.R., Watson, A.R., Welti, J.C., Silva, R.G., Robinson, S.D., Da Violante, G., Gourlaouen, M., Salih, M., Jones, M.C., et al. (2009). Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors. Nature Medicine 15, 392–400.
Rockey, D.C., Bell, P.D., and Hill, J.A. (2015). Fibrosis — A Common Pathway to Organ Injury and Failure. New England Journal of Medicine 372, 1138–1149.
Rodriguez-Nieves, J.A., and Macoska, J.A. (2013). Prostatic fibrosis, lower urinary tract symptoms and BPH. Nature Reviews Urology 10, 546–550.
Rowedder, J.E., Ludbrook, S.B., and Slack, R.J. (2017). Determining the True Selectivity Profile of αv Integrin Ligands Using Radioligand Binding: Applying an Old Solution to a New Problem. SLAS DISCOVERY: Advancing Life Sciences R＆D 22, 962–973.
Seguin, L., Desgrosellier, J.S., Weis, S.M., and Cheresh, D.A. (2015). Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance. Trends in Cell Biology 25, 234–240.
Steri, V., Ellison, T.S., Gontarczyk, A.M., Weilbaecher, K., Schneider, J.G., Edwards, D., Fruttiger, M., Hodivala-Dilke, K.M., and Robinson, S.D. (2014). Acute Depletion of Endothelial β3-Integrin Transiently Inhibits Tumor Growth and Angiogenesis in MiceNovelty and Significance. Circulation Research 114, 79–91.
Takada, Y., Ye, X., and Simon, S. (2007). The integrins. Genome Biology 8, 215.
Weller, M., Nabors, L.B., Gorlia, T., Leske, H., Rushing, E., Bady, P., Hicking, C., Perry, J., Hong, Y.-K., Roth, P., et al. (2016). Cilengitide in newly diagnosed glioblastoma: biomarker expression and outcome. Oncotarget 7.
Wolfenson, H., Lavelin, I., and Geiger, B. (2013). Dynamic Regulation of the Structure and Functions of Integrin Adhesions. Developmental Cell 24, 447–458.
Wynn, T. (2008). Cellular and molecular mechanisms of fibrosis. The Journal of Pathology 214, 199–210.
Wynn, T.A., and Ramalingam, T.R. (2012). Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nature Medicine 18, 1028–1040.
Xiong, J.-P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S.L., and Arnaout, M.A. (2002). Crystal Structure of the Extracellular Segment of Integrin αVβ3 in Complex with an Arg-Gly-Asp Ligand. 296, 6.
何艾樺 (2017). 設計具有專一性辨識整合蛋白αIIbβ3與低出血性風險之去整合突變蛋白 = Design of Integrin αIIbβ3-Specific Disintegrin Variants with a Low Risk of Bleeding.
陳柔瑞 (2016). 去整合蛋白的KGD迴圈、C端區域以及二聚體對於整合蛋白交互作用所扮演的角色 = The Roles of the KGD Loop, C-terminus, and Dimeric Forms of Disintegrins in the Interactions of Integrins.
張耀宗 (2014). 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 = Structure-activity relationships of the RGD loop, linker region, and C-terminus of Rhodostomin mutants in the recognition of integrins.
楊智凱 (2014). 探討C端區域在馬來腹蛇蛇毒蛋白中對於整合蛋白辨識所扮演的角色 = The Role of C-terminal Region of Rhodostomin in the Recognition of Integrins.
詹秉澤 (2012). 利用馬來腹蛇蛇毒蛋白作為架構設計針對整合蛋白αvβx與α5β1之專一性拮抗物 = Development of Integrins αvβx and α5β1-specific Antagonists Using Rhodostomin as a Scaffold.
梁天豪 (2012). 利用馬來腹蛇蛇毒蛋白探討去整合蛋白C端序列對於整合蛋白的辨識及受質誘導結合位的表現 = The Use of Rhodostomin to Study the Effect of C-terminal Region of Disintegrin on Recognition and Ligand-Induced Binding Site of Integrins.
郭芷歆 (2010). 探討ARGDMX motif在Rhodostomin中對於辨識整合蛋白αvβ3、αIIbβ3和α5β1所扮演的角色 = The Role of the ARGDMX Motif of Rhodostomin in Recognition of Integrins αvβ3, αIIbβ3 and α5β1.