臺灣博碩士論文加值系統

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。

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.

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

Adachi, M., Taki, T., Higashiyama, M., Kohno, N., Inufusa, H., and Miyake, M. (2000). Significance of integrin alpha5 gene expression as a prognostic factor in node-negative non-small cell lung cancer. Clinical cancer research 6, 96-101.
Altieri, D.C., Plescia, J., and Plow, E.F. (1993). The structural motif glycine 190-valine 202 of the fibrinogen gamma chain interacts with CD11b/CD18 integrin (alphaM beta 2, Mac-1) and promotes leukocyte adhesion. J Biol Chem 268, 1847-1853.
Anangi, R., Chen, C.Y., Cheng, C.H., Chen, Y.C., Chen, C.C., Chu, Y.P., Chang, C.H., Jeng, W.Y., Shiu, J.H., Chuang, W.J. (2007). Expression of snake venom toxins in Pichia pastoris. Toxin Reviews 26, 169-187.
Atkinson, R. A., Saudek, V., and Pelton, J.T. (1994). Echistatin: the refined structure of a disintegrin in solution by 1H NMR and restrained molecular dynamics. Int J Pept Protein Res. 43, 563-572
Berman, A.E., Kozlova, N.I., and Morozevich, G.E. (2003). Integrins: structure and signaling. Biochemistry (Mosc) 68, 1284-1299.
Bilgrami, S., Tomar, S., Yadav, S., Kaur, P., Kumar, J., Jabeen, T., Sharma, S., and Singh, T.P. (2004). Crystal structure of schistatin, a disintegrin homodimer from saw-scaled viper (Echis carinatus) at 2.5 A resolution. Journal of molecular biology 341, 829-837.
Bilgrami, S., Yadav, S., Kaur, P., Sharma, S., Perbandt, M., Betzel, C., and Singh, T.P. (2005). Crystal structure of the disintegrin heterodimer from saw-scaled viper (Echis carinatus) at 1.9 A resolution. Biochemistry 44, 11058-11066.
Blobel, C.P., and White, J.M. (1992) Structure, function and evolutionary relationship of proteins containing a disintegrin domain. Curr Opin Cell Biol. 4, 760-5.
Brunger, A.T. (1992). X-PLOR :version 3.1 : a system for x-ray crystallography and NMR. New Haven, Yale University Press.
Calvete, J.J. (2005). Structure-function correlations of snake venom disintegrins. Current pharmaceutical design 11, 829-835.
Calvete JJ, Moreno-Murciano MP, Theakston RD, Kisiel DG, Marcinkiewicz C. (2003). Snake venom disintegrins: novel dimeric disintegrins and structural diversification by disulphide bond engineering. Biochem J. 372, 725-34.
Calvete, J.J., Marcinkiewicz, C., Monleon, D., Esteve, V., Celda, B., Juarez, P., and Sanz, L. (2005). Snake venom disintegrins: evolution of structure and function. Toxicon 45, 1063-1074.
Carr, P.A., Erickson, H.P., and Palmer, A.G., 3rd (1997). Backbone dynamics of homologous fibronectin type III cell adhesion domains from fibronectin and tenascin. Structure 5, 949-959.
Chen, Y., Pitzenberger, S.M., Garsky, V.M., Lumma, P.K., Sanyal, G., and Baum, J. (1991). Proton NMR assignments and secondary structure of the snake venom protein echistatin. Biochemistry 30, 11625-11636.
Chen, Y., Suri, A.K., Kominos, D., Sanyal, G., Naylor, A.M., Pitzenberger, S.M., Garsky, V.M., Levy, R.M., and Baum, J. (1994). Three-dimensional structure of echistatin and dynamics of the active site. J Biomol NMR 4, 307-324.
Chen, C.H. (2010). The roles of rhodostomin mutants with an RXD motif in integrin recognition and anti-melanoma tumor cell activity. In Department of Biochemistry and Molecular Biology (Tainan, National Cheng Kung University).
Chen, C.Y. (2005). Use of rhodostomin to study the Integrin recognition sequences and to establish a method for preparing amino-acid-type selective isotope labeling of proteins. In The Institute of Basic Medical Sciences (Tainan, National Cheng Kung University).
Chen, C.Y., Cheng, C.H., Chen, Y.C., Lee, J.C., Chou, S.H., Huang, W., Chuang, W.J. (2006). Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris. Proteins 62, 279-287.
Chen, C.Y., Shiu, J.H., Hsieh, Y.H., Liu, Y.C., Chen, Y.C., Chen, Y.C., Jeng, W.Y.,
Tang, M.J., Lo, S.J., and Chuang, W.J. (2009). Effect of D to E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: importance of the interactions between the D residue and integrin. Proteins 76, 808-821.
Chen, J., Alexander, J. S., and Orr, A.W. (2012). Integrins and Their Extracellular Matrix Ligands in Lymphangiogenesis and Lymph Node Metastasis. International Journal of Cell Biology 2012, 1-12.
Cherny, R.C., Honan, M.A., and Thiagarajan, P. (1993). Site-directed mutagenesis of the arginine-glycine-aspartic acid in vitronectin abolishes cell adhesion. J Biol Chem 268, 9725-9729.
Chuang, L.C., Chen, P.Y., Chen, C., Huang, T.H., Wang, K.T., Chiou, S.H., and Wu, S.H. (1996). Structural analysis of a biologically active echistatin analogue des(46-49)-[Ala 8,37] -echistatin gamma with three disulfide bonds by 2D-NMR and computer graphics. Biochem Biophys Res Commun. 220, 246-254.
Clore, G. M., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C. and Gronenborn, A. M. (1990). Deviations from the simple two parameter model free approach to the interpretation of 15N nuclear magnetic relaxation of proteins. J. Am. Chem. Soc. 112, 4989-4991.
Cooke, R.M., Carter, B.G., Martin, D.M., Murray-Rust, P., and Weir, M.P. (1991). Nuclear magnetic resonance studies of the snake toxin echistatin. 1H resonance assignments and secondary structure. Eur J Biochem. 202, 323-328.
Cooke, R.M., Carter, B.G., Murray-Rust, P., Hartshorn, M.J., Herzyk, P., and Hubbard, R.E. (1992). The solution structure of echistatin: evidence for disulphide bond rearrangement in homologous snake toxins. Protein Eng. 5, 473-477.
Cole, R., and Loria, J.P. (2003). FAST-Modelfree: a program for rapid automated analysis of solution NMR spin-relaxation data. J Biomol NMR 26, 203-213.
Cox, D., Brennan, M., and Moran, N. (2010). Integrins as therapeutic targets: lessons and opportunities. Nature reviews Drug discovery 9, 804-820.
Dalvit, C., Widmer, H., Bovermann, G., Breckenridge, R., and Metternich, R. (1991). 1H NMR studies of echistatin in solution. Sequential resonance assignments and secondary structure. Eur J Biochem. 202, 315-321.
Demain, A.L., and Vaishnav, P. (2009). Production of recombinant proteins by microbes and higher organisms. Biotechnol. Adv. 27, 297-306.
Dennis, M.S., Carter, P., and Lazarus, R.A. (1993). Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: analysis by site-directed mutagenesis. Proteins 15, 312-321.
Dennis, M.S., Henzel, W.J., Pitti, R.M., Lipari, M.T., Napier, M.A., Deisher, T.A., Bunting, S., and Lazarus, R.A. (1990). Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of platelet-aggregation inhibitors. Proceedings of the National Academy of Sciences of the United States of America 87, 2471-2475.
Desgrosellier, J.S., and Cheresh, D.A. (2010). Integrins in cancer: biological implications and therapeutic opportunities. Nature Rev Cancer 10, 9-22.
Diemand, A.V., and Scheib, H. (2004). iMolTalk: an interactive, internet-based protein structure analysis server. Nucleic Acids Res 32, W512-516.
Dominguez, C., Boelens, R., and Bonvin, A.M. (2003). HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125, 1731-1737.
Fiser, A., and Sali, A. (2003). Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol 374, 461-491.
Friedlander, M., Brooks, P.C., Shaffer, R.W., Kincaid, C.M., Varner, J.A., and Cheresh, D.A. (1995). Definition of two angiogenic pathways by distinct alpha v integrins. Science 270, 1500-1502.
Fujii, Y., Okuda, D., Fujimoto, Z., Horii, K., Morita, T., and Mizuno, H. (2003). Crystal structure of trimestatin, a disintegrin containing a cell adhesion recognition motif RGD. J Mol Biol 332, 1115-1122.
Gan, Z. R., Gould, R. J., Jacobs, J. W., Friedman, P. A. and Polokoff, M. A. (1988). Echistatin: a potent platelet aggregation inhibitor from the venom of the viper, Echis carinatus. J. Biol. Chem. 263, 19827–19832.
Gan, Z.R., Condra, J.H., Gould, R.J., Zivin, R.A., Bennett, C.D., Jacobs, J.W., Friedman, P.A., and Polokoff, M.A. (1989) High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin. Gene 79,159-66.
Garsky, V.M., Lumma, P.K., Freidinger, R.M., Pitzenberger, S.M., Randall, W.C., Veber, D.F., Gould, R.J., and Friedman, P.A. (1989). Chemical synthesis of echistatin, a potent inhibitor of platelet aggregation from Echis carinatus: synthesis and biological activity of selected analogs. Proc Natl Acad Sci USA. 86, 4022-4026.
Gilchrist, I.C. (2003). Platelet glycoprotein IIb/IIIa inhibitors in percutaneous coronary intervention: focus on the pharmacokinetic-pharmacodynamic relationships of eptifibatide. Clinical pharmacokinetics 42, 703-720.
Gould, R.J., Polokoff, M.A., Friedman, P.A., Huang, T.F., Holt, J.C., Cook, J.J., and Niewiarowski, S. (1990). Disintegrins: a family of integrin inhibitory proteins from viper venoms. Proceedings of the Society for Experimental Biology and Medicine Society for Experimental Biology and Medicine (New York, NY) 195, 168-171.
Gray, W.R. (1993). Echistatin disulfide bridges: selective reduction and linkage assignment. Protein Sci. 2,1749-1755.
Guo, R.T., Chou, L.J., Chen, Y.C., Chen, C.Y., Pari, K., Jen, C.J., Lo, S.J., Huang, S.L., Lee, C.Y., Chang, T.W., et al. (2001). Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin. Proteins 43, 499-508.
Hantgan, R.R., Stahle, M.C., Connor, J.H., Lyles, D.S., Horita, D.A., Rocco, M., Nagaswami, C., Weisel, J.W., and McLane, M.A. (2004). The disintegrin echistatin stabilizes integrin alphaIIbbeta3's open conformation and promotes its oligomerization. J Mol Biol 342, 1625-1636.
Hein, T.W., Platts, S.H., Waitkus-Edwards, K.R., Kuo, L., Mousa, S.A., and Meininger, G.A. (2001). Integrin-binding peptides containing RGD produce coronary arteriolar dilation via cyclooxygenase activation. Am J Physiol Heart Circ Physiol 281, H2378-2384.
Hermosilla, T., Munoz, D., Herrera-Molina, R., Valdivia, A., Munoz, N., Nham, S.U., Schneider, P., Burridge, K., Quest, A.F., and Leyton, L. (2008). Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication. Biochim Biophys Acta 1783, 1111-1120.
Hirsch, E., Iglesias, A., Potocnik, A.J., Hartmann, U., and Fassler, R. (1996). Impaired migration but not differentiation of haematopoietic stem cells in the absence of beta1 integrins. Nature 380, 171-175.
Hogg, P.J. (2003). Disulfide bonds as switches for protein function. Trends Biochem. Sci. 28, 210-214.
Horton, M.A. (1995). Interactions of connective tissue cells with the extracellular matrix. Bone 17, 51S-53S.
Huang, T.F. (1998). What have snakes taught us about integrins? Cellular and molecular life sciences : Cell Mol Life Sci 54, 527-540.
Huang, T.F., Sheu, J.R., Teng, C.M., Chen, S.W., and Liu, C.S. (1991). Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex. J Biochem 109, 328-334.
Huang, T.F., Wu, Y.J., and Ouyang, C. (1987). Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom. Biochim Biophys Acta 925, 248-257.
Humphries, J.D., Byron, A., and Humphries, M.J. (2006). Integrin ligands at a glance. J Cell Sci 119, 3901-3903.
Humphries, M.J. (1990). The molecular basis and specificity of integrin-ligand interactions. J Cell Sci 97, 585-592.
Hynes, R.O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell 110, 673-687.
Jacobson, M.A., Forma, F.M., Buenaga, R.F., Hofmann, K.J., Schultz, L.D., Gould, R.J., and Friedman, P.A. (1989). Expression and secretion of biologically active echistatin in Saccharomyces cerevisiae. Gene 85, 511-516.
Juliano, D., Wang, Y., Marcinkiewicz, C., Rosenthal, L.A., Stewart, G.J. and Niewiarowski, S. (1996) Disintegrin interaction with αvβ3 integrin on human umbilical vein endothelial cells: expression of ligand-induced binding site on β3 subunit. Exp Cell Res 225, 132-142.
Kay, L.E., Muhandiram, D.R., Farrow, N.A., Aubin, Y., and Forman-Kay, J.D. (1996). Correlation between dynamics and high affinity binding in an SH2 domain interaction. Biochemistry 35, 361-368.
Kim, S., Bell, K., Mousa, S.A., and Varner, J.A. (2000). Regulation of angiogenesis in vivo by ligation of integrin alpha5beta1 with the central cell-binding domain of fibronectin. Am J Pathol 156, 1345-1362.
Kim, S.I., Kim, K.S., Kim, H.S., Choi, M.M., Kim, D.S., Chung, K.H., and Park, Y.S. (2004). Inhibition of angiogenesis by salmosin expressed in vitro. Oncol Res 14, 227-233.
Klaus, W., Broger, C., Gerber, P., and Senn, H. (1993). Determination of the disulphide bonding pattern in proteins by local and global analysis of nuclear magnetic resonance data. Application to flavoridin. J Mol Biol 232, 897-906.
Kleckner, I.R., and Foster, M.P. (2011). An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta 1814, 942-968.
Koh, D.C., Armugam, A., and Jeyaseelan, K. (2006). Snake venom components and their applications in biomedicine. Cell Mol Life Sci 63, 3030-3041.
Koivunen, E., Wang, B., and Ruoslahti, E. (1995). Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins. Biotechnology (N Y) 13, 265-270.
Koradi, R., Billeter, M., and Wuthrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph 14, 51-55, 29-32.
Krieger, F., Moglich, A., and Kiefhaber, T. (2005). Effect of proline and glycine residues on dynamics and barriers of loop formation in polypeptide chains. J Am Chem Soc 127, 3346-3352.
Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26, 283-291.
Lee, L.K., Rance, M., Chazin, W.J., and Palmer, A.G., 3rd (1997). Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13C alpha nuclear spin relaxation. J Biomol NMR 9, 287-298.
Lipari, G., and Szabo, A. (1982). Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J Am Chem Soc 104, 4546-4559.
Liu, C.Z., Wang, Y.W., Shen, M.C., and Huang, T.F. (1994). Analysis of human platelet glycoprotein IIb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry. Thromb Haemost 72, 919-925.
Lu, X., Lu, D., Scully, M.F., and Kakkar, V.V. (2003). Modulation of integrin-binding selectivity by mutation within the RGD-loop of snake venom proteins: a novel drug development approach. Curr Med Chem Cardiovasc Hemato Agents 1, 189-196.
Lu X, Lu D, Scully MF, Kakkar VV. (2005) Snake venom metalloproteinase containing a disintegrin-like domain, its structure-activity relationships at interacting with integrins. Curr Med Chem Cardiovasc Hematol Agents. 3, 249-60.
Mandel, A.M., Akke, M., and Palmer, A.G., 3rd (1995). Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme. J Mol Biol 246, 144-163.
Marcinkiewicz, C., Vijay-Kumar, S., McLane, M.A., and Niewiarowski, S. (1997). Significance of RGD loop and C-terminal domain of echistatin for recognition of alphaIIb beta3 and alpha(v) beta3 integrins and expression of ligand-induced binding site. Blood 90, 1565-1575.
Marcinkiewicz, C. (2005). Functional characteristic of snake venom disintegrins: potential therapeutic implication. Curr Phar Des 11, 815-827.
Mas-Moruno, C., Beck, J.G., Doedens, L., Frank, A.O., Marinelli, L., Cosconati, S., Novellino, E., and Kessler, H. (2011). Increasing alphavbeta3 selectivity of the anti-angiogenic drug cilengitide by N-methylation. Angew Chem Int Ed Eng 50, 9496-9500.
Mas-Moruno, C., Rechenmacher, F., and Kessler, H. (2010). Cilengitide: the first anti-angiogenic small molecule drug candidate design, synthesis and clinical evaluation. Anticancer Agents Med Chem 10, 753-768.
Matter, M.L., Zhang, Z., Nordstedt, C., and Ruoslahti, E. (1998). The alpha5 beta1 integrin mediates elimination of amyloid-beta peptide and protects against apoptosis. J Cell Biol 141, 1019-1030.
McLane, M.A., Kowalska, M.A., Silver, L., Shattil, S.J., and Niewiarowski, S. (1994). Interaction of disintegrins with the alpha IIb beta 3 receptor on resting and activated human platelets. Biochem J. 301, 429-436.
McLane, M.A., Vijay-Kumar, S., Marcinkiewicz, C., Calvete, J.J., and Niewiarowski, S. (1996). Importance of the structure of the RGD-containing loop in the disintegrins echistatin and eristostatin for recognition of alpha IIb beta 3 and alphav beta3 integrins. FEBS Lett. 391, 139-143.
McLane MA, Kuchar MA, Brando C, Santoli D, Paquette-Straub CA, Miele ME. (2001). New insights on disintegrin-receptor interactions: eristostatin and melanoma cells. Haemostasis 31, 177-82.
McLane, M.A., Sanchez, E.E., Wong, A., Paquette-Straub, C., and Perez, J.C. (2004). Disintegrins. Curr Drug Targets Cardiovasc Haematol Disord 4, 327-355.
Minea, R., Swenson, S., Costa, F., Chen, T.C., and Markland, F.S. (2005). Development of a novel recombinant disintegrin, Contortrostatin, as an effective anti-tumor and anti- angiogenic agent. Pathophysiol Haemost Thromb 34, 177–183.
Miyata, S., Koshikawa, N., Yasumitsu, H., and Miyazaki, K. (2000). Trypsin stimulates integrin alpha(5)beta(1)-dependent adhesion to fibronectin and proliferation of human gastric carcinoma cells through activation of proteinase-activated receptor-2. J Biol Chem 275, 4592-4598.
Mizejewski, G.J. (1999). Role of Integrins in Cancer: Survey of Expression Patterns. Proc Soc Exp Biol Med 222, 124-138.
Moiseeva, N., Bau, R., Swenson, S.D., Markland, F.S., Jr., Choe, J.Y., Liu, Z.J., and Allaire, M. (2008). Structure of acostatin, a dimeric disintegrin from Southern copperhead (Agkistrodon contortrix contortrix), at 1.7 A resolution. Acta Crystallogr D Biol Crystallogr 64, 466-470.
Monleón, D., Moreno-Murciano, M.P., Kovacs, H., Marcinkiewicz, C., Calvete, J.J. and Celda, B. (2003). Concerted Motions of the Integrin-binding Loop and the C-terminal Tail of the Non-RGD Disintegrin Obtustatin. J Biol Chem 278, 45570-45576.
Monleon, D., Esteve, V., Kovacs, H., Calvete, J.J., and Celda, B. (2005). Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR. Biochem J 387, 57-66.
Morton, A., and Matthews, B.W. (1995). Specificity of ligand binding in a buried nonpolar cavity of T4 lysozyme: linkage of dynamics and structural plasticity. Biochemistry 34, 8576-8588.
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, 131-140.
Pang, J.T., Fort, S., Della Siega, A., and Cohen, E.A. (2002). Emergency coronary artery bypass surgery in the era of glycoprotein IIb/IIIa receptor antagonist use. J Card Surg 17, 425-431.
Pierschbacher, M.D., and Ruoslahti, E. (1984). Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309, 30-33.
Pouwels, J., Nevo, J., Pellinen, T., Yla¨nne, J., and Ivaska, J. (2012). Negative regulators of integrin activity. Journal of Cell Science 125, 1–10.
Rahman, S., Aitken, A., Flynn, G., Formstone, C and Savidge, G.F. (1998). Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes. Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation. Biochem J 335, 247–257.
Rata, I.A., Li, Y., and Jakobsson, E. (2010). Backbone statistical potential from local sequence-structure interactions in protein loops. J Phys Chem B 114, 1859-1869.
Reidhaar-Olson, J.F., and Sauer, R.T. (1990). Functionally acceptable substitutions in two alpha-helical regions of lambda repressor. Proteins 7, 306-316.
Ross, F.P., and Teitelbaum, S.L. (2005). alphavbeta3 and macrophage colony- stimulating factor: partners in osteoclast biology. Immunol Rev 208, 88-105.
Ruoslahti, E. (1991). Integrins. J Clinic Invest 87, 1-5.
Ruoslahti, E. (1996). RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 12, 697-715.
Ruoslahti, E., and Pierschbacher, M.D. (1987). New perspectives in cell adhesion: RGD and integrins. Science 238, 491-497.
Scarborough, R.M., Rose, J.W., Naughton, M.A., Phillips, D.R., Nannizzi, L., Arfsten, A., Campbell, A.M., and Charo, I.F. (1993). Characterization of the integrin specificities of disintegrins isolated from American pit viper venoms. J Biol Chem 268, 1058-1065.
Schagger, H., and von Jagow, G. (1987). Tricine-sodium dodecyl sulfate- polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368-379.
Schrodinger, L.L.C. (2010). The PyMOL Molecular Graphics System, Version 1.3r1.
Selistre-de-Araujo, H. S., Pontes, C. L. S., Montenegro, C. F., and Martin, A. C. B. M. (2010). Snake Venom Disintegrins and Cell Migration.Toxins 2, 2606-2621.
Shen, F.C. (2010). The role of the XRGD motif of rhodostomin in integrins recognition. In Department of Biochemistry and Molecular Biology (Tainan National Cheng Kung University).
Sheu, J.R., Yen, M.H., Kan, Y.C., Hung, W.C., Chang, P.T., and Luk, H.N. (1997). Inhibition of angiogenesis in vitro and in vivo: comparison of the relative activities of triflavin, an Arg-Gly-Asp-containing peptide and anti-alpha(v)beta3 integrin monoclonal antibody. Biochim Biophys Acta 1336, 445-454.
Shiu, J.H., Chen, C.Y., Chen, Y.C., Chang, Y.T., Chang, Y.S., Huang, C.H., and Chuang, W.J. (2012). Effect of P to A mutation of the N-terminal residue adjacent to the Rgd motif on rhodostomin: importance of dynamics in integrin recognition. PLoS One. 7, 28833.
Singh, P., Reimer, C.L., Peters, J.H., Stepp, M.A., Hynes, R.O., and Van De Water, L. (2004). The spatial and temporal expression patterns of integrin alpha9 beta1 and one of its ligands, the EIIIA segment of fibronectin, in cutaneous wound healing. J Invest Dermato 123, 1176-1181.
Springer, T.A., and Wang, J.H. (2004). The three-dimensional structure of integrins and their ligands, and conformational regulation of cell adhesion. Advances in Protein Chemistry 68, 29-63.
Subramanian, G.M., Fiscella, M., Lamouse-Smith, A., Zeuzem, S., and McHutchison, J.G. (2007). Albinterferon alpha-2b: a genetic fusion protein for the treatment of chronic hepatitis C. Nature biotechnology 25, 1411-1419.
Swenson S, Ramu S, Markland FS. (2007) Anti-angiogenesis and RGD-containing snake venom disintegrins. Curr Pharm Des. 13, 2860-71.
Takagi, J. (2007). Structural basis for ligand recognition by integrins. Curr Op Cell Biol 19, 557-564.
Takagi, J., Petre, B.M., Walz, T., and Springer, T.A. (2002). Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110, 599-511.
Takagi, J., and Springer, T.A. (2002). Integrin activation and structural rearrangement. Immunol Rev 186, 141-163.
Teilum, K., Olsen, J.G., and Kragelund, B.B. (2009). Functional aspects of protein flexibility. Cell Mol Life Sci 66, 2231-2247.
Vale, R., and Kreis, T. (1999). Guidebook to the Extracellular Matrix, Anchor and Adhesion Proteins, 2nd edn (Oxford, New York : Oxford University Press).
Wattam, B., Shang, D., Rahman, S., Egglezou, S., Scully, M., Kakkar, V., and Lu, X. (2001). Arg-Tyr-Asp (RYD) and Arg-Cys-Asp (RCD) motifs in dendroaspin promote selective inhibition of beta1 and beta3 integrins. Biochem J 356, 11-17.
White, C.E., Kempi, N.M., and Komives, E.A.(1994). Expression of highly disulfide- bonded proteins in Pichia pastoris. Structure 2, 1003-1005.
Wickstrom, S.A., Alitalo, K., and Keski-Oja, J. (2002). Endostatin associates with integrin alpha5 beta1 and caveolin-1, and activates Src via a tyrosyl phosphatase- dependent pathway in human endothelial cells. Cancer Res 62, 5580-5589.
Wickstrom, S.A., Alitalo, K., and Keski-Oja, J. (2004). An endostatin-derived peptide interacts with integrins and regulates actin cytoskeleton and migration of endothelial cells. J Biol Chem 279, 20178-20185.
Wider, G. (2000). Structure Determination of Biological Macromolecules in Solution Using NMR spectroscopy. BioTechniques 29, 1278–1294.
Wierzbicka-Patynowski, I., Niewiarowski, S., Marcinkiewicz, C., Calvete, J.J., Marcinkiewicz, M.M., and McLane, M.A. (1999). Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins. J Biol Chem. 274, 37809-37814.
Wishart, D. (2005). NMR spectroscopy and protein structure determination: applications to drug discovery and development. Curr Pharm Biotechnol 6, 105-120.
Wright, P.S., Saudek, V., Owen, T.J., Harbeson, S.L., and Bitonti, A.J. (1993): An echistatin C-terminal peptide activates GPIIbIIIa binding to fibrinogen, fibronectin, vitronectin and collagen type I and type IV. Biochem J 293, 263-267.
Xiao, T., Takagi, J., Coller, B.S., Wang, J.H., and Springer, T.A. (2004). Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature 432, 59-67.
Xiong, J.P., Stehle, T., Diefenbach, B., Zhang, R., Dunker, R., Scott, D.L., Joachimiak, A., Goodman, S.L., and Arnaout, M.A. (2001). Crystal structure of the extracellular segment of integrin alphaVbeta3. Science 294, 339-345.
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 alphaVbeta3 in complex with an Arg-Gly-Asp ligand. Science 296, 151-155.
Yahalom, D., Wittelsberger, A., Mierke, D.F., Rosenblatt, M., Alexander, J.M., and Chorev, M. (2002). Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study. Biochemistry 41, 8321-8331.
Yeh, C.H., Peng, H.C., and Huang, T.F. (1998). Accutin, a new disintegrin, inhibits angiogenesis in vitro and in vivo by acting as integrin alphavbeta3 antagonist and inducing apoptosis. Blood 92, 3268-3276.
Yeh, C.H., Peng, H.C., Yang, R.S., and Huang, T.F. (2001). Rhodostomin, a snake venom disintegrin, inhibits angiogenesis elicited by basic fibroblast growth factor and suppresses tumor growth by a selective alpha(v)beta(3) blockade of endothelial cells. Mol Pharm 59, 1333-1342.
Zhang, X.P., Kamata, T., Yokoyama, K., Puzon-McLaughlin, W., and Takada, Y. (1998). Specific interaction of the recombinant disintegrin-like domain of MDC-15 (metargidin, ADAM-15) with integrin alphavbeta3. J Biol Chem 273: 7345-7350.
Zhou, X., Murphy, F.R., Gehdu, N., Zhang, J., Iredale, J.P., and Benyon, R.C. (2004). Engagement of alphavbeta3 integrin regulates proliferation and apoptosis of hepatic stellate cells. The Journal of biological chemistry 279, 23996-24006.
Zhou, Y., Hagood, J.S., Lu, B., Merryman, W.D., and Murphy-Ullrich, J.E. (2010). Thy-1-integrin alphavbeta5 interactions inhibit lung fibroblast contraction-induced latent transforming growth factor-beta1 activation and myofibroblast differentiation. J Biol Chem 285, 22382-22393.