在過去研究中，人類嗜酸性血球陽離子蛋白（human eosinophil cationic protein, ECP）中所發現的十個胺基酸所組成的小片段胜肽（GBP¬¬ECP）具有與肝素結合能力和細胞穿膜能力，但其機制尚未明確，而此片段中的色氨酸可能是胜肽與細胞膜表面結合的關鍵。本研究將先建立一個仿生模型，利用肝素作為細胞膜表面的醣類以及十二烷基磷酸膽鹼（Dodecylphosphocholine,DPC）所製造的單層微胞粒（micelle）來模擬細胞膜，並利用核磁共振技術，鑑定胜肽上單一胺基酸與其他物質之間的關係。除此之外，本研究也利用同源蛋白衍生出的其他十個胺基酸的小片段胜肽GBPEDN以及GBPECP(W4R)，探討胺基酸的差異所造成與肝素結合能力和穿膜效應的影響，同時，本研究也利用螢光實驗探討色胺酸在穿膜機制中所扮演的角色，證實此胺基酸的側鏈具有進入到微胞粒中的效應。本論文將此小片段胜肽與肝素以及微胞粒作用的胺基酸建立出來，探討其關係有助於未來開發及設計新型穿膜胜肽。

A 10-residue glycosaminoglycan binding peptide derived from human eosinophil cationic protein, GBPECP, has been recently designated as a potent cell-penetrating peptide. Intracellular penetration of GBPECP highly depends on the existence of a tryptophan residue in the sequence. To realize the cell-penetrating mechanism in a model system mimicking peptide, glycan, and membrane environment, Dodecylphosphocholine (DPC) lipid micelle and heparin fragment were titrated into GBPECP solution to induce chemical shift perturbations in nuclear magnetic resonance (NMR) study. Our data showed that charged residues of R5 and K7 played substantial roles in recognizing heparin while R3 had less effect. The only aromatic residue W4, however, acted as an irreplaceable moiety for lipid membrane insertion, suggesting its critical role in related to GBPECP cell entry. Interestingly, although replacement of W4 with R4 significantly improved heparin binding activity of the peptide, such modification abolished cell penetration. Furthermore, in the absence of heparin, binding between GBPECP and DPC micelle still took place. The side chain of W4 adopts an opposite orientation from those of R5 and R7, with W4 responsible for cell penetrating and both R5 and K7 for GAG binding. These two effects appeared to synergistically promote GBPECP penetration through macropinocytosis and further modulate microenvironment of cell surface. More recently we have discovered that GBPECP treatment suppressed tumor cell migration and invasion. GBPECP, therefore, shows high potential as novel therapeutics through rapid and effective internalization, and interference with cell motility.

1. Fang, S.L., et al., A novel cell-penetrating peptide derived from human eosinophil cationic protein. PLoS One, 2013. 8(3): p. e57318.
2. Dyer, K.D. and H.F. Rosenberg, The RNase a superfamily: generation of diversity and innate host defense. Molecular diversity, 2006. 10(4): p. 585-597.
3. Cho, S., J.J. Beintema, and J. Zhang, The ribonuclease A superfamily of mammals and birds: identifying new members and tracing evolutionary histories. Genomics, 2005. 85(2): p. 208-20.
4. Yang, D., et al., Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation. The Journal of Immunology, 2004. 173(10): p. 6134-6142.
5. Boix, E., et al., Structural determinants of the eosinophil cationic protein antimicrobial activity. Biol Chem, 2012. 393(8): p. 801-15.
6. Lien, P.C., et al., In silico prediction and in vitro characterization of multifunctional human RNase3. Biomed Res Int, 2013. 2013: p. 170398.
7. García-Mayoral, M.F., et al., Insights into the glycosaminoglycan-mediated cytotoxic mechanism of eosinophil cationic protein revealed by NMR. ACS chemical biology, 2012. 8(1): p. 144-151.
8. Chien-Jung Chen, K.-C.T., Ping-Hsueh Kuo, Pei-Lin Chang, Wen-Ching Wang,Yung-Jen Chuang, and Margaret Dah-Tsyr Chang, A heparan sulfate-binding cell penetrating peptide for tumor and migration inhibition. BioMed Research International, 2014.
9. Yu, S.J., et al., Correction: Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein Inhibits Mite Allergen Der p 2 Induced Inflammasome Activation. PLoS One, 2015. 10(4): p. e0127255.
10. Cardin, A.D. and H.J. Weintraub, Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis, 1989. 9(1): p. 21-32.
11. Fan, T.C., et al., A Heparan Sulfate‐Facilitated and Raft‐Dependent Macropinocytosis of Eosinophil Cationic Protein. Traffic, 2007. 8(12): p. 1778-1795.
12. Koren, E. and V.P. Torchilin, Cell-penetrating peptides: breaking through to the other side. Trends in molecular medicine, 2012. 18(7): p. 385-393.
13. Lindgren, M., et al., Cell-penetrating peptides. Trends in pharmacological sciences, 2000. 21(3): p. 99-103.
14. Frankel, A.D. and C.O. Pabo, Cellular uptake of the tat protein from human immunodeficiency virus. Cell, 1988. 55(6): p. 1189-1193.
15. Joliot, A., et al., Antennapedia homeobox peptide regulates neural morphogenesis. Proc Natl Acad Sci U S A, 1991. 88(5): p. 1864-8.
16. Gautam, A., et al., CPPsite: a curated database of cell penetrating peptides. Database (Oxford), 2012. 2012: p. bas015.
17. Fosgerau, K. and T. Hoffmann, Peptide therapeutics: current status and future directions. Drug discovery today, 2015. 20(1): p. 122-128.
18. Koren, E. and V.P. Torchilin, Cell-penetrating peptides: breaking through to the other side. Trends Mol Med, 2012. 18(7): p. 385-93.
19. Ter-Avetisyan, G., et al., Cell entry of arginine-rich peptides is independent of endocytosis. J Biol Chem, 2009. 284(6): p. 3370-8.
20. Erazo-Oliveras, A., et al., Improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges. Pharmaceuticals (Basel), 2012. 5(11): p. 1177-209.
21. Bechara, C., et al., Tryptophan within basic peptide sequences triggers glycosaminoglycan-dependent endocytosis. The FASEB Journal, 2013. 27(2): p. 738-749.
22. Mohan, C.G., et al., The crystal structure of eosinophil cationic protein in complex with 2', 5'-ADP at 2.0 Å resolution reveals the details of the ribonucleolytic active site. Biochemistry, 2002. 41(40): p. 12100-12106.
23. Carreras, E., et al., Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein. Biochemistry, 2003. 42(22): p. 6636-6644.
24. Torrent, M., et al., Topography studies on the membrane interaction mechanism of the eosinophil cationic protein. Biochemistry, 2007. 46(3): p. 720-733.
25. García-Mayoral, M.F., et al., NMR structural determinants of eosinophil cationic protein binding to membrane and heparin mimetics. Biophysical journal, 2010. 98(11): p. 2702-2711.
26. Torrent, M., et al., Bactericidal and membrane disruption activities of the eosinophil cationic protein are largely retained in an N-terminal fragment. Biochemical Journal, 2009. 421(3): p. 425-434.
27. Torrent, M., et al., Eosinophil cationic protein aggregation: identification of an N-terminus amyloid prone region. Biomacromolecules, 2010. 11(8): p. 1983-1990.
28. Pulido, D., et al., Antimicrobial action and cell agglutination by the eosinophil cationic protein are modulated by the cell wall lipopolysaccharide structure. Antimicrobial agents and chemotherapy, 2012. 56(5): p. 2378-2385.
29. Fan, T.-c., et al., Characterization of molecular interactions between eosinophil cationic protein and heparin. Journal of Biological Chemistry, 2008. 283(37): p. 25468-25474.
30. Torrent, M., M. Nogués, and E. Boix, Eosinophil cationic protein (ECP) can bind heparin and other glycosaminoglycans through its RNase active site. Journal of Molecular Recognition, 2011. 24(1): p. 90-100.
31. Bechara, C. and S. Sagan, Cell-penetrating peptides: 20 years later, where do we stand? FEBS Letters, 2013. 587(12): p. 1693-1702.
32. Swaminathan, G.J., et al., Atomic resolution (0.98 Å) structure of eosinophil-derived neurotoxin. Biochemistry, 2002. 41(10): p. 3341-3352.
33. Boix, E., et al., Crystal structure of eosinophil cationic protein at 2.4 Å resolution. Biochemistry, 1999. 38(51): p. 16794-16801.
34. Schrodinger, LLC, The PyMOL Molecular Graphics System, Version 1.8. 2015.
35. Sorrentino, S., The eight human “canonical” ribonucleases: molecular diversity, catalytic properties, and special biological actions of the enzyme proteins. FEBS letters, 2010. 584(11): p. 2194-2200.
36. Holm, T., Cell-penetrating peptides: Uptake, stability and biological activity. 2011.
37. Delaglio, F., et al., NMRPipe: A multidimensional spectral processing system based on UNIX pipes. Journal of Biomolecular NMR, 1995. 6.
38. Hyberts, S.G., et al., Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling. Journal of biomolecular NMR, 2012. 52(4): p. 315-327.
39. Goddard, T.D. and D.G. Kneller, SPARKY. 1995: p. SPARKY 3, University of California, San Francisco.
40. Bax, A. and D.G. Davis, MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. Journal of Magnetic Resonance (1969), 1985. 65(2): p. 355-360.
41. Piotto, M., V. Saudek, and V. Sklenář, Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. Journal of biomolecular NMR, 1992. 2(6): p. 661-665.
42. Sklenar, V., et al., Gradient-tailored water suppression for 1 H-15 N HSQC experiments optimized to retain full sensitivity. Journal of Magnetic Resonance, Series A, 1993. 102(2): p. 241-245.
43. Bax, A. and D.G. Davis, Practical aspects of two-dimensional transverse NOE spectroscopy. Journal of Magnetic Resonance (1969), 1985. 63(1): p. 207-213.
44. Palmer, A.G., et al., Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy. Journal of Magnetic Resonance (1969), 1991. 93(1): p. 151-170.
45. Kay, L., P. Keifer, and T. Saarinen, Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. Journal of the American Chemical Society, 1992. 114(26): p. 10663-10665.
46. Schleucher, J., et al., A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients. Journal of biomolecular NMR, 1994. 4(2): p. 301-306.
47. Persson, D., et al., Application of a novel analysis to measure the binding of the membrane-translocating peptide penetratin to negatively charged liposomes. Biochemistry, 2003. 42(2): p. 421-429.
48. Wishart, D., B. Sykes, and F. Richards, The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry, 1992. 31(6): p. 1647-1651.
49. Metzler, W.J., et al., Characterization of the three-dimensional solution structure of human profilin: proton, carbon-13, and nitrogen-15 NMR assignments and global folding pattern. Biochemistry, 1993. 32(50): p. 13818-13829.
50. Wishart, D.S. and B.D. Sykes, The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. Journal of biomolecular NMR, 1994. 4(2): p. 171-180.
51. Williamson, M.P., Using chemical shift perturbation to characterise ligand binding. Progress in nuclear magnetic resonance spectroscopy, 2013. 73: p. 1-16.
52. Reissmann, S., Cell penetration: scope and limitations by the application of cell‐penetrating peptides. Journal of Peptide Science, 2014. 20(10): p. 760-784.
53. Acid, S.A., Lehninger principles of biochemistry. 2004: p. 254.
54. Campbell, I.D. and R.A. Dwek, Biological spectroscopy. 1984: Benjamin/Cummings Pub. Co.
55. Orfi, L., M. Lin, and C.K. Larive, Measurement of SDS micelle-peptide association using 1H NMR chemical shift analysis and pulsed-field gradient NMR spectroscopy. Analytical chemistry, 1998. 70(7): p. 1339-1345.
56. Mäler, L., Solution NMR studies of cell-penetrating peptides in model membrane systems. Advanced drug delivery reviews, 2013. 65(8): p. 1002-1011.
57. Glover, K.J., et al., Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules. Biophysical Journal, 2001. 81(4): p. 2163-2171.
58. Björnerås, J., M. Nilsson, and L. Mäler, Analysing DHPC/DMPC bicelles by diffusion NMR and multivariate decomposition. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2015. 1848(11): p. 2910-2917.
59. Hyberts, S.G., H. Arthanari, and G. Wagner, Applications of non-uniform sampling and processing. Topics in Current Chemistry, 2012. 316: p. 125-148.
60. Stejskal, E.O. and J.E. Tanner, Spin diffusion measurements: spin echoes in the presence of a time‐dependent field gradient. The journal of chemical physics, 1965. 42(1): p. 288-292.
61. Morris, K.F. and C.S. Johnson Jr, Diffusion-ordered two-dimensional nuclear magnetic resonance spectroscopy. Journal of the American Chemical Society, 1992. 114(8): p. 3139-3141.
62. Miller, C.C., The Stokes-Einstein Law for Diffusion in Solution. Proceedings of the Royal Society of London. Series A, Containing Papers of a Mathematical and Physical Character, 1924. 106(740): p. 724-749.