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研究生:陳亞萍
研究生(外文):Chen, Ya-Ping
論文名稱:Structural difference between PF4/CXCL4 and PF4v1/CXCL4L1 revealed by NMR
論文名稱(外文):利用核磁共振技術探討PF4/CXCL4和PF4v1/CXCL4L1的結構差異性
指導教授:蘇士哲蘇士哲引用關係
指導教授(外文):Sue, Shih-Che
口試委員:吳文桂黃維寧
口試日期:2011-6-24
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:48
中文關鍵詞:第四型血小板因子變異型第四型血小板因子
外文關鍵詞:PF4PF4v1
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Chemokines were described for the ability to recruit leukocytes and affect tumor growth. Platelet factor-4 (PF4, also known as CXCL4) was the first chemokine demonstrated to be able to inhibit angiogenesis. The feature makes PF4/CXCL4 to be a potent target for tumor therapy. A nonallelic PF4/CXCL4 variant, designated PF4v1/CXCL4L1 was isolated from activated human platelets. PF4v1/CXCL4L1 differs in only three amino acids located in the carboxy-terminal part, but the variant contains higher potent in anti-angiogenesis. To more realize the structural basis in regulating the anti-angiogenic activity, we identified the structure difference between two molecules in the study. PF4/CXCL4 monomer carries two intramolecular disulfide bonds and forms a stable tetramer comprising two asymmetric dimers in solution. In contrast to PF4/CXCL4, the majority state of PF4v1/CXCL4L1 is a dimer and it shows the ability to form intermolecular disulfide bond, indicating the presence of free cysteines. Well-separated resonances in NMR 1H-15N HSQC spectrum reveal a folded symmetric dimer of PF4v1/CXCL4L1. However under reduced condition, PF4v1/CXCL4L1 behaves like a molten globule while PF4/CXCL4 still enables to spontaneously fold itself without the aid of intramolecular disulfide bonds. In final, the three mutations are equally important in keeping the structural difference. Reintroducing any PF4/CXCL4 sequence back to the three mutations won’t recovery the PF4/CXCL4 structural property in PF4v1/CXCL4L1. Taken together, the three mutations change PF4/CXCL4 oligomerization states, disulfide bond connectivity as well as protein folding property. The difference might cause different anti-angiogenic activity in vivo.
趨化激素(Chemokine)可吸引白血球至發炎部位,也會影響腫瘤的生長。第四型血小板因子(PF4,又稱CXCL4)是第一個被發現可以抑制血管新生 (angiogenesis)的趨化激素,此特性可使PF4/CXCL4做為治療腫瘤的標靶蛋白。從人體血小板中分離出變異型的PF4/CXCL4,稱為PF4v1或CXCL4L1。其與PF4/CXCL4的不同在於羧基端(carboxy-terminal)部位的三個胺基酸,此三個胺基酸的不同造成PF4v1/CXCL4L1比PF4/CXCL4更具有抑制血管新生的能力。為了更加了解這兩個蛋白質對於抗血管新生作用上的調節,我們針對其在結構上的差異性來進行討論。PF4/CXCL4的單體具有兩對分子內的雙硫鍵,會形成一個穩定的四聚體,此四聚體由兩個不對稱的二聚體所構成。反觀,PF4v1/CXCL4L1為二聚體,此二聚體由分子間的雙硫鍵所形成,此現象也說明了PF4v1/CXCL4L1可能具有游離的半胱氨酸(cysteine)。核磁共振1H-15N HSQC光譜中訊號的分散顯示了PF4v1/CXCL4L1為摺疊且結構對稱的二聚體。但在還原條件下,PF4v1/CXCL4L1顯示像熔球蛋白(molten globule)的結構特性,而少了分子內雙硫鍵的PF4/CXCL4其結構是可以自發性的摺疊。此外,此三個胺基酸對於保留結構差異性都是很重要的,在PF4v1/CXCL4L1中三個胺基酸任何一個置換成PF4/CXCL4的殘基,都使得其無法回復成PF4/CXCL4的結構特性。總而言之,此三個氨基酸的突變改變了PF4/CXCL4的聚合態、雙硫鍵的連接、甚至是摺疊的特性。這些差異也許是造成「抗血管新生」活性調節上不同的原因。
Abstract 1
中文摘要 2
Abbreviations. 3
1. Introduction. 5
1.1 Angiogensis 5
1.2 CXC chemokines 5
a) Angiogenic CXC chemokines 6
b) Angiostatic CXC chemokines 6
1.3 Platelet factor-4 (PF4/CXCL4) 7
1.4 Crystal structure of PF4/CXCL4 8
1.5 PF4/CXCL4 variant, PF4v1/CXCL4L1 8
1.6 Aim of study 10
2. Material and Methods. 17
2.1 Cloning and expression 17
2.2 Purification of PF4/CXCL4 18
2.3 Purification of PF4v1/CXCL4L1 and mutations of PF4v1-L58P, PF4v1-E66K and PF4v1-H67L 18
2.4 15N-labeled sample for NMR 19
2.5 HSQC experiment 19
2.6 Circular dichroism spectroscopy 19
3. Results. 22
3.1 Helical context reduced in of PF4v1/CXCL4L1 22
3.2 Quaternary structure of PF4/CXCL4 22
3.3 Dimeric PF4v1/CXCL4L1 with symmetric structural packing 23
3.4 High entropic glutamic acid enhances PF4v1/CXCL4L1 stability 24
3.5 Considerable effect of each mutation site 25
3.6 Intermolecular disulfide bond in purified PF4v1/CXCL4L1 26
3.7 Separate two kinds of PF4v1/CXCL4L1 dimer 27
3.8 Disulfide linkages in protein folding 28
4. Discussion. 39
4.1 C-terminal helix produces structure differences between PF4/CXCL4 and PF4v1/CXCL4L1 39
4.2 Disulfide linkage in PF4v1/CXCL4L1 40
4.3 Importance of individual mutations in PF4v1/CXCL4L1 structure 41
4.4 Future approach of PF4v1/CXCL4L1 43
4.5 Summary 44
5. References. 46

1. Folkman, J. (1971) Tumor angiogenesis: therapeutic implications, N Engl J Med 285, 1182-1186.
2. Zetter, B. R. (1998) Angiogenesis and tumor metastasis, Annu Rev Med 49, 407-424.
3. Risau, W. (1997) Mechanisms of angiogenesis, Nature 386, 671-674.
4. Gastl, G., Hermann, T., Steurer, M., Zmija, J., Gunsilius, E., Unger, C., and Kraft, A. (1997) Angiogenesis as a target for tumor treatment, Oncology 54, 177-184.
5. Carmeliet, P. (2005) Angiogenesis in life, disease and medicine, Nature 438, 932-936.
6. Proudfoot, A. E., Handel, T. M., Johnson, Z., Lau, E. K., LiWang, P., Clark-Lewis, I., Borlat, F., Wells, T. N., and Kosco-Vilbois, M. H. (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines, Proc Natl Acad Sci U S A 100, 1885-1890.
7. Belperio, J. A., Keane, M. P., Arenberg, D. A., Addison, C. L., Ehlert, J. E., Burdick, M. D., and Strieter, R. M. (2000) CXC chemokines in angiogenesis, J Leukoc Biol 68, 1-8.
8. Luster, A. D. (1998) Chemokines--chemotactic cytokines that mediate inflammation, N Engl J Med 338, 436-445.
9. Strieter, R. M., Polverini, P. J., Kunkel, S. L., Arenberg, D. A., Burdick, M. D., Kasper, J., Dzuiba, J., Van Damme, J., Walz, A., Marriott, D., and et al. (1995) The functional role of the ELR motif in CXC chemokine-mediated angiogenesis, J Biol Chem 270, 27348-27357.
10. Struyf, S., Burdick, M. D., Proost, P., Van Damme, J., and Strieter, R. M. (2004) Platelets release CXCL4L1, a nonallelic variant of the chemokine platelet factor-4/CXCL4 and potent inhibitor of angiogenesis, Circ Res 95, 855-857.
11. Shellenberger, T. D., Wang, M., Gujrati, M., Jayakumar, A., Strieter, R. M., Burdick, M. D., Ioannides, C. G., Efferson, C. L., El-Naggar, A. K., Roberts, D., Clayman, G. L., and Frederick, M. J. (2004) BRAK/CXCL14 is a potent inhibitor of angiogenesis and a chemotactic factor for immature dendritic cells, Cancer Res 64, 8262-8270.
12. Maione, T. E., Gray, G. S., Petro, J., Hunt, A. J., Donner, A. L., Bauer, S. I., Carson, H. F., and Sharpe, R. J. (1990) Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides, Science 247, 77-79.
13. Vandercappellen, J., Van Damme, J., and Struyf, S. (2011) The role of the CXC chemokines platelet factor-4 (CXCL4/PF-4) and its variant (CXCL4L1/PF-4var) in inflammation, angiogenesis and cancer, Cytokine Growth Factor Rev 22, 1-18.
14. Gupta, S. K., and Singh, J. P. (1994) Inhibition of endothelial cell proliferation by platelet factor-4 involves a unique action on S phase progression, J Cell Biol 127, 1121-1127.
15. Perollet, C., Han, Z. C., Savona, C., Caen, J. P., and Bikfalvi, A. (1998) Platelet factor 4 modulates fibroblast growth factor 2 (FGF-2) activity and inhibits FGF-2 dimerization, Blood 91, 3289-3299.
16. Gengrinovitch, S., Greenberg, S. M., Cohen, T., Gitay-Goren, H., Rockwell, P., Maione, T. E., Levi, B. Z., and Neufeld, G. (1995) Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms, J Biol Chem 270, 15059-15065.
17. Sato, Y., Abe, M., and Takaki, R. (1990) Platelet factor 4 blocks the binding of basic fibroblast growth factor to the receptor and inhibits the spontaneous migration of vascular endothelial cells, Biochem Biophys Res Commun 172, 595-600.
18. Borgstrom, P., Discipio, R., and Maione, T. E. (1998) Recombinant platelet factor 4, an angiogenic marker for human breast carcinoma, Anticancer Res 18, 4035-4041.
19. Hansell, P., Maione, T. E., and Borgstrom, P. (1995) Selective binding of platelet factor 4 to regions of active angiogenesis in vivo, Am J Physiol 269, H829-836.
20. Hagedorn, M., Zilberberg, L., Lozano, R. M., Cuevas, P., Canron, X., Redondo-Horcajo, M., Gimenez-Gallego, G., and Bikfalvi, A. (2001) A short peptide domain of platelet factor 4 blocks angiogenic key events induced by FGF-2, FASEB J 15, 550-552.
21. Jouan, V., Canron, X., Alemany, M., Caen, J. P., Quentin, G., Plouet, J., and Bikfalvi, A. (1999) Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action, Blood 94, 984-993.
22. Sharpe, R. J., Byers, H. R., Scott, C. F., Bauer, S. I., and Maione, T. E. (1990) Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4, J Natl Cancer Inst 82, 848-853.
23. Lozano, R. M., Redondo-Horcajo, M., Jimenez, M. A., Zilberberg, L., Cuevas, P., Bikfalvi, A., Rico, M., and Gimenez-Gallego, G. (2001) Solution structure and interaction with basic and acidic fibroblast growth factor of a 3-kDa human platelet factor-4 fragment with antiangiogenic activity, J Biol Chem 276, 35723-35734.
24. Zhang, X., Chen, L., Bancroft, D. P., Lai, C. K., and Maione, T. E. (1994) Crystal structure of recombinant human platelet factor 4, Biochemistry 33, 8361-8366.
25. Mayo, K. H., and Chen, M. J. (1989) Human platelet factor 4 monomer-dimer-tetramer equilibria investigated by 1H NMR spectroscopy, Biochemistry 28, 9469-9478.
26. Bikfalvi, A., Dubrac, A., Quemener, C., Lacazette, E., Lopez, D., Zanibellato, C., Wu, W. G., and Prats, H. (2010) Functional divergence between 2 chemokines is conferred by single amino acid change, Blood 116, 4703-4711.
27. Vandercappellen, J., Liekens, S., Bronckaers, A., Noppen, S., Ronsse, I., Dillen, C., Belleri, M., Mitola, S., Proost, P., Presta, M., Struyf, S., and Van Damme, J. (2010) The COOH-terminal peptide of platelet factor-4 variant (CXCL4L1/PF-4var47-70) strongly inhibits angiogenesis and suppresses B16 melanoma growth in vivo, Mol Cancer Res 8, 322-334.
28. Lasagni, L., Grepin, R., Mazzinghi, B., Lazzeri, E., Meini, C., Sagrinati, C., Liotta, F., Frosali, F., Ronconi, E., Alain-Courtois, N., Ballerini, L., Netti, G. S., Maggi, E., Annunziato, F., Serio, M., Romagnani, S., Bikfalvi, A., and Romagnani, P. (2007) PF-4/CXCL4 and CXCL4L1 exhibit distinct subcellular localization and a differentially regulated mechanism of secretion, Blood 109, 4127-4134.
29. Struyf, S., Salogni, L., Burdick, M. D., Vandercappellen, J., Gouwy, M., Noppen, S., Proost, P., Opdenakker, G., Parmentier, M., Gerard, C., Sozzani, S., Strieter, R. M., and Van Damme, J. (2011) Angiostatic and chemotactic activities of the CXC chemokine CXCL4L1 (platelet factor-4 variant) are mediated by CXCR3, Blood 117, 480-488.
30. Holzwarth, G., and Doty, P. (1965) The Ultraviolet Circular Dichroism of Polypeptides, J Am Chem Soc 87, 218-228.
31. Greenfield, N., and Fasman, G. D. (1969) Computed circular dichroism spectra for the evaluation of protein conformation, Biochemistry 8, 4108-4116.
32. Venyaminov, S., Baikalov, I. A., Shen, Z. M., Wu, C. S., and Yang, J. T. (1993) Circular dichroic analysis of denatured proteins: inclusion of denatured proteins in the reference set, Anal Biochem 214, 17-24.
33. Greenfield, N. J. (2006) Using circular dichroism spectra to estimate protein secondary structure, Nature Protocols 1, 2876-2890.
34. Ross, P. D., and Subramanian, S. (1981) Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20, 3096-3102.

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