抗血管新生趨化素CXCL4及CXCL4L1其受體鍵結專一性及寡聚態研究

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研究生：	吳欣勵 Wu, Hsin Li
論文名稱：	抗血管新生趨化素CXCL4及CXCL4L1其受體鍵結專一性及寡聚態研究 Receptor binding specificity and oligomerization status of anti-angiogenic chemokines, CXCL4 and CXCL4L1
指導教授：	蘇士哲 Sue, Shih Che
口試委員:	鄭惠春 Cheng, Hui Chun 徐駿森 Hsu, Chun Hua
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	54
中文關鍵詞：	趨化素、寡聚態
外文關鍵詞：	CXCL4, CXCL4L1, chemokine, oligomerization
相關次數：	點閱：1 下載：0
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趨化因子 (chemokine) 與其受體 (chemokine receptor) 之間的交互作用廣泛地參與發炎反應、血液凝集以及癌症轉移。大部分的趨化因子具有不同的寡聚態，而寡聚態的不同具有不同的生理意義。CXCL4 (PF4) 為最初被發現具有抑制血管新生活性的趨化因子，主要是透過CXCL4的受體¬－CXCR3來媒介CXCL4的抗血管新生活性。我們想更深入的了解不同的CXCL4寡聚態與CXCR3之間的調控機制，監測CXCR3的N端序列 (18-36) 與CXCL4的四聚體、二聚體以及單體之間的交互作用。我們利用pH值、氯化鈉濃度的不同以及突變的方式去調控CXCL4的寡聚態。藉由滴定CXCR3的N端胜肽到CXCL4，並且探討酪胺酸硫酸化 (tyrosine sulfation) 轉譯後修飾對趨化因子作用的重要性。酪胺酸硫酸化CXCR3胜肽對CXCL4的四聚體、二聚體及單體的作用範圍皆有所不同。
除此之外，我們也將注意集中在CXCL4L1，是CXCL4的變異蛋白，有三個殘基的不同，分別是P58L、K66E、L67H。CXCL4L1擁有比CXCL4更高的抗血管新生能力。CXCL4L1的C端螺旋結構在水溶液可能是個不穩定的結構。因為C端螺旋結構是穩定二聚體重要的角色，所以CXCL4L1比CXCL4更容易從四聚體分離成單體。我們認為不同的寡聚態與CXCL4跟CXCL4L1的抗血管新生活性相關。

Interactions of chemokines and chemokine receptors are extensively involved in blood coagulation, inflammatory and cancer metastasis. Most chemokines have distinct oligomerization states that are correlated with their biological functions. CXCL4 (PF4) is the first chemokine identified to be anti-angiogenic chemokine and its activity is mediated through its receptor CXCR3. To understand the detail mechanism of different CXCL4 oligomers in regulating CXCR3 function, we prepared CXCL4 monomer, dimer and tetramer and monitored the interaction with CXCR3 N-terminal sequence (residue 18-36). CXCL4 oligomer status was controlled by pH, NaCl concentration and mutation(s). By titrating the CXCR3 N-terminus-derived peptide into CXCL4 solutions, we characterized the critical role of post-translational modification of tyrosine sulfation for chemokine interaction. The sulfated CXCR3 N-terminus-derived peptide showed different extents in interacting CXCL4 monomer, dimer and tetramer. Furthermore, we focused on CXCL4L1, a CXCL4 variant that has three substitutions at P58L, K66E and L67H. CXCL4L1 possesses higher anti-angiogenetic activity than CXCL4. We revealed that CXCL4L1 contained a C-terminal helix with disordered property. Because of the role of C-terminal helix in stabilizing dimer interface, CXCL4L1 have greater ability to dissociate itself into monomer. We believe the different oligomer tendencies to change CXCL4 and CXCL4L1 anti-angiogensis activity.

Contents    I
Abstract    III
中文摘要    IV
Abbreviations    V
   Introduction    1
1 Chemokines    1
2 Chemokine interacts with GAG and GPCR    2
2.1 Chemokine: Receptor model    2
2.2 Role for receptor post-translation modifications    3
3 Human CXCL4 and CXCL4L1    4
3.1 CXCL4 (PF4)    4
3.2 CXCL4L1 (PF4v1)    5
3.3 Structural difference between CXCL4 and CXCL4L1    6
3.4 Regulation of angiostasis    6
4 CXCR3, the CXCL4 and CXCL4L1 downstream receptor    7
5 Aim of the study    8
   Materials and Methods    16
1 Protein Expression and Purification    16
2 Preparation 15N-labeled Protein for NMR    17
3 Peptide Synthesis    18
4 NMR Experiment    18
4.1 NMR Titrations of Protein with CXCR3-derived Peptide    18
5 Size exclusion Experiment    21
6 Dynamic Light Scattering    21
   Results    23
1 Oligomer equilibrium of CXCL4    23
2 NaCl stabilizes the repulsive force between two α-helixes    24
3 Interaction of CXCL4 to CXCR3 sulfopeptide    25
3.1 Sulfation of Tyrosine improves CXCR3 peptide binding affinity with CXCL4    25
4 Properties of sulfopeptide binding to CXCL4 in different oligomer states    26
4.1 Sulfopeptide binding to CXCL4 monomer    27
4.2 Sulfopeptide binding to CXCL4 dimer    28
4.3 Sulfopeptide binding to CXCL4 tetramer    29
5 Oligomer equilibrium of CXCL4L1    30
5.1 Oligomer states of CXCL4L1 under different pH and ionic strength    30
5.2 Monomer structural comparison between CXCL4 and CXCL4L1    31
6 Temperature dependent conformational change    32
7 E28A mutant of CXCL4L1    32
8 Sulfopeptide binding to monomer CXCL4L1    33
   Discussion    47
1 Interaction between CXCR3-sulfopeptide and CXCL4    47
2 Conformational properties of CXCL4L1    49
References    52

                                

1. Balkwill, F., Cancer and the chemokine network. Nat Rev Cancer, 2004. 4(7): p. 540-50.
2. Salanga, C. and T. Handel, Chemokine oligomerization and interactions with receptors and glycosaminoglycans: the role of structural dynamics in function. Experimental cell research, 2011. 317(5): p. 590-601.
3. Abdallah, M., T. Michel, and L. Kohidai, Autism Spectrum Disorders and Circulating Chemokines. 2014: p. 1627-1642.
4. Carlson, J., et al., The heterodimerization of platelet-derived chemokines. Biochim Biophys Acta, 2013. 1834(1): p. 158-68.
5. Handel, T.M. and E.K. Lau, Chemokine Structure and Receptor Interactions, in Chemokine Roles in Immunoregulation and Disease, P.M. Murphy and R. Horuk, Editors. 2004, Springer Berlin Heidelberg: Berlin, Heidelberg. p. 101-124.
6. Sepuru, K.M. and K. Rajarathnam, CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine: STRUCTURAL EVIDENCE FOR TWO DISTINCT NON-OVERLAPPING BINDING DOMAINS. J Biol Chem, 2016. 291(8): p. 4247-55.
7. Wang, L., et al., Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses. Nat Immunol, 2005. 6(9): p. 902-10.
8. Loetscher, P. and I. Clark-Lewis, Agonistic and antagonistic activities of chemokines. Journal of leukocyte biology, 2001. 69(6): p. 881-884.
9. Elisseeva, E.L., et al., NMR studies of active N-terminal peptides of stromal cell-derived factor-1 structural basis for receptor binding. Journal of Biological Chemistry, 2000. 275(35): p. 26799-26805.
10. Qin, L., et al., Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine. Science, 2015. 347(6226): p. 1117-1122.
11. Millard, C.J., et al., Structural basis of receptor sulfotyrosine recognition by a CC chemokine: The N-terminal region of CCR3 bound to CCL11/eotaxin-1. Structure, 2014. 22(11): p. 1571-1581.
12. Ludeman, J.P. and M.J. Stone, The structural role of receptor tyrosine sulfation in chemokine recognition. British journal of pharmacology, 2014. 171(5): p. 1167-1179.
13. Kleist, A.B., et al., New paradigms in chemokine receptor signal transduction: Moving beyond the two-site model. Biochemical pharmacology, 2016.
14. Ziarek, J.J., et al., Sulfopeptide probes of the CXCR4/CXCL12 interface reveal oligomer-specific contacts and chemokine allostery. ACS chemical biology, 2013. 8(9): p. 1955-1963.
15. Tan, J.H., et al., Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1). Journal of Biological Chemistry, 2013. 288(14): p. 10024-10034.
16. Deuel, T.F., et al., Amino acid sequence of human platelet factor 4. Proceedings of the National Academy of Sciences, 1977. 74(6): p. 2256-2258.
17. Kowalska, M.A., L. Rauova, and M. Poncz, Role of the platelet chemokine platelet factor 4 (PF4) in hemostasis and thrombosis. Thrombosis research, 2010. 125(4): p. 292-296.
18. Aidoudi, S. and A. Bikfalvi, Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis. Thrombosis & Haemostasis, 2010. 104(5): p. 941.
19. Auerbach, D.J., et al., Identification of the platelet-derived chemokine CXCL4/PF-4 as a broad-spectrum HIV-1 inhibitor. Proceedings of the National Academy of Sciences, 2012. 109(24): p. 9569-9574.
20. Zhang, X., et al., Crystal structure of recombinant human platelet factor 4. Biochemistry, 1994. 33(27): p. 8361-8366.
21. Li, J., et al., The roles and potential therapeutic implications of CXCL4 and its variant CXCL4L1 in the pathogenesis of chronic liver allograft dysfunction. Cytokine & growth factor reviews, 2015. 26(1): p. 67-74.
22. Vandercappellen, J., J. Van Damme, and S. Struyf, 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 reviews, 2011. 22(1): p. 1-18.
23. Vandercappellen, J., et al., The COOH-terminal peptide of platelet factor-4 variant (CXCL4L1/PF-4var47-70) strongly inhibits angiogenesis and suppresses B16 melanoma growth in vivo. Molecular Cancer Research, 2010. 8(3): p. 322-334.
24. Kuo, J.-H., et al., Alternative C-Terminal Helix Orientation Alters Chemokine Function STRUCTURE OF THE ANTI-ANGIOGENIC CHEMOKINE, CXCL4L1. Journal of Biological Chemistry, 2013. 288(19): p. 13522-13533.
25. Hagedorn, M., et al., Domain swapping in a COOH-terminal fragment of platelet factor 4 generates potent angiogenesis inhibitors. Cancer research, 2002. 62(23): p. 6884-6890.
26. Maione, T.E., et al., Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science, 1990. 247(4938): p. 77-79.
27. Struyf, S., et al., Platelets release CXCL4L1, a nonallelic variant of the chemokine platelet factor-4/CXCL4 and potent inhibitor of angiogenesis. Circulation research, 2004. 95(9): p. 855-857.
28. Flier, J., et al., Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. The Journal of pathology, 2001. 194(4): p. 398-405.
29. Struyf, S., et al., Angiostatic and chemotactic activities of the CXC chemokine CXCL4L1 (platelet factor-4 variant) are mediated by CXCR3. Blood, 2011. 117(2): p. 480-488.
30. Colvin, R.A., et al., CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis. Molecular and cellular biology, 2006. 26(15): p. 5838-5849.
31. Booth, V., et al., The CXCR3 binding chemokine IP-10/CXCL10: structure and receptor interactions. Biochemistry, 2002. 41(33): p. 10418-10425.
32. Salanga, C.L. and T.M. Handel, Chemokine oligomerization and interactions with receptors and glycosaminoglycans: the role of structural dynamics in function. Exp Cell Res, 2011. 317(5): p. 590-601.
33. Vandercappellen, J., J. Van Damme, and S. Struyf, 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, 2011. 22(1): p. 1-18.
34. Balkwill, F., Cancer and the chemokine network. Nature Reviews Cancer, 2004. 4(7): p. 540-550.

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