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研究生:鍾毅
研究生(外文):CHUNG, YI
論文名稱:利用電腦模擬運算重新設計胜肽以抑制細胞激素與其接受器之交互作用
論文名稱(外文):De Novo Designed Peptides by Molecular Simulations Inhibit the Interactions between Cytokines and Their Cognate Receptos
指導教授:許豪仁
指導教授(外文):HSU, HAO-JEN
口試委員:蔡惠旭江信仲
口試委員(外文):TSAI, HUI-HSUJIANG, SHINN-JONG
口試日期:2016-09-02
學位類別:碩士
校院名稱:慈濟大學
系所名稱:生物化學碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:105
語文別:英文
論文頁數:96
中文關鍵詞:敗血症細胞激素胜肽鏈電腦模擬設計表面電漿共振儀分子動態模擬
外文關鍵詞:sepsiscytokinespeptidesin silico designedsurface plasmon resonancemolecular dynamics simulations
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在發炎反應中,敗血症,又稱血液中毒症,是一種全身性系統的發炎反應症狀,通常伴隨著高發病率與高死亡率。當發炎反應中重要調控發炎的細胞激素大量表現時,身體免疫系統會啟動一連串的反應,但當細胞激素總量超出身體機制調控時,就可能會導致敗血性休克,多重器官衰竭,甚至是死亡的發生。目前為止的研究結果顯示,許多針對敗血症設計之治療藥物正在進行臨床期測試,但是整體敗血症胜肽藥物設計領域仍在努力克服(1)提高存活率、(2)下降副作用與(3)胜肽藥物效力最大化。目前了解參與發炎反應主要的促發炎細胞調節因子,例如:腫瘤壞死因子[tumor necrosis factor-α (TNF-α)]、白細胞介素6[interleukin-6(IL-6)]和白細胞介素1 -亞型[interleukin-1beta (IL-1β)]通常來自lipopolysaccharide誘導的巨噬細胞所釋放,在臨床上可以發現上述的細胞激素在敗血性休克與多重器官衰竭的嚴重性發炎反應扮演重要的角色。
本篇目的就在於利用分子嵌合與動態模擬運算技術,設計一段胜肽鏈抑制細胞激素TNF-α, IL-1β和IL-6與其細胞表面的接受器進行嵌合,使嚴重敗血症病人血液中的細胞激素濃度得以下降並且干擾細胞激素與接受器嵌合的下游訊息傳遞路徑中,初始辨識的過程。而胜肽鏈的設計將從已解出的蛋白質結晶與水溶液中的結構複合體資訊為基礎,挑選並利用電腦軟體(Discovery studio 4.5)將挑選後的胜肽鏈與細胞激素做嵌合運算,並運算蛋白質表面以了解嵌合位置的特性。三種細胞激素分析後將匯集統整嵌合位置的特性,統整後設計一條18個氨基酸,擁有較佳親和力的胜肽鏈(KCF18),再將設計的蛋白質胜肽鏈利用電腦分子動態模擬運算約200 ns以確定整體嵌合的結構穩定性。利用分子動態模擬當中獲得的200 ns軌跡檔案運算嵌合自由能(MM/PBSA binding free energy),從獲得嵌合自由能數值來比較設計前後複合體的能量結果。而本篇設計之蛋白質胜肽鏈與原始嵌合的複合體比較確實有較好的嵌合能量,其意義在於有較高的親合力。在體外與細胞實驗的部分我們利用表面電漿共振儀(surface plasmon resonance)來證明上述嵌合運算與嵌合自由能的結果,偵測細胞激素與胜肽鏈是否有嵌合,而結果證明設計的胜肽鏈確實能夠在流體中與細胞激素作用。在細胞實驗的部分我們也利用monocyte binding assay和trans-well assay證明此設計胜肽鏈具有抑制發炎的效果。WST-1的實驗分析證實所設計的胜肽鏈與原生截斷的胜肽鏈(SEM18)並不具有細胞毒殺的毒性。我們期望在未來這個設計的胜肽鏈能夠應用並設計成一種吸收器的形式,可以幫助敗血症病人降低血液中細胞激素濃度與干擾發炎反應的下游訊息傳遞。

Sepsis, blood poisoning, is a systemic inflammatory response syndrome (SIRS) with high incidence and mortality. An overwhelming systemic response brought about by the release of various inflammatory mediators, which can lead to shock, multiple organ damage and death. Up to date, several therapeutic agents have been tested in clinical trails of sepsis, but the significant survival advantage is still limited. Some pro-inflammatory cytokines, such as TNF-α released from LPS-activated macrophages, IL-6, and IL-1β, also have much influence on the septic shock and multiple organ dysfunction syndrome (MODS).
The aim of this study is using the molecular docking and molecular dynamics methods to design a preferable peptide to inhibit the cytokines, such as TNF-α, IL-6, and IL-1β binding to their cognate receptors and reduce their concentrations in the blood of patients with severe sepsis to interrupt the initial cytokine cascade. The target peptides determined from the binding sites of resolved cytokine-receptor complex structures were firstly docked to validate the selected peptide-cytokine binding site and peptides property, and then they were combined as a preferable peptide with 18 amino acids. 200 ns molecular dynamics simulations were performed to confirm the stability of complexes. The MM/PBSA binding free energies of these peptide-cytokine complexes were also calculated for comparison. The results showed that the designed peptide (KCF18) has better binding affinity than truncated peptide (SEM18) and original receptor (TNFR1) in TNF-α, and also has negative values in IL-1β and IL-6. Surface plasmon resonance (SPR) detection was performed to validate the docking and binding free energy calculations. The results indicated that the KCF18 peptide is able to capture the cytokines consistent with our predictions (KD: 60.9 μM and 111.5 μM in TNF-α and IL-6, respectively). The monocyte binding assay and trans-well assay also indicated that the SEM18 could inhibit the TNF-α-induced monocyte binding to the HMEC-1 cells. KCF18 peptide could inhibit the inflammatory response of three kinds of cytokine-induced monocyte adhesion and transmigration. The WST-1 assay indicated the designed peptide (KCF18) and truncated peptide (SEM18) were not cause cytotoxicity in HMEC-1 cells. The outcome of the research may be applied to manufacture a cytokines adsorber for treating patients with severe sepsis in the future.

CONTENTS
致謝 I
CHINESE ABSTRACT V
ABSTRACT VII
CONTENTS IX
LIST OF FIGURES XII
LIST OF TABLES XIV
LIST OF ABBREVIATIONS XV
CHAPTER 1 INTRODUCTION 1
1.1 SEPSIS…. 1
1.2 CYTOKINES 1
1.2.1 TUMOR NECROSIS FACTOR-ALPHA (TNF-α) 3
1.2.2 INTERLEUKIN-1 BETA (IL-1β) 4
1.2.3 INTERLEUKIN-6 (IL-6) 5
1.3 PEPTIDE DRUG DESIGN 6
1.4 THE AIM OF THE STUDY 7
CHAPTER 2 MATERIALS AND METHODS 8
2.1 MATERIALS 8
2.1.1 THE SOURCE OF PROTEIN STRUCTURES 8
2.1.2 THE SOURCE OF RECOMBINANT PROTEINS 8
2.1.3 PEPTIDE SYNTHESIS 8
2.2 METHODS 9
2.2.1 HOMOLOGY MODELING OF THE N-TERMINUS OF IL-6 9
2.2.2 MOLECULAR DOCKING 9
2.2.3 MOLECULAR DYNAMICS (MD) SIMULATIONS 10
2.2.4 SURFACE PLASMON RESONANCE (SPR) 11
2.2.5 CELL CULTURE 12
2.2.6 CELL VIABILITY ASSAY 12
2.2.7 MONOCYTE BINDING ASSAY 12
2.2.8 TRANSMIGRATION ASSAY 13
2.2.9 CIRCULAR DICHROISM 13
FLOWCHART 14
2.3 ANALYSIS 15
2.3.1 ROOT MEAN SQUARE DEVIATION (RMSD) 15
2.3.2 DEFINITION OF SECONDARY STRUCTURE OF PROTEINS (DSSP) 15
2.3.3 BINDING FREE ENERGY ("ΔGbind " ) 16
2.3.4 FULL CORRELATION ANALYSIS (FCA) AND FREE ENERGY LANDSCAPE (FEL) 17
2.3.5 SOLVENT ACCESSIBLE SURFACE AREA (SASA) 18
2.3.6 THE NUMBER OF HYDROGEN BOND 18
2.3.7 RADIUS OF GYRATION 18
CHAPTER 3 RESULTS 19
3.1 THE INTERACTIONS BETWEEN CYTOKINE AND ITS COGNATE RECEPTOR 20
3.1.1 TNF-ALPHA COMPLEX 20
3.1.2 IL-1 BETA COMPLEX 22
3.1.3 IL-6 COMPLEX 23
3.1.4 THE INTERACTIONS BETWEEN CYTOKINES AND TRUNCATED PEPTIDE OR NEW PEPTIDE 24
3.2 DETECTING THE BINDING AFFINITY BETWEEN PEPTIDE AND CYTOKINE BY SURFACE PLASMON RESONANCE MEASUREMENT 54
3.3 TREATING PEPTIDES TO CHECK CELL VIABILITY IN WST-1 ASSAY 57
3.4 PEPTIDES INHIBIT CYTOKINES INDUCED MONOCYTE BINDING AND TRANSMIGRATION TO ENDOTHELIAL CELLS 58
CHAPTER 4 DISCUSSION 64
CHAPTER 5 CONCLUSION 68
CHAPTER 6 FUTURE PROSPECTS 69
CHAPTER 7 REFERENCE 70


CHAPTER 7 REFERENCE

1. Cai B, Deitch EA, Ulloa L (2010) Novel insights for systemic inflammation in sepsis and hemorrhage. Mediators Inflamm 2010: 642462.

2. Hattori Y, Takano K-i, Teramae H, Yamamoto S, Yokoo H, et al. (2010) Insights Into Sepsis Therapeutic Design Based on the Apoptotic Death Pathway. J. Pharm. Sci. 114: 354-365.

3. Paul M, Shani V, Muchtar E, Kariv G, Robenshtok E, et al. (2010) Systematic review and meta-analysis of the efficacy of appropriate empiric antibiotic therapy for sepsis. Antimicrob Agents Chemother 54: 4851-4863.

4. Gutsmann T, Razquin-Olazaran I, Kowalski I, Kaconis Y, Howe J, et al. (2010) New antiseptic peptides to protect against endotoxin-mediated shock. Antimicrob Agents Chemother 54: 3817-3824.

5. Hashimoto M, Furuyashiki M, Kaseya R, Fukada Y, Akimaru M, et al. (2007) Evidence of immunostimulating lipoprotein existing in the natural lipoteichoic acid fraction. Infect Immun 75: 1926-1932.

6. Yang S, Tamai R, Akashi S, Takeuchi O, Akira S, et al. (2001) Synergistic effect of muramyldipeptide with lipopolysaccharide or lipoteichoic acid to induce inflammatory cytokines in human monocytic cells in culture. Infect Immun 69: 2045-2053.

7. Pini A, Falciani C, Mantengoli E, Bindi S, Brunetti J, et al. (2010) A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo. FASEB J 24: 1015-1022.

8. Evans ME, Pollack M (1993) Effect of antibiotic class and concentration on the release of lipopolysaccharide from Escherichia coli. J Infect Dis 167: 1336-1343.

9. Bozza FA, Salluh JI, Japiassu AM, Soares M, Assis EF, et al. (2007) Cytokine profiles as markers of disease severity in sepsis: a multiplex analysis. Crit Care 11: R49.


10. Surbatovic M, Popovic N, Vojvodic D, Milosevic I, Acimovic G, et al. (2015) Cytokine profile in severe Gram-positive and Gram-negative abdominal sepsis. Sci Rep 5: 11355.

11. Burkovskiy I, Sardinha J, Zhou J, Lehmann C (2013) Cytokine release in sepsis. Adv Biosci Biotechnol 04: 860-865.

12. Cavaillon JM, Adib-Conquy M, Fitting C, Adrie C, Payen D (2003) Cytokine cascade in sepsis. Scand J Infect Dis 35: 535-544.

13. Devi Ramnath R, Weing S, He M, Sun J, Zhang H, et al. (2009) Inflammatory mediators in sepsis: Cytokines, chemokines, adhesion molecules and gases. J Organ Dysfunct 2: 80-92.

14. Zhu J, Mohan C (2010) Toll-like receptor signaling pathways--therapeutic opportunities. Mediators Inflamm 2010: 781235.

15. Park WS, Jung WK, Lee DY, Moon C, Yea SS, et al. (2010) Cilostazol protects mice against endotoxin shock and attenuates LPS-induced cytokine expression in RAW 264.7 macrophages via MAPK inhibition and NF-kappaB inactivation: not involved in cAMP mechanisms. Int Immunopharmacol 10: 1077-1085.

16. Hietbrink F, Koenderman L, Rijkers G, Leenen L (2006) Trauma: the role of the innate immune system. World J Emerg Surg 1: 15.

17. Dhainaut JF, Yan SB, Joyce DE, Pettila V, Basson B, et al. (2004) Treatment effects of drotrecogin alfa (activated) in patients with severe sepsis with or without overt disseminated intravascular coagulation. J Thromb Haemost 2: 1924-1933.

18. Jeong YI, Jung ID, Lee CM, Chang JH, Chun SH, et al. (2009) The novel role of platelet-activating factor in protecting mice against lipopolysaccharide-induced endotoxic shock. PLoS One 4: e6503.

19. Tang CW, Feng WM, Du HM, Bao Y, Zhu M (2011) Delayed Administration of D-Ala2-D-Leu5-Enkephalin, a Delta-Opioid Receptor Agonist, Improves Survival in a Rat Model of Sepsis. Tohoku J. Exp. Med. 224: 69-76.
20. Vanhee P, van der Sloot AM, Verschueren E, Serrano L, Rousseau F, et al. (2011) Computational design of peptide ligands. Trends Biotechnol 29: 231-239.

21. Brunetti J, Lelli B, Scali S, Falciani C, Bracci L, et al. (2014) A novel phage-library-selected peptide inhibits human TNF-alpha binding to its receptors. Molecules 19: 7255-7268.

22. Ma L, Gong H, Zhu H, Ji Q, Su P, et al. (2014) A novel small-molecule tumor necrosis factor alpha inhibitor attenuates inflammation in a hepatitis mouse model. J Biol Chem 289: 12457-12466.

23. Richter F, Liebig T, Guenzi E, Herrmann A, Scheurich P, et al. (2013) Antagonistic TNF receptor one-specific antibody (ATROSAB): receptor binding and in vitro bioactivity. PLoS One 8: e72156.

24. Song MY, Park SK, Kim CS, Yoo TH, Kim B, et al. (2008) Characterization of a novel anti-human TNF-alpha murine monoclonal antibody with high binding affinity and neutralizing activity. Exp Mol Med 40: 35-42.

25. Eck MJ, Sprang SR (1989) The structure of tumor necrosis factor-alpha at 2.6 A resolution. Implications for receptor binding. J Biol Chem 264: 17595-17605.

26. Bodmer JL, Schneider P, Tschopp J (2002) The molecular architecture of the TNF superfamily. Trends Biochem Sci 27: 19-26.

27. Locksley RM, Killeen N, Lenardo MJ (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104: 487-501.

28. Zhang G (2004) Tumor necrosis factor family ligand-receptor binding. Curr Opin Struct Biol 14: 154-160.

29. Lewis AK, Valley CC, Sachs JN (2012) TNFR1 signaling is associated with backbone conformational changes of receptor dimers consistent with overactivation in the R92Q TRAPS mutant. Biochemistry 51: 6545-6555.

30. Reis CR, van Assen AH, Quax WJ, Cool RH (2011) Unraveling the binding mechanism of trivalent tumor necrosis factor ligands and their receptors. Mol Cell Proteomics 10: M110 002808.
31. Day ES, Cote SM, Whitty A (2012) Binding efficiency of protein-protein complexes. Biochemistry 51: 9124-9136.

32. Auron PE (1998) The interleukin 1 receptor: ligand interactions and signal transduction. Cytokine Growth Factor Rev 9: 221-237.

33. Issafras H, Corbin JA, Goldfine ID, Roell MK (2014) Detailed mechanistic analysis of gevokizumab, an allosteric anti-IL-1beta antibody with differential receptor-modulating properties. J Pharmacol Exp Ther 348: 202-215.

34. Vigers GP, Anderson LJ, Caffes P, Brandhuber BJ (1997) Crystal structure of the type-I interleukin-1 receptor complexed with interleukin-1beta. Nature 386: 190-194.

35. Hailey KL, Capraro DT, Barkho S, Jennings PA (2013) Allosteric switching of agonist/antagonist activity by a single point mutation in the interluekin-1 receptor antagonist, IL-1Ra. J Mol Biol 425: 2382-2392.

36. Heidary DK, Roy M, Daumy GO, Cong Y, Jennings PA (2005) Long-range coupling between separate docking sites in interleukin-1beta. J Mol Biol 353: 1187-1198.

37. Wang D, Zhang S, Li L, Liu X, Mei K, et al. (2010) Structural insights into the assembly and activation of IL-1beta with its receptors. Nat Immunol 11: 905-911.

38. Chou TH, Chuang CY, Wu CM (2010) Quantification of Interleukin-6 in cell culture medium using surface plasmon resonance biosensors. Cytokine 51: 107-111.
39. Savino R, Lahm A, Giorgio M, Cabibbo A, Tramontano A, et al. (1993) Saturation mutagenesis of the human interleukin 6 receptor-binding site: implications for its three-dimensional structure. Proc Natl Acad Sci U S A 90: 4067-4071.

40. Simpson RJ, Hammacher A, Smith DK, Matthews JM, Ward LD (1997) Interleukin-6: structure-function relationships. Protein Sci 6: 929-955.

41. Xu GY, Yu HA, Hong J, Stahl M, McDonagh T, et al. (1997) Solution structure of recombinant human interleukin-6. J Mol Biol 268: 468-481.

42. Boulanger MJ, Chow D-c, Brevnova E, Martick M, Sandford G, et al. (2004) Molecular Mechanisms for Viral Mimicry of a Human Cytokine: Activation of gp130 by HHV-8 Interleukin-6. J Mol Biol 335: 641-654.

43. Boulanger MJ, Chow DC, Brevnova EE, Garcia KC (2003) Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex. Science 300: 2101-2104.

44. Deckert F, Legay F (2000) Development and validation of an IL-6 immuno-receptor assay based on surface plasmon resonance. J Pharm Biomed Anal 23: 403-412.

45. Kalai M, Montero-Julian FA, Grotzinger J, Fontaine V, Vandenbussche P, et al. (1997) Analysis of the human interleukin-6/human interleukin-6 receptor binding interface at the amino acid level: proposed mechanism of interaction. Blood 89: 1319-1333.

46. Fosgerau K, Hoffmann T (2015) Peptide therapeutics: current status and future directions. Drug Discov Today 20: 122-128.

47. Ozkirimli E, Sariyar B (2012) Protein-Peptide Interactions Revolutionize Drug Development. 10.5772/48418.

48. Brandenburg K, Andra J, Garidel P, Gutsmann T (2011) Peptide-based treatment of sepsis. Appl Microbiol Biotechnol 90: 799-808.

49. Hruby VJ (2002) Designing peptide receptor agonists and antagonists. Nat Rev Drug Discov 1: 847-858.

50. Kozakov D, Grove LE, Hall DR, Bohnuud T, Mottarella SE, et al. (2015) The FTMap family of web servers for determining and characterizing ligand-binding hot spots of proteins. Nat Protoc 10: 733-755.

51. Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4: 363-371.



52. Veverka V, Baker T, Redpath NT, Carrington B, Muskett FW, et al. (2012) Conservation of functional sites on interleukin-6 and implications for evolution of signaling complex assembly and therapeutic intervention. J Biol Chem 287: 40043-40050.

53. Wang J, Cieplak P, Kollman PA (2000) How Well Does a Restrained Electrostatic Potential (RESP) Model Perform in Calculating Conformational Energies of Organic and Biological Molecules? J COMPUT CHEM 21: 1049-1074.

54. Moal IH, Jimenez-Garcia B, Fernandez-Recio J (2015) CCharPPI web server: computational characterization of protein-protein interactions from structure. Bioinformatics 31: 123-125.

55. Paissoni C, Spiliotopoulos D, Musco G, Spitaleri A (2014) GMXPBSA 2.0: A GROMACS tool to perform MM/PBSA and computational alanine scanning. Comput Phys Commun. 185: 2920-2929.

56. Sittel F, Jain A, Stock G (2014) Principal component analysis of molecular dynamics: on the use of Cartesian vs. internal coordinates. J Chem Phys 141: 014111.

57. Lange OF, Grubmuller H (2008) Full correlation analysis of conformational protein dynamics. Proteins 70: 1294-1312.

58. Masetti M, Falchi F, Recanatini M (2014) Protein dynamics of the HIF-2alpha PAS-B domain upon heterodimerization and ligand binding. PLoS One 9: e94986.


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