介白素-1乙型是一調控發炎及免疫反應的前驅物，實驗中我們探討經環形序列重組後之介白素-1乙型-環形序列重組蛋白36，其特性以及生物活性。環形序列重組是一蛋白質生物工程之技術，在於改變蛋白質序列，惟因三級結構通常不會有太大改變而可保有蛋白質原先的生物功能。在本篇研究中，圓二色光譜顯示環形序列重組蛋白在加溫後訊號沒有太大改變，但野生型介白素-1乙型其光譜在加溫至55度時訊號明顯減弱，結果顯示環形序列重組蛋白較野生型介白素-1乙型擁有較高的耐熱性。在血漿皮質醇含量檢測上，基於圓二色光譜結果顯示環形序列重組36蛋白在加熱至50度時較野生型穩定，結果顯示蛋白質加熱後注射至動物體內環形序列重組36蛋白其皮質醇含量在加溫至65度顯示仍具有約84個百分比的生物活性，而野生型介白素-1乙型在加溫到45至50度時皮質醇量有所減少，證明環形序列重組36蛋白較野生型介白素-1乙型更為穩定。

介白素-1受體拮抗蛋白是介白素-1天然的抑制者，具有與其競爭介白素-1受體的功能。基於介白素-1受體拮抗蛋白與介白素-1競爭進而降低由介白素-1引起的免疫反應，目前在人類方面已被用於抗發炎治療上。在此我們證實介白素-1受體拮抗蛋白其活體實驗上的生物活性以及二級和三級結構上的特徵。實驗上，圓二色光譜顯示雞之介白素-1受體拮抗蛋白其蛋白質結構為三葉草型蛋白，而晶體結構方面顯示其具有4個α螺旋和12個β鏈，以上結果顯示雞之介白素-1受體拮抗蛋白在結構方面與人的介白素-1受體拮抗蛋白相似，推測其在功能上具有與介白素-1競爭之功能。將蛋白質注射至雞翼靜脈研究雞之介白素-1受體拮抗蛋白是否與介白素-1進行競爭，結果顯示血漿皮質醇含量在注射介白素-1受體拮抗蛋白連同介白素-1乙型與單只注射介白素-1相比，注射後兩小時其皮質醇含量較單一注射介白素-1少，證明雞之介白素-1受體拮抗蛋白具有競爭功能，使腎上腺在抑制由介白素-1乙型引發的免疫反應上不須產生過多的皮質醇來進行抑制。我們也發現人的介白素-1受體拮抗蛋白其與受體結合的氨基酸與雞之介白素-1受體拮抗蛋白大致相同，五個胺基酸中只有一個與人的介白素-1受體拮抗蛋白不同，推測介白素-1受體拮抗蛋白的結合胺基酸也保留在其他物種上，以相同方式調節免疫及發炎反應。

Interleukin-1 beta (IL-1β) acts a precursor in the regulation of inflammatory and immune responses. Here, we describe the character and bioactivity on a circular permutation of chicken interleukin-1 beta, CP36. Circular permutation is a bioengineering method that changes the protein’s amino acid sequence but allows the protein to retain its original bioactivity, usually due to the conserved tertiary structure. In this study, circular dichroism showed that spectrum of CP36 has no difference when the protein is heated to 55℃, but the spectrum signal of wild-type chicken IL-1β decreased when the protein is heated to 55℃. These results indicated CP36 is more stable than WT chicken IL-1β. As observed in thermal resistance, result of CD spectrum showed the greater tolerance of CP36 in chemical resistance. On the functional assay of plasma cortisol level, according to the thermal stability, CP36 is more stable than WT when protein heats to 50℃. We injected heat-treatment protein, cortisol level showing CP36 retains about 84% bioactivity even heats to 65℃, while WT chicken IL-1β decreased when the temperature heats to 45-50℃, demonstrated CP36 is more stable than WT chicken IL-1β.

Interleukin-1 receptor antagonist (IL-1Ra) is a naturally inhibitor of interleukin-1 (IL-1) to compete interleukin-1 receptor (IL-1R). According to the effetely IL-1Ra competing with IL-1 to reduce the immune response, the treatment of IL-1Ra has been used on anti-inflammatory therapy on human. Here, we confirm the in vivo bioactivity and determine the characters on secondary and tertiary structure of IL-1Ra. In this study, the result of circular dichroism far-UV spectrum indicated chicken IL-1Ra is a β-trefoil protein. Crystal structure indicated chicken IL-1Ra contains 4 α-helixes and 12 β-strands. These results show that chicken IL-1Ra and human IL-1Ra are identical in structure, speculating chicken IL-1Ra has the ability to compete with IL-1. Protein was injected into wing vein to investigate whether IL-1Ra compete with IL-1. Results showed the plasma cortisol in the level on the group injected IL-1Ra with IL-1β decreased than that only IL-1β injected lasted two hours, confirming that chicken IL-1Ra has the ability to compete with IL-1β. The production of cortisol produced by adrenal gland decreased enough to inhibit the immune response stimulated by IL-1β. We also found the receptor binding residues in human IL-1Ra conserved in chicken IL-1Ra, there is only one different of five, suggesting the binding residues is conserved even in other species.

Abstract I
Part I: Structural and functional effects of circular permutation on chicken Interleukin-1 beta. I
Part II: Structural and functional assays of chicken Interleukin-1 receptor antagonist. II
中文摘要 III
Part I: 1
Structural and functional effects of circular permutation on chicken Interleukin-1 beta. 1
Introduction 2
1. Circular permutation 2
2. Interleukin-1 beta 2
3. Aim of the study 3
Materials and methods 5
1. Materials 5
2. Methods 6
a Circular permutation 6
b Protein construct 7
c Protein expression 7
d Protein purification 8
e Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 8
f Bradford protein-binding assay 9
g Circular dichroism 9
h Plasma cortisol levels 10
i Fluorescence spectroscopy 10
Results 11
1. Circular permutation of chicken IL-1β 11
2. Circular dichroism (CD) 11
a Secondary structure 12
b Thermal stability 12
c Chemical denaturation 12
3. Functional assay 13
4. Fluorescence 13
a Intrinsic fluorescence 14
b ANS-binding assay 14
Conclusion and Discussion 15
Figures 16
Part II: 29
Structural and functional assays of chicken Interleukin-1 receptor antagonist 29
Introduction 30
1. Interleukin-1 receptor antagonist 30
2. Interleukin-1 beta 30
3. Aim of the study 31
Materials and methods 32
1. Materials 32
2. Methods 33
a Protein construct 33
b Protein expression 33
c Protein purification 34
d Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 34
e Bradford protein-binding assay 35
f Circular dichroism 35
g Functional assay 35
h Chicken interleukin-1 receptor antagonist crystallization 36
i X-ray diffraction data collection 36
j Structure determination of chicken IL-1Ra 37
Results 38
1. Protein purification 38
2. Functional assay 38
3. Binding interface between IL-1Ra and IL-1R 39
4. Circular dichroism (CD) 39
5. Crystal structure of IL-1Ra 40
Conclusion and Discussion 42
Figures 43
Table 60
Reference 64

1. Spencer Bliven, A.P., Circular Permutation in Proteins. PLoS Computational Biology, 2012. 8(3).

2. Lo, W.C., et al., CPred: a web server for predicting viable circular permutations in proteins. Nucleic Acids Res, 2012. 40(Web Server issue): p. W232-7.

3. Cheng, C.S., et al., Structural and functional comparison of cytokine interleukin-1 beta from chicken and human. Mol Immunol, 2011. 48(6-7): p. 947-55.

4. Stephen, P., et al., Circular permutation of the starch-binding domain: inversion of ligand selectivity with increased affinity. Chem Commun (Camb), 2012. 48(20): p. 2612-4.

5. Mizobata, T., et al., Probing the Functional Mechanism of Escherichia coli GroEL Using Circular Permutation. PLoS One, 2011. 6(10): p. e26462.

6. BRUCE A. CUNNINGHAM, et al., Favin versus concanavalin A: Circularly permuted amino acid sequences. Proc Natl Acad Sci U S A, 1979. 76(7): p. 3218-3222.

7. Zhen Qian and S. Lutz, Improving the Catalytic Activity of Candida antarctica Lipase B by Circular Permutation. J. AM. CHEM. SOC, 2005. 127: p. 13466-13467.

8. Capraro, D.T., et al., Folding Circular Permutants of IL-1b: Route Selection Driven by Functional Frustration. PLoS One, 2012. 7(6): p. e38512.

9. O'Shea, E., et al., 3,4-Methylenedioxymethamphetamine increases pro-interleukin-1beta production and caspase-1 protease activity in frontal cortex, but not in hypothalamus, of Dark Agouti rats: role of interleukin-1beta in neurotoxicity. Neuroscience, 2005. 135(4): p. 1095-105.

10. Orio, L., et al., 3,4-Methylenedioxymethamphetamine increases interleukin-1beta levels and activates microglia in rat brain: studies on the relationship with acute hyperthermia and 5-HT depletion. J Neurochem, 2004. 89(6): p. 1445-53.

11. Franco-Barraza, J., et al., Actin cytoskeleton participation in the onset of IL-1beta induction of an invasive mesenchymal-like phenotype in epithelial MCF-7 cells. Arch Med Res, 2010. 41(3): p. 170-81.

12. Norman W. Baylor, W.E., Paul Richman, Aluminum salts in vaccines—US perspective. Vaccine, 2002. 20: p. S18–S23.

13. Hogenesch, H., Mechanism of immunopotentiation and safety of aluminum adjuvants. Front Immunol, 2012. 3: p. 406.

14. Zhan, N., et al., Enhancement of humoral immunity in mice by coupling pUCpGs10 and aluminium to the HCV recombinant immunogen. Virol J, 2011. 8: p. 507.

15. Tsung-Teng Huang1, 3,4, David M. Ojcius1,5, John D. Young1,3,6,7, Yi-Hui Wu8, Yun-Fei Ko7, Tsui- and Yin Wong1, 4, Cheng-Yeu Wu1,3,4, Chia-Chen Lu9, Hsin-Chih Lai1,2,4*, The Anti-Tumorigenic Mushroom Agaricus blazei Murill Enhances IL-1b Production and Activates the NLRP3 Inflammasome in Human Macrophages. PLoS ONE, 2012. 7(7): p. e41383.

16. Lo, W.C., et al., Deciphering the preference and predicting the viability of circular permutations in proteins. PLoS One, 2012. 7(2): p. e31791.

17. Whitehead, T.A., L.M. Bergeron, and D.S. Clark, Tying up the loose ends: circular permutation decreases the proteolytic susceptibility of recombinant proteins. Protein Eng Des Sel, 2009. 22(10): p. 607-13.

18. Greenfield, N.J., Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc, 2006. 1(6): p. 2527-35.

19. ADRIANA DEL REY, H.B., ERNST SORKIN, and CHARLES A. DINARELLO, Interleukin-1 and Glucocorticoid Hormones Integrate an Immunoregulatory Feedback Circuit. Ann N Y Acad Sci. , 1987. 496: p. 85-90.

20. Adamek, D.H., et al., Structural and energetic consequences of mutations in a solvated hydrophobic cavity. J Mol Biol, 2005. 346(1): p. 307-18.

21. Shela Gorinstein, I.G., Snejana Moncheva, Marina Zemser, Moshe Weisz, Abraham Caspi, Imanuel Libman, Henry Tzvi Lerner, Simon Trakhtenberg, and Olga Martín-Belloso, Intrinsic Tryptophan Fluorescence of Human Serum Proteins and Related Conformational Changes. Journal of Protein Chemistry, 2000. 19(8): p. 637-624.

22. RJ Evans, J.B., JD Childs, GP Vigers, BJ Brandhuber, JJ Skalicky, RC Thompson and SP Eisenberg, Mapping receptor binding sites in interleukin (IL)-1 receptor antagonist and IL-1 beta by site-directed mutagenesis. Identification of a single site in IL-1ra and two sites in IL-1 beta. J. Biol. Chem., 1995. 270: p. 11477-11483.

23. Qian, L., et al., Recombinant human interleukin-1 receptor antagonist reduces acute lethal toxicity and protects hematopoiesis from chemotoxicity in vivo. Biomed Pharmacother, 2013. 67(2): p. 108-15.

24. Perrier, S., F. Darakhshan, and E. Hajduch, IL-1 receptor antagonist in metabolic diseases: Dr Jekyll or Mr Hyde? FEBS Lett, 2006. 580(27): p. 6289-94.

25. Akash, M.S., et al., Interleukin-1 receptor antagonist improves normoglycemia and insulin sensitivity in diabetic Goto-Kakizaki-rats. Eur J Pharmacol, 2013. 701(1-3): p. 87-95.

26. Mizutani, H., et al., Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up-regulates inflammatory cytokines, prostaglandin E synthesis and osteoclast formation in interleukin-1 receptor antagonist-deficient mice. J Periodontal Res, 2013.

27. Shi, X.L., et al., Effect evaluation of interleukin-1 receptor antagonist nanoparticles for mesenchymal stem cell transplantation. World J Gastroenterol, 2013. 19(12): p. 1984-91.

28. Dinarello, C.A., Biologic Basis for Interleukin-l in Disease. American Society of Hematology, 1996. 87: p. 2095-2147.

29. Omar Touzani, H.B., Julien Chuquet, Nancy Rothwell, Potential mechanisms of interleukin-1 involvement in cerebral ischaemia. Journal of Neuroimmunology, 1999. 100: p. 203-215.

30. Jones, R.C., Avian reovirus infections. Revue scientifique et technique 2000. 19(2): p. 614-625.

31. Schiff, M.H., Role of interleukin 1 and interleukin 1 receptor antagonist in the mediation of rheumatoid arthritis. Ann Rheum Dis, 2000. 59: p. i103-i108.

32. Gibson, M.S., et al., Identification, cloning, and functional characterization of the IL-1 receptor antagonist in the chicken reveal important differences between the chicken and mammals. J Immunol, 2012. 189(2): p. 539-50.

33. ADRIANA DELR EY, H.B., ERNST SORKIN, AND CHARLES A. DINARELLO Interleukin-1 and glucocorticoid hormones integrate an immunoregulatory feedback circuit. Ann N Y Acad Sci, 1987. 796: p. 85-90.

34. Latypov, R.F., et al., Biophysical characterization of structural properties and folding of interleukin-1 receptor antagonist. J Mol Biol, 2007. 368(4): p. 1187-201.