摘要
IL-1α(介白素,Interleukin-1α)為一具有多樣生物活性的訊息傳遞分子，其經由非典型路徑分泌出細胞後，會與細胞表面的受器作用並引發訊息傳遞。如引起發燒、刺激肝臟製造急性蛋白、增大淋巴細胞的回應、誘發關節退化、增加骨髓細胞數目等等。且IL-1α也是造成類風濕性關節炎與退化性關節炎的主因之一，其在人體中扮演了重要的角色。
□已有研究指出，Suramin藉由干擾IL-1α與其受器的結合而抑制軟骨被破壞，進而減緩類風濕性關節炎的症狀。在本篇論文中，我們探討一個有機化合物Suramin藉由與IL-1α結合干擾蛋白質與其受器的交互作用，最後達到抑制軟骨被破壞的效果
□首先，我們利用1H-15N HSQC擾動實驗來找出IL-1α與Suramin作用的胺基酸以描繪出結合位置。由穩定態變溫實驗發現 Suramin能夠穩定IL-1α分子。此外，為了瞭解結合態中IL-1α與Suramin的相對位置與方向性，我們使用三維共振的NMR實驗;包括HNCA、HCCONH、CCONH 、HCCH-TOCSY 與13C-edited及13C ＆15N-filtered的實驗來得到骨架與側鏈的資訊，利用HADDOCK對IL-1α與Suramin進行分子對應模擬。此計算結果說明Suramin如何影響IL-1α與IL-1RΙ的結合。

目錄
Abstract…………………………………………………………………1
摘要………………………………………………………………………3
第一章 前言
1.1 Human Interleukin-1 alpha(IL-1α)之特性……………………4
1.2 Human Interleukin-1 alpha(IL-1α)之結構……………………5
1.3 Human Interleukin-1 alpha(IL-1α)在訊息傳遞扮演的角色…6
1.4 Human Interleukin-1 alpha(IL-1α)在病理與生理的重要性.10
1.5實驗動機……………………………………………………………11
1.6生物核磁共振技術的介紹…………………………………………13
1.6.1 蛋白質分子的NMR光譜循序判定………………………………14
1.6.1.1蛋白質分子的骨架循序判定…………………………………14
1.6.1.2蛋白質分子的支鏈循序判定…………………………………15
1.7 HADDOCK(high ambiguity driven docking)介紹……………16
第二章 材料與方法
2.1 Human IL-1α的取得………………………………………………20
2.1.1蛋白質之表現……………………………………………………20
2.1.2 IL-1α蛋白質的純化……………………………………………22
2.1.3 IL-1α蛋白質的濃縮與去鹽……………………………………23
2.1.4 IL-1α蛋白質的濃度測定………………………………………25
2.2 IL-1α蛋白質的基本性質鑑定……………………………………26
2.2.1 IL-1α蛋白質質量鑑定…………………………………………26
2.2.2螢光放射光譜(Fluoresence spectrum)………………………27
2.3 IL-1α與 Suramin的恆溫滴定熱卡計實驗(VP-ITC) …………29
2.4 IL-1α蛋白質的同位素標定實驗…………………………………30
2.5 IL-1α蛋白質的分子的三維核磁共振實驗………………………32
第三章 結果與討論
3.1 IL-1α蛋白質的表現及純化………………………………………33
3.1.1異丙基硫化半乳糖(IPTG)對大腸桿菌的誘導作用……………33
3.1.2 從大腸桿菌中取得IL-1α蛋白質………………………………34
3.1.3 IL-1α蛋白質的純化與分離……………………………………35
3.1.4 IL-1α蛋白質的濃縮與去鹽……………………………………36
3.2 IL-1α蛋白質之基本性質鑑定……………………………………38
3.2.1 IL-1α蛋白質的分子量鑑定……………………………………38
3.2.2 IL-1α蛋白質的螢光光譜………………………………………39
3.3 IL-1α蛋白質的同位素標定實驗…………………………………40
3.4 Interleukin-1α與Suramin交互作用之探討……………………41
3.4.1蛋白質折疊熱力學………………………………………………41
3.4.2穩定態螢光實驗(Steady State Fluorescence Experiment)43
3.5 Interleukin-1α與Suramin交互作用位置與結構之探討………44
3.5.1 比較滴定前後HSQC變化………………………………………44
3.5.2 ANS螢光反應……………………………………………………46
3.5.3 IL-1α與 Suramin的恆溫滴定熱卡計實驗(VP-ITC) ………49
3.6以Isotope Filter NMR 幫助HADDOCK預測複合物分子對應……49
結論……………………………………………………………………57
參考文獻………………………………………………………………58

參考文獻
(1)Dinarello, C. (1988). "Biology of interleukin 1." FASEB J. 2(2): 108-115.

(2)Furutani, Y., M. Notake, et al. (1986). "Complete nucleotide sequence of the gene for human interleukin 1 alpha." Nucl. Acids Res. 14(8): 3167-3179.

(3)Graves, B. J., M. H. Hatada, et al. (1990). "Structure of interleukin 1 .alpha. at 2.7-.ANG. resolution." Biochemistry 29(11): 2679-2684.

(4)Dinarello, C. (1994). "The interleukin-1 family: 10 years of discovery." FASEB J. 8(15): 1314-1325.

(5)Fleenor, D. L., I.-H. Pang, et al. (2003). "Involvement of AP-1 in Interleukin-1{alpha}-Stimulated MMP-3 Expression in Human Trabecular Meshwork Cells." Invest. Ophthalmol. Vis. Sci. 44(8): 3494-3501.

(6)Tak, P. P. and G. S. Firestein (2001). "NF-κB: a key role in inflammatory diseases." The Journal of Clinical Investigation 107(1): 7-11.

(7)Vincenti, M. and C. Brinckerhoff (2002).
"Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific
transcription factors." Arthritis Res 4(3): 157 - 164.

(8)Gronich, J., M. Konieczkowski, et al. (1994) Interleukin 1 alpha causes rapid activation of cytosolic phospholipase A2 by phosphorylation in rat mesangial cells. J. Clin. Invest. 93：1224-1233

(9)Luo, M., N. Flamand, et al. "Metabolism of arachidonic acid to eicosanoids within the nucleus.
" Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1761(5-6): 618-625.

(10)Wolfram J., Emmanuelle P. et al.(2001) AP-1 in mouse development and tumorigenesis. Oncogene 20：2401-2412
(11)Karin, M. (1995). "The Regulation of AP-1 Activity by Mitogen-activated Protein Kinases." Journal of Biological Chemistry 270(28): 16483-16486.

(12)Mengshol, J. A., M. P. Vincenti, et al. (2000). "Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-jun N-terminal kinase, and nuclear factor kappaB: Differential regulation of collagenase 1 and collagenase 3." Arthritis & Rheumatism 43(4): 801-811.

(13)Dayer, J.-M. (2003). "The pivotal role of interleukin-1 in the clinical manifestations of rheumatoid arthritis." Rheumatology 42(suppl_2): ii3-10.

(14)Shibakawa, A., K. Yudoh, et al. (2005). "The role of subchondral bone resorption pits in osteoarthritis : MMP production by cells derived from bone marrow.
"Osteoarthritis and cartilage / OARS Osteoarthritis Research Society 13(8): 679-687
.
(15)Beutler, B. and A. Cerami (1986). "Cachectin and tumour necrosis factor as two sides of the same biological coin. " Nature 320(6063): 584-588

(16)Dinarello, C. A. (1986) Interleukin-1: Amino acid sequences, multiple biological activities and comparison with tumor necrosis factor. Year Immunol. 2：68-89

(17)Lewis, C., A. Frazer, et al. (1999). "The effect of suramin on the resorption of bovine nasal cartilage." Inflammopharmacology 7(4): 387-400.

(18)Clore, G. and A. Gronenborn (1991). "Structures of larger proteins in solution: three- and four-dimensional heteronuclear NMR spectroscopy." Science 252(5011): 1390-1399.
(19)Gronenborn, A. M. and G. M. Clore (1995). "Structures of Protein Complexes by Multidimensional Heteronuclear Magnetic Resonance Spectroscopy." Critical Reviews in Biochemistry and Molecular Biology 30(5): 351 - 385

(20)Clore, G. M. and A. M. Gronenborn (1998). "Determining the structures of large proteins and protein complexes by NMR." Trends in Biotechnology 16: 22-34

(21)Salzmann, M., G. Wider, et al. (1999). "TROSY-type Triple-Resonance Experiments for Sequential NMR Assignments of Large Proteins." Journal of the American Chemical Society 121(4): 844-848.

(22)Meissner, A., oslash, et al. (2001). "A Sequential HNCA NMR Pulse Sequence for Protein Backbone Assignment." Journal of Magnetic Resonance 150: 100-104.

(23)Dominguez, C., R. Boelens, et al. (2003). "HADDOCK:  A
Protein−Protein Docking Approach Based on Biochemical or
Biophysical Information." Journal of the American Chemical Society 125(7): 1731-1737.

(24)Gasymov, O. K. and B. J. Glasgow (2007). "ANS fluorescence: Potential to augment the identification of the external binding sites of proteins." Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 1774(3): 403-411.