人類S100A9鈣離子結合蛋白 (鈣粒蛋白B)為S100蛋白質家族的一員，通常伴隨著S100A8鈣離子結合蛋白 (鈣粒蛋白A) 出現，此系列蛋白在結構上具有高度相似性，通常S100A9以同質二聚體形式存在於骨髓細胞中，在發炎反應發生時，扮演著重要的調節角色。人類RAGE 蛋白質 (Receptors for the Advanced Glycation End products) 是一種免疫球蛋白，可以與多種配體結合 (如S100蛋白家族)，誘導細胞產生訊息傳遞，進而促進發炎反應、糖尿病及一些慢性疾病甚至是細胞增生的現象。
CHAPS為目前商業量產的藥物，在結構上具有助於結合細胞膜蛋白疏水性區域，而在本實驗發現對於mS100A9蛋白具有良好的活性抑制效果，因此我們希望進一步了解mS100A9-RAGE V domain、mS100A9-CHAPS之間的交互作用，並利用HADDOCK模擬在生物體內蛋白質錯合物的結構。
在本篇論文中，我們利用三維核磁共振實驗來完成蛋白質化學位移判定，將實驗所得距離、二面角、氫鍵限制條件經由軟體計算，計算解出mutant S100A9蛋白質在水溶液的二聚體結構，並搭配二維核磁共振滴定實驗，研究mS100A9蛋白與RAGE V domain蛋白以及與CHAPS之間的反應結合位置，並藉由二維光譜上的交叉峰位移，來推測mS100A9及RAGE V domain的解離常數(Kd)為5.7 μM。最後根據WST-1 Assay實驗的結果證實mS100A9是經由與RAGE上的 V domain結合後影響，並使細胞活性提升。同時CHAPS分子也能夠與RAGE V domain競爭mS100A9的結合，成功地抑制細胞活性。
此篇研究有助於了解S100A9蛋白質與RAGE V domain的反應情形，並對於未來的抑制細胞增生藥物發展有更進一步的幫助。然而後續仍須研究生物性的實驗，探討此反應是否可應用在癌症及相關疾病的治療。

Human S100A9 (Calgranulin B) is a Ca2+-binding protein from the S100 family that often accompanies human the S100A8 protein (Calgranulin A). S100A9 presents as a homodimer in myeloid cells and emerges as an important mediator during inflammation after calcium binds to its EF hand motifs. Human receptors for the advanced glycation end products (RAGE) protein is one of the target proteins for S100A9 binding to its hydrophobic surface. Interaction between these two proteins will trigger signaling transduction cascades that promote cell growth, proliferation, and tumorigenesis. Here, we solved the solution structure of the mS100A9 homodimer by conducting multi-dimensional NMR experiments. We further characterize the solution interactions between mS100A9 and the RAGE V domain as well as S100A9 with the CHAPS molecule via NMR spectroscopy. Finally, using the HADDOCK program, we demonstrate that CHAPS molecules play a crucial role in blocking the interaction between mS100A9 and the RAGE V domain. WST-1 assay results also support that CHAPS inhibit the bioactivity of mS100A9. This report will help to inform new drug development against cell proliferation.

1. Schneider M, Fu X, Keating AE. X-ray vs. Nmr structures as templates for computational protein design. Proteins 2009:77(1): 97-110.
2. Bax A, Ikura M. An efficient 3d nmr technique for correlating the proton and 15n backbone amide resonances with the alpha-carbon of the preceding residue in uniformly 15n/13c enriched proteins. J Biomol NMR 1991:1(1): 99-104.
3. Brutscher B, Cordier F, Simorre JP, Caffrey M, Marion D. High-resolution 3d hncoca experiment applied to a 28 kda paramagnetic protein. J Biomol NMR 1995:5(2): 202-206.
4. Meissner A, Sorensen OW. Sequential hncacb and cbcanh protein nmr pulse sequences. J Magn Reson 2001:151(2): 328-331.
5. Arbogast L, Majumdar A, Tolman JR. Hnco-based measurement of one-bond amide 15n-1h couplings with optimized precision. J Biomol NMR 2010:46(2): 175-189.
6. Logan TM, Olejniczak ET, Xu RX, Fesik SW. A general method for assigning nmr spectra of denatured proteins using 3d hc(co)nh-tocsy triple resonance experiments. J Biomol NMR 1993:3(2): 225-231.
7. Wang H, Zuiderweg ER. Hcch-tocsy spectroscopy of 13c-labeled proteins in h2o using heteronuclear cross-polarization and pulsed-field gradients. J Biomol NMR 1995:5(2): 207-211.
8. Fletcher CM, Jones DN, Diamond R, Neuhaus D. Treatment of noe constraints involving equivalent or nonstereoassigned protons in calculations of biomacromolecular structures. J Biomol NMR 1996:8(3): 292-310.
9. Cornilescu G, Delaglio F, Bax A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 1999:13(3): 289-302.
10. Wishart D. Chemical shift index. In. Chemical shift index. Encyclopedia of biophysics: Springer; 2013.
11. Rieping W, Habeck M, Bardiaux B, Bernard A, Malliavin TE, Nilges M. Aria2: Automated noe assignment and data integration in nmr structure calculation. Bioinformatics 2007:23(3): 381-382.
12. Linge JP, Williams MA, Spronk CA, Bonvin AM, Nilges M. Refinement of protein structures in explicit solvent. Proteins 2003:50(3): 496-506.
13. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al. Crystallography & nmr system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 1998:54(Pt 5): 905-921.
14. Schäfer BW, Heizmann CW. The s100 family of ef-hand calcium-binding proteins: Functions and pathology. Trends in biochemical sciences 1996:21(4): 134-140.
15. Leclerc E, Fritz G, Vetter SW, Heizmann CW. Binding of s100 proteins to rage: An update. Biochim Biophys Acta 2009:1793(6): 993-1007.
16. Ostendorp T, Leclerc E, Galichet A, Koch M, Demling N, Weigle B, Heizmann CW, Kroneck PM, Fritz G. Structural and functional insights into rage activation by multimeric s100b. The EMBO journal 2007:26(16): 3868-3878.
17. Kiryushko D, Novitskaya V, Soroka V, Klingelhofer J, Lukanidin E, Berezin V, Bock E. Molecular mechanisms of ca2+ signaling in neurons induced by the s100a4 protein. Molecular and cellular biology 2006:26(9): 3625-3638.
18. Leukert N, Vogl T, Strupat K, Reichelt R, Sorg C, Roth J. Calcium-dependent tetramer formation of s100a8 and s100a9 is essential for biological activity. Journal of molecular biology 2006:359(4): 961-972.
19. Moroz O, Dodson G, Wilson K, Lukanidin E, Bronstein I. Multiple structural states of s100a12: A key to its functional diversity. Microscopy research and technique 2003:60(6): 581-592.
20. Zimmer DB, Chessher J, Wilson GL, Zimmer WE. S100a1 and s100b expression and target proteins in type i diabetes 1. Endocrinology 1997:138(12): 5176-5183.
21. Eue I, Konig S, Pior J, Sorg C. S100a8, s100a9 and the s100a8/a9 heterodimer complex specifically bind to human endothelial cells: Identification and characterization of ligands for the myeloid-related proteins s100a9 and s100a8/a9 on human dermal microvascular endothelial cell line-1 cells. Int Immunol 2002:14(3): 287-297.
22. Guozheng W, Zhang S, Fernig DG, Spiller D, Martin-Fernandez M, Zhang H, Yi D, Zihe R, Rudland PS, Barraclough R. Heterodimeric interaction and interfaces of s100a1 and s100p. Biochemical Journal 2004:382(1): 375-383.
23. Zong H, Madden A, Ward M, Mooney MH, Elliott CT, Stitt AW. Homodimerization is essential for the receptor for advanced glycation end products (rage)-mediated signal transduction. J Biol Chem 2010:285(30): 23137-23146.
24. Donato R. Functional roles of s100 proteins, calcium-binding proteins of the ef-hand type. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1999:1450(3): 191-231.
25. Narumi K, Miyakawa R, Ueda R, Hashimoto H, Yamamoto Y, Yoshida T, Aoki K. Proinflammatory proteins s100a8/s100a9 activate nk cells via interaction with rage. J Immunol 2015:194(11): 5539-5548.
26. Leanderson T, Ivars F. S100a9 and tumor growth. Oncoimmunology 2012:1(8): 1404-1405.
27. Foell D, Roth J. Proinflammatory s100 proteins in arthritis and autoimmune disease. Arthritis Rheum 2004:50(12): 3762-3771.
28. Baillet A. [s100a8, s100a9 and s100a12 proteins in rheumatoid arthritis]. Rev Med Interne 2010:31(6): 458-461.
29. Averill MM, Kerkhoff C, Bornfeldt KE. S100a8 and s100a9 in cardiovascular biology and disease. Arterioscler Thromb Vasc Biol 2012:32(2): 223-229.
30. Markowitz J, Carson WE, 3rd. Review of s100a9 biology and its role in cancer. Biochim Biophys Acta 2013:1835(1): 100-109.
31. Lim SY, Raftery MJ, Geczy CL. Oxidative modifications of damps suppress inflammation: The case for s100a8 and s100a9. Antioxid Redox Signal 2011:15(8): 2235-2248.
32. Sakurai S, Yonekura H, Yamamoto Y, Watanabe T, Tanaka N, Li H, Rahman AK, Myint KM, Kim CH, Yamamoto H. The age-rage system and diabetic nephropathy. J Am Soc Nephrol 2003:14(8 Suppl 3): S259-263.
33. Schmidt AM, Vianna M, Gerlach M, Brett J, Ryan J, Kao J, Esposito C, Hegarty H, Hurley W, Clauss M et al. Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface. J Biol Chem 1992:267(21): 14987-14997.
34. Averill MM, Barnhart S, Becker L, Li X, Heinecke JW, Leboeuf RC, Hamerman JA, Sorg C, Kerkhoff C, Bornfeldt KE. S100a9 differentially modifies phenotypic states of neutrophils, macrophages, and dendritic cells: Implications for atherosclerosis and adipose tissue inflammation. Circulation 2011:123(11): 1216-1226.
35. Hyogo H, Yamagishi S. Advanced glycation end products (ages) and their involvement in liver disease. Curr Pharm Des 2008:14(10): 969-972.
36. Hori O, Brett J, Slattery T, Cao R, Zhang J, Chen JX, Nagashima M, Lundh ER, Vijay S, Nitecki D et al. The receptor for advanced glycation end products (rage) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system. J Biol Chem 1995:270(43): 25752-25761.
37. Yan SD, Bierhaus A, Nawroth PP, Stern DM. Rage and alzheimer's disease: A progression factor for amyloid-beta-induced cellular perturbation? J Alzheimers Dis 2009:16(4): 833-843.
38. Yamagishi S-i. Role of advanced glycation end products (ages) and receptor for ages (rage) in vascular damage in diabetes. Experimental gerontology 2011:46(4): 217-224.
39. Zong H, Ward M, Stitt AW. Ages, rage, and diabetic retinopathy. Current Diabetes Reports 2011:11(4): 244-252.
40. Ishibashi Y, Matsui T, Takeuchi M, Yamagishi S. Metformin inhibits advanced glycation end products (ages)-induced renal tubular cell injury by suppressing reactive oxygen species generation via reducing receptor for ages (rage) expression. Hormone and metabolic research= Hormon-und Stoffwechselforschung= Hormones et metabolisme 2012:44(12): 891-895.
41. Park H, Boyington JC. The 1.5 crystal structure of human receptor for advanced glycation endproducts (rage) ectodomains reveals unique features determining ligand binding. Journal of Biological Chemistry 2010:285(52): 40762-40770.
42. Ott C, Jacobs K, Haucke E, Navarrete Santos A, Grune T, Simm A. Role of advanced glycation end products in cellular signaling. Redox Biol 2014:2(411-429.
43. Deane R, Du Yan S, Submamaryan RK, LaRue B, Jovanovic S, Hogg E, Welch D, Manness L, Lin C, Yu J et al. Rage mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nat Med 2003:9(7): 907-913.
44. Sims GP, Rowe DC, Rietdijk ST, Herbst R, Coyle AJ. Hmgb1 and rage in inflammation and cancer. Annu Rev Immunol 2010:28(367-388.
45. Yatime L, Andersen GR. Structural insights into the oligomerization mode of the human receptor for advanced glycation end-products. Febs j 2013:280(24): 6556-6568.
46. Ramasamy R, Yan SF, Schmidt AM. Receptor for age (rage): Signaling mechanisms in the pathogenesis of diabetes and its complications. Annals of the New York Academy of Sciences 2011:1243(1): 88-102.
47. Fritz G. Rage: A single receptor fits multiple ligands. Trends in biochemical sciences 2011:36(12): 625-632.
48. Xie J, Reverdatto S, Frolov A, Hoffmann R, Burz DS, Shekhtman A. Structural basis for pattern recognition by the receptor for advanced glycation end products (rage). Journal of Biological Chemistry 2008:283(40): 27255-27269.
49. Basta G, Lazzerini G, Massaro M, Simoncini T, Tanganelli P, Fu C, Kislinger T, Stern DM, Schmidt AM, De Caterina R. Advanced glycation end products activate endothelium through signal-transduction receptor rage a mechanism for amplification of inflammatory responses. Circulation 2002:105(7): 816-822.
50. Tsoporis J, Izhar S, Leong-Poi H, Desjardins J-F, Huttunen H, Parker T. S100b interaction with the receptor for advanced glycation end products (rage) a novel receptor-mediated mechanism for myocyte apoptosis postinfarction. Circulation Research 2010:106(1): 93-101.
51. Bierhaus A, Stern DM, Nawroth PP. Rage in inflammation: A new therapeutic target? Current opinion in investigational drugs (London, England: 2000) 2006:7(11): 985-991.
52. Matsumoto S, Yoshida T, Murata H, Harada S, Fujita N, Nakamura S, Yamamoto Y, Watanabe T, Yonekura H, Yamamoto H et al. Solution structure of the variable-type domain of the receptor for advanced glycation end products: New insight into age-rage interaction. Biochemistry 2008:47(47): 12299-12311.
53. Foell D, Kucharzik T, Kraft M, Vogl T, Sorg C, Domschke W, Roth J. Neutrophil derived human s100a12 (en-rage) is strongly expressed during chronic active inflammatory bowel disease. Gut 2003:52(6): 847-853.
54. Koch M, Chitayat S, Dattilo BM, Schiefner A, Diez J, Chazin WJ, Fritz G. Structural basis for ligand recognition and activation of rage. Structure 2010:18(10): 1342-1352.
55. Sorci G, Bianchi R, Riuzzi F, Tubaro C, Arcuri C, Giambanco I, Donato R. S100b protein, a damage-associated molecular pattern protein in the brain and heart, and beyond. Cardiovasc Psychiatry Neurol 2010:2010(
56. Gianni K, Vitolo MI, Shapiro P, Weber DJ. The role of the map kinase transduction pathway in regulating s100b in melanoma. The FASEB Journal 2009:23(1_MeetingAbstracts): 710.710.
57. Ishisaka R, Utsumi T, Kanno T, Arita K, Katsumura N, Akiyama J, Utsumi K. Participation of a cathepsin l-type protease in the activation of caspase-3. Cell structure and function 1999:24(6): 465-470.
58. Businaro R, Leone S, Fabrizi C, Sorci G, Donato R, Lauro G, Fumagalli L. S100b protects lan‐5 neuroblastoma cells against aβ amyloid‐induced neurotoxicity via rage engagement at low doses but increases aβ amyloid neurotoxicity at high doses. Journal of neuroscience research 2006:83(5): 897-906.
59. Heizmann CW. S100 proteins. In. S100 proteins. Encyclopedia of molecular pharmacology: Springer; 2008.
60. Mohan SK, Gupta AA, Yu C. Interaction of the s100a6 mutant (c3s) with the v domain of the receptor for advanced glycation end products (rage). Biochemical and biophysical research communications 2013:434(2): 328-333.
61. Srikrishna G, Nayak J, Weigle B, Temme A, Foell D, Hazelwood L, Olsson A, Volkmann N, Hanein D, Freeze HH. Carboxylated n-glycans on rage promote s100a12 binding and signaling. J Cell Biochem 2010:110(3): 645-659.
62. Hermani A, De Servi B, Medunjanin S, Tessier PA, Mayer D. S100a8 and s100a9 activate map kinase and nf-κb signaling pathways and trigger translocation of rage in human prostate cancer cells. Experimental cell research 2006:312(2): 184-197.
63. Leclerc E, Fritz G, Weibel M, Heizmann CW, Galichet A. S100b and s100a6 differentially modulate cell survival by interacting with distinct rage (receptor for advanced glycation end products) immunoglobulin domains. Journal of Biological Chemistry 2007:282(43): 31317-31331.
64. Huttunen HJ, Kuja-Panula J, Sorci G, Agneletti AL, Donato R, Rauvala H. Coregulation of neurite outgrowth and cell survival by amphoterin and s100 proteins through receptor for advanced glycation end products (rage) activation. Journal of Biological Chemistry 2000:275(51): 40096-40105.
65. Gasic-Milenkovic J, Loske C, Münch G. Advanced glycation endproducts cause lipid peroxidation in the human neuronal cell line sh-sy5y. Journal of Alzheimer's Disease 2003:5(1): 25-30.
66. Sharma RK, Makino CL, Hicks D, Duda T. Ros-gc interlocked ca(2+)-sensor s100b protein signaling in cone photoreceptors: Review. Front Mol Neurosci 2014:7(
67. Arcuri C, Bianchi R, Brozzi F, Donato R. S100b increases proliferation in pc12 neuronal cells and reduces their responsiveness to nerve growth factor via akt activation. J Biol Chem 2005:280(6): 4402-4414.
68. Joo JH, Yoon SY, Kim JH, Paik SG, Min SR, Lim JS, Choe IS, Choi I, Kim JW. S100a6 (calcyclin) enhances the sensitivity to apoptosis via the upregulation of caspase-3 activity in hep3b cells. J Cell Biochem 2008:103(4): 1183-1197.
69. Leclerc E, Fritz G, Weibel M, Heizmann CW, Galichet A. S100b and s100a6 differentially modulate cell survival by interacting with distinct rage (receptor for advanced glycation end products) immunoglobulin domains. J Biol Chem 2007:282(43): 31317-31331.
70. Martindale JL, Holbrook NJ. Cellular response to oxidative stress: Signaling for suicide and survival. J Cell Physiol 2002:192(1): 1-15.
71. Mori T, Koyama N, Arendash GW, Horikoshi-Sakuraba Y, Tan J, Town T. Overexpression of human s100b exacerbates cerebral amyloidosis and gliosis in the tg2576 mouse model of alzheimer's disease. Glia 2010:58(3): 300-314.
72. Wen XH, Duda T, Pertzev A, Venkataraman V, Makino CL, Sharma RK. S100b serves as a ca(2+) sensor for ros-gc1 guanylate cyclase in cones but not in rods of the murine retina. Cell Physiol Biochem 2012:29(3-4): 417-430.
73. Jensen JL, Indurthi VS, Neau DB, Vetter SW, Colbert CL. Structural insights into the binding of the human receptor for advanced glycation end products (rage) by s100b, as revealed by an s100b-rage-derived peptide complex. Acta Crystallogr D Biol Crystallogr 2015:71(Pt 5): 1176-1183.
74. Itou H, Yao M, Fujita I, Watanabe N, Suzuki M, Nishihira J, Tanaka I. The crystal structure of human mrp14 (s100a9), a ca(2+)-dependent regulator protein in inflammatory process. J Mol Biol 2002:316(2): 265-276.