Basic fibroblast growth factor 2 (FGF2) 是一個被廣為研究的蛋白質,它一般存在於血管細胞的胞外基質(extracellular matrix)上，並與其上的硫酸乙醯肝素（heparan sulfate proteoglycans, HSPGs）結合。當受到刺激時，FGF2會與受體結合來開啟傷口愈合、血管新生重要的生理功能。但它作為一個膜外蛋白，卻沒有一般secretory protein 該有的signal peptide，無法藉由一般蛋白的分泌機制到達細胞外。近期的文獻提出一個全新的分泌模型來解釋FGF2是如何穿越細胞膜與硫酸乙醯肝素結合。此模型闡述Phosphatidylinositol 4,5-bisphosphate (PIP2)會將FGF2吸引至細胞內膜附近，FGF2在此受磷酸化修飾並形成多聚體後，在細胞膜上形成一個孔洞。再藉由與硫酸乙醯肝素的結合離開細胞膜。在western blot的實驗中更呈現若磷酸化FGF2在帶有PIP2的脂質膜上，會提升FGF2的多聚化現象。但在分子層級上， FGF2是如何形成多聚體？又磷酸化是一個出膜標記，或其以某種方式直接/間接地促使FGF2形成多聚體？故本篇論文嘗試結合結構生物學及分子動力學模擬（molecular dynamics simulations）為主的電腦方法來探索此全新分泌模型部分分子層面的細節。我們首先在FGF2晶體結構的crystal packing中找到兩個接觸較緊密的二聚體結構dimer31、dimer33。模擬結果顯示dimer31結構有高穩定性，且始終維持原交界面。我們將dimer31磷酸化進行模擬，找出磷酸化Y73 (pY73)直接與另一個次單元的R60形成鹽橋，改變原有的交界面，使其二聚化更加的穩定。我們更發現pY73不僅直接形成鹽橋，還會改變蛋白的結構使R60向外被推出，讓R60有更大的機會與另一個次單元的pY73形成分子間鹽橋。此現象亦可幫助解釋為何磷酸化的FGF2可以形成更高的多聚體。我們也建構了FGF2-PIP2 複合體的結構模型，明確指出PIP2的結合位，並發現其正確地吻合了NMR的chemical shift結果。我們認為PIP2先將FGF2吸引到細胞內膜上，使得局部濃度升高。這讓FGF2較容易被膜上激酶磷酸化並且形成較穩定的多聚體，進而穿過細胞膜。

The major function of fibroblast growth factor2 (FGF2) is to induce the wound healing signaling and angiogenesis. Because FGF2 is a secretory protein without containing a signal peptide, it cannot pass cell membrane through the known ER/Golgi-dependent secretory pathway. An unconventional secretion mechanism of FGF2 has been reported. First, FGF2 must be recruited by Phosphatidylinositol 4,5-bisphosphate(PIP2) and phosphorylated by tyrosinekinase at Y73 . FGF2 forms a homo-oligomeric structure and then is inserted into membrane. The heparan sulfate proteoglycans in ECM pulls out the oligomeric FGF2. However, the molecular details of this unconventional secretion pathway is unknown, e.g. how does the FGF2 form oligomer? or what is the role of phosphorylation? We propose a high resolution FGF2-PIP2 complex structure that highly agrees with the NMR chemical shift data by molecular dynamics simulations. Furthermore, we found a stable dimeric structure “dimer31” from two identified FGF2 crystal packing forms. Based on this dimer31 structure, we find out that the major function of phosphorylated Y73 is to form an intermolecular salt bridge with R60 of the other subunit. Phosphorylated Y73 helps not only to form salt bridge directly but also promote R60 to find the Y73 in the other subunit. The results has helped us gain insights in unconventional secretion pathways.

Darden T, York D, Pedersen L. 1993. Particle Mesh Ewald - an N.Log(N) Method for Ewald Sums in Large Systems. J Chem Phys 98: 10089-92
Ebert AD, Laussmann M, Wegehingel S, Kaderali L, Erfle H, et al. 2010. Tec-kinase-mediated phosphorylation of fibroblast growth factor 2 is essential for unconventional secretion. Traffic 11: 813-26
Faham S, Hileman RE, Fromm JR, Linhardt RJ, Rees DC. 1996. Heparin structure and interactions with basic fibroblast growth factor. Science 271: 1116-20
Feller SE, Zhang YH, Pastor RW, Brooks BR. 1995. Constant-Pressure Molecular-Dynamics Simulation - the Langevin Piston Method. J Chem Phys 103: 4613-21
Florkiewicz RZ, Majack RA, Buechler RD, Florkiewicz E. 1995. Quantitative export of FGF-2 occurs through an alternative, energy-dependent, non-ER/Golgi pathway. Journal of cellular physiology 162: 388-99
Humphrey W, Dalke A, Schulten K. 1996. VMD: visual molecular dynamics. Journal of molecular graphics 14: 33-8, 27-8
Kastrup JS, Eriksson ES, Dalboge H, Flodgaard H. 1997. X-ray structure of the 154-amino-acid form of recombinant human basic fibroblast growth factor. comparison with the truncated 146-amino-acid form. Acta crystallographica. Section D, Biological crystallography 53: 160-8
Lin X. 2004. Functions of heparan sulfate proteoglycans in cell signaling during development. Development 131: 6009-21
Muller HM, Steringer JP, Wegehingel S, Bleicken S, Munster M, et al. 2015. Formation of Disulfide Bridges Drives Oligomerization, Membrane Pore Formation and Translocation of Fibroblast Growth Factor 2 to Cell Surfaces. The Journal of biological chemistry
Musumeci M, Coppola V, Addario A, Patrizii M, Maugeri-Sacca M, et al. 2011. Control of tumor and microenvironment cross-talk by miR-15a and miR-16 in prostate cancer. Oncogene 30: 4231-42
Nickel W. 2011. The unconventional secretory machinery of fibroblast growth factor 2. Traffic 12: 799-805
Nickel W, Rabouille C. 2009. Mechanisms of regulated unconventional protein secretion. Nature reviews. Molecular cell biology 10: 148-55
Nugent MA, Iozzo RV. 2000. Fibroblast growth factor-2. The international journal of biochemistry & cell biology 32: 115-20
Rothman JE. 1994. Mechanisms of intracellular protein transport. Nature 372: 55-63
Rothman JE, Wieland FT. 1996. Protein sorting by transport vesicles. Science 272: 227-34
Schlessinger J, Plotnikov AN, Ibrahimi OA, Eliseenkova AV, Yeh BK, et al. 2000. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol Cell 6: 743-50
Steringer JP, Bleicken S, Andreas H, Zacherl S, Laussmann M, et al. 2012. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion. The Journal of biological chemistry 287: 27659-69
Steringer JP, Muller HM, Nickel W. 2015. Unconventional Secretion of Fibroblast Growth Factor 2-A Novel Type of Protein Translocation across Membranes? Journal of molecular biology 427: 1202-10
Temmerman K, Ebert AD, Muller HM, Sinning I, Tews I, Nickel W. 2008. A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2. Traffic 9: 1204-17
Thisse B, Thisse C. 2005. Functions and regulations of fibroblast growth factor signaling during embryonic development. Developmental biology 287: 390-402
Venkataraman G, Sasisekharan V, Herr AB, Ornitz DM, Waksman G, et al. 1996. Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation. Proceedings of the National Academy of Sciences of the United States of America 93: 845-50
Xie J, Supekova L, Schultz PG. 2007. A genetically encoded metabolically stable analogue of phosphotyrosine in Escherichia coli. ACS chemical biology 2: 474-8
Zehe C, Engling A, Wegehingel S, Schafer T, Nickel W. 2006. Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2. Proceedings of the National Academy of Sciences of the United States of America 103: 15479-84