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.

摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 vi
表目錄 vii
1. 緒論 1
1.1 Basic fibroblast growth factor 2 (FGF2) 1
1.2 ER/Golgi-dependent secretory pathway 2
1.3 Unconventional secretory model for FGF2 3
2. 方法 4
2.1 Molecular Dynamics Simulations 4
2.1.1 FGF2 單體結構 4
2.1.2 FGF2 二聚體結構 4
2.1.3 FGF2 Binding with PIP2結構 5
2.1.4 磷酸化FGF2 7
2.1.5 加氫與加水 7
2.1.6 模擬設定及方法 9
2.2 主成分分析（Principal Component Analysis, PCA） 11
2.3 Root Mean Square Fluctuations (RMSF) 12
2.4 Root Mean Square Deviation (RMSD) 12
2.5 Overlap 13
2.6 B-factor 與RMSF 的換算公式 13
2.7 接觸個數分析 13
3. 結果 14
3.1 Monomer模擬結果分析 14
3.2 比較Phosphorylated 與 Unphosphorylated FGF2 Monomer 之差異 16
3.3 FGF2 Monomer Binding with PIP2 結果分析 18
3.4 FGF2 Dimer31 模擬結果分析 20
3.5 FGF2 Dimer33 模擬結果分析 24
3.6 Phosphorylated FGF2 Dimer31模擬結果分析 25
3.7 Phosphorylated FGF2 Dimer31 Binding with PIP2模擬結果分析 29
3.8 FGF2與FGF4序列比對 31
4. 討論 32
4.1 分子動力學模擬首次發現的高解析度FGF2-PIP2複合體結構吻合NMR chemical shift結果 32
4.2 Dimer31 packing 可形成穩定的FGF2二聚體 33
4.3 磷酸化Y73與R60形成分子間鹽橋以穩定dimer31結構 33
4.4 磷酸化Y73將R60推向另一個次單元加速分子間鹽橋的形成 35
5. 結論 36
6. 參考文獻 38

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