Bcl-2 associated X protein (BAX)，屬於Bcl-2 家族蛋白中的促凋亡蛋白，在細胞凋亡 (apoptosis) 過程中會受到BH3-only蛋白活化而進行粒線體外膜通透化反應 (MOMP) 進而造成細胞死亡。近年來，BAX常被用來當作調控腫瘤細胞死亡與否的主要藥理學靶標。聚乙二醇修飾 (PEGylation) 是近年來在研究蛋白質藥物動力學的調控方面很熱門的一種方式。這次研究中我們利用聚乙二醇 (PEG) 修飾BAX的各種mutant，研究PEGylation 對BAX穩定性及生物學上活性的探討。結果顯示PEG可增強蛋白質的穩定性，並且由於PEG的屏蔽效應，在cytochrome c release assay實驗中，兩種凋亡刺激 (BimBH3胜肽和cBid蛋白)會對BAX的活化產生不一樣的激活效果。其中我們發現BAX Y164C是個很特別的位點，此BAX mutant在不具有任何BH3-only 蛋白活化下，本身就有自行活化 (auto-activated)引發MOMP的能力，而在PEGylation後，BAX 164-PEG表現出增強的穩定性，並且保持無活性的單體狀態。此外，在正常一般BH3-only 蛋白 (cBid蛋白) 的活化下，BAX 164-PEG無法表現正常細胞凋亡的功能，只有在BimBH3激活肽的誘發下才會有MOMP的能力。我們在mitochondria-like脂質組成的GUV上進行光漂白螢光恢復實驗 (FRAP-GUV) 也得到與cytochrome c release assay一樣的結果。此外，由time-resolved ESR (TR-ESR)實驗及double electron-electron resonance (DEER) 技術量測的結構證據中證明在BimBH3 peptide的hit and run 活化模型下，BAX 164-PEG能夠與BAX wt進行相似的寡聚化過程並且共同形成能夠引起MOMP的凋亡寡聚體。綜合以上結果， 顯示BAX 164-PEG的成孔活性可不需依賴BCL2家族蛋白調控的凋亡途徑，可以僅利用合成胜肽來完成活化結果。此發現在未來研究調控癌症細胞標靶藥物的設計上可能有幫助的作用。

Bcl-2 associated X (BAX) is a member of the Bcl-2 protein family, acting as a critical gatekeeper to control mitochondrial apoptosis. During apoptosis, BAX is induced by BH3-only protein to cause mitochondrial outer membrane permeability (MOMP) that leads to cell death. In the recent years, it has become a major pharmacological target for modulating death of tumor cells. PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein therapeutics. Here, we modified various single-cysteine variants of BAX protein with polyethylene glycols (PEG) in order to enhance protein stability and modulate the pore-inducing activity of BAX. Due to the conjugation of PEG, many of the PEGylated BAX proteins studied become relatively stable. We found that PEG may somehow influence the BAX activity in response to the apoptotic stimuli such as BimBH3 peptide and cBid (Caspase-8 cleaved Bid) protein. One exception we found is BAX 164-PEG. BAX 164C is auto-activated to cause MOMP in the absence of BH3-only protein, exhibiting instability. However, after PEGylation, BAX 164-PEG exhibits an improved stability and remains as an inactive monomer. Moreover, BAX 164-PEG is resistant to stimulus of cBid, but it can still be activated by BimBH3 activator peptide to promote MOMP. In our further structure-based investigation, BAX 164-PEG is able to assemble with wild-type BAX (BAX wt) to form an apoptotic oligomer which is induced by BimBH3 peptide through a hit-and-run mechanism (a well-known model for the BAX activation). In conclusion, we report a new finding that the pore-inducing activity of BAX 164-PEG is independent from the Bcl2-mediated apoptotic pathways, but it can be administrated by the synthetic BH3 peptides. As such, the result suggests a therapeutic approach for modulating the pore-inducing activity of BAX that is crucial to the regulation of cell death.

摘要
致謝辭
目錄
第一章 序論…………………………………1
1-1. 細胞凋亡 (Apoptosis)…………………………………1
1-2. Bcl-2家族蛋白 (Bcl-2 family proteins)…………………………………2
1-3. Bcl-2 Associated X (BAX) protein介紹…………………………………2
1-4. BimBH3 peptide介紹…………………………………4
1-5. Bid protein介紹…………………………………4
1-6. BAX 活化位置…………………………………5
1-7. 聚乙二醇修飾 (PEGylation)…………………………………5
1-8. 巨型單層微脂體 (Giant Unilamellar Vesicle, GUV)…………………………………6
1-9. 研究目的與動機…………………………………6
第二章 儀器介紹與實驗原理…………………………………8
2-1. 電子自旋共振光譜…………………………………8
2-1-1. 電子自旋共振光譜儀 (Electron Spin Resonance, ESR)……………………………8
2-1-2. 連續波型電子自旋共振光譜原理簡介 (CW-ESR)…………………………………9
2-1-2-1. 賽曼效應 (Zeeman effect)…………………………………9
2-1-2-2. 超微細偶合作用 (Hyperfine interaction)…………………………………10
2-1-2-3. 定位自旋標記 (site-directed spin label, SDSL)……………………………10
2-1-2-4. CW-ESR光譜線型與自旋標記的轉動動力學關係…………………………………11
2-1-3. 雙重電子電子共振光譜 (Double Electron-Electron Resonance, DEER)…………………………………12
2-2. 聚乙二醇修飾 (PEGylation)…………………………………15
2-3. 固相胜肽合成法 (solid phase peptide synthesis, SPPS)………………………15
2-4. 圓二色光譜儀 (Circular Dichroism, CD)…………………………………17
2-5. 液相層析儀 (liquid chromatography)…………………………………18
2-5-1. 高效液相層析法 (high performance liquid chromatography, HPLC) …………………………………18
2-5-2. 快速液態層析儀 (fast performance liquid chromatography, FPLC) …………………………………19
2-6. 蛋白質標記轉移法 (Label transfer protein interaction analysis) …………………………………19
2-7. 共軛焦顯微鏡 (confocal microscopes)…………………………………21
2-8. 螢光漂白恢復技術 (Fluorescence recovery after photobleaching) …………………………………21
第三章 樣品製備與實驗測量方法…………………………………………………………22
3-1. 蛋白質的純化及修飾…………………………………22
3-1-1. BAX蛋白質的表現及純化…………………………………22
3-1-2. mPEG-MAL和BAX胺基酸cysteine修飾反應…………………………………24
3-1-3. Bid蛋白質的表現及純化…………………………………24
3-1-4. cBid蛋白質 (cleaved Bid) 的製備…………………………………25
3-2. BimBH3 peptide的合成及純化…………………………………26
3-3. 定位自旋標記樣品製備及量測…………………………………28
3-3-1. 雙重電子-電子共振光譜 ( DEER ) 量測…………………………………28
3-3-2.連續波型-電子自旋共振光譜 ( time-resolved CW-ESR ) 量測………………29
3-4. 圓二色光譜 ( Circular Dichroism, CD )量測…………………………………30
3-5. Cytochrome c release assay…………………………………30
3-6. Label Transfer 實驗…………………………………30
3-7. Fluorescence recovery after photobleaching (FRAP) 實驗………………31
3-7-1. GUV 製備…………………………………31
3-7-2. FRAP實驗…………………………………32
第四章 結果與討論 …………………………………………………………………………35
4-1. BAX蛋白進行聚乙二醇修飾 (PEGylation) 的位點挑選…………………………………35
4-2. BAX mutant與聚乙二醇化的BAX mutant-PEG 之CD光譜…………………………………35
4-3. BAX mutant及BAX mutant-PEG的cytochrome c release assay結果……38
4-4. BAX mutant及BAX mutant-PEG在GUV系統下的 FRAP實驗結果………………………44
4-5. BAX Y164C與BAX 164-PEG之變溫CD光譜…………………………………46
4-6. cBid蛋白與BAX 164-PEG的label transfer結果…………………………………47
4-7.以 time-resolved cw-ESR 研究 BimBH3-R1對 BAX 164-PEG寡聚化之表現 …………………………………49
4-8. 以DEER 量測BAX 164-PEG寡聚物的結構…………………………………53
4-8-1. 在BimBH3 peptide的活化下，利用label transfer實驗驗證BAX wt與BAX 164-PEG 會共同形成BAX Oligomer…………………………………54
4-8-2. 以 DEER 量測BAX wt-R1 及BAX 164-PEG寡聚物的結構……………………………55
第五章 結論………………………………………………………………………………58
參考文獻……………………………………………………………………………………60

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