- 學位論文
孕烯醇酮衍生物探針用於顯影及純化其相對結合蛋白質的研究
Pregnenolone Derivatized Probes to Image and Purify the Corresponding Binding Proteins
摘要
孕烯醇酮（Pregnenolone, P5）是類固醇生合成路徑中的第一個產物，同時也 是一種神經類固醇，具有改善記憶力和神經系統修復的功能。在之前實驗室和中 研院鍾邦柱博士實驗室合作的研究中發現孕烯醇酮可以穩定斑馬魚胚胎微管的 結構，並且影響胚胎外包過程中的細胞移動。由此推論斑馬魚胚胎中具有和孕烯 醇酮結合的蛋白質受體。但此蛋白質在過去並未被確認，因此吾人設計並合成一 系列孕烯醇酮衍生物探針，希望可以純化並確認此結合蛋白的生理功能。 此論文研究運用三種不同的策略來探測孕烯醇酮結合蛋白(見圖):第一個策 略利用具有螢光團的孕烯醇酮衍生物，對細胞內的孕烯醇酮結合蛋白進行顯影。 分子設計上，分別從孕烯醇酮C3 或C7 的位置接上連接端，並於末端接上 TokyoGreen 螢光團。再將螢光探針滲透至HeLa 細胞裡，由雷射掃瞄共軛焦顯微鏡（confocal microscope）所得影像判斷螢光探針所在位置。實驗結果顯示這些螢光探針主要累積在粒線體中而非在細胞微管上。推測原因可能是微管上的結合蛋白與孕烯醇酮親合性作用力並不強，因此在螢光染色的清洗步驟中被移除掉。第二個策略是利用二氧化矽奈米粒子當作固相載體，在表面修飾由不同位置修飾連結的孕烯醇酮衍生物，希望藉由多重作用(multivalency interactions)機制，可以有效和孕烯醇酮結合蛋白作用並達到純化分離的目的，同時結合電泳與質譜可以進一步鑑定結合蛋白質的分子量。第三個策略是分別從孕烯醇酮不同位置或位向（分別為C3、C7a、C7b或C20）進行衍生化，同時改變連接鏈的長度且引入光活化反應基團benzophenone 與diazirine，以期於辨識後經由照光活化使得化合物和結合蛋白形成共價性的巨分子。在測試光親和性探針對於促進微管蛋白聚合的實驗中，發現從孕烯醇酮的C7 接出連接端的位向與長度會影響其探針促進微管生成的活性。而在對斑馬魚胚胎細胞液進行光標定實驗中，成功地共價性標定到孕烯醇酮結合蛋白，經由LC-MS/MS 鑑定後，得知此孕烯醇酮結合蛋白的身份是cytoplasmic linker protein 170 (CLIP-170)。之後，由鍾邦柱研究員實驗室翁瑞霞博士利用各種CLIP-170 的缺失型突變體（depletion mutant）分別進行光標定的實驗，發現 CLIP-170 中的胺基酸920-970 片段對於與孕烯醇酮的辨識結合扮演重要的角色。而由這些不同衍生化的孕烯醇酮光親和性探針對CLIP-170 的光標定實驗中，亦可推知孕烯醇酮的3b-hydroxy與20-ketone是與CLIP-170辨識與結合的重要官能基團。
並列摘要
Pregnenolone is the first product of steroidogenesis as well as a kind of neurosteroids, which can improve memory and neurological recovery. Based on previous research, pregnenolone had been proven to stabilize microtubules and promote zebrafish embryonic cell movement. According to these findings, a pregnenolone binding protein, which can modulate the formation of microtubules in zebrafish embryo, has been assumed to exist. Therefore, a series of pregnenolone derivatized probes have been designed and synthesized to identify this corresponding binding protein. Three strategies have been developed to study the unknown pregnenolone binding protein. The first strategy had employed fluorescent pregnenolone derivatives to image the corresponding binding protein in cells. The fluorophore unit, i.e., TokyoGreen, had been designed to connect with pregnenolone moiety at its C3 or C7 position, respectively. The result showed that the TokyoGreen-incorporated pregnenolone probes can penetrate into the HeLa cells and accumulate mainly in mitochondria. However, the fluorescence signal on microtubule is weak due to low affinity interaction, which is liable to be washed-out. The second strategy had employed silica nanoparticles modified with pregnenolone derivatives to purify corresponding binding proteins via multivalency interaction. Utilizing mass spectrometry and polyacrylamide gel electrophoresis, the identity of the isolated protein will be exactly verified. The third strategy is to join photoreactive groups (benzophenone and diazirine) with pregnenolone to establish the ligand-directed photoaffinity probes, which can capture the corresponding binding protein via the formation of a covalent bond upon activation by light. Various spacer segments with different chain lengths had been grafted on C3, C7a, C7b or C20 position of the pregnenolone derivatives, respectively, and linked with benzophenone/diazirine and biotin tag on the other end. In vitro tubulin polymerization experiment indicated that the stereo-orientation and spatial arrangement at the C7 position of pregnenolone are essential for the probe to function. This pregnenolone photoaffinity probes had then been employed to label corresponding binding proteins from zebrafish embryo lysates and the pregnenolone binding protein, cytoplasmic linker protein 170 (CLIP-170), had ultimately been found by LC-MS/MS identification. The photolabeling experiments of CLIP-170 or its various depletion mutants showed that the aa 920-970 fragment of CLIP-170 plays an important role in the binding event with pregnenolone. Furthermore, the photolabeling experiments of various pregnenolone photoaffinity derivatives towards CLIP-170 imply that the ketone group at C20 position and the hydroxy group at C3 position of pregnenolone are vital for the binding of pregnenolone with CLIP-170.