臺灣博碩士論文加值系統

人類基因體裡包含兩百種以上的絲胺酸水解酶成員，然而目前仍有將近半數的此類酵素，並不清楚其基質與可能參與的生理功能。近年來亦有一些文獻發現某些未知絲胺酸水解酶活性的上升，會與癌症的發生與轉移有關。因此，系統化地去鑑定某群跟疾病相關的絲胺酸水解酶具有相當的重要性。本篇研究主要在探討經過親水性修飾後製備之fluorophosphonate (FP) 探針(包括FP-edo-biotin, FP-edo-fluorescein)，是否能利用其與活化態絲胺酸形成共價鍵的特性，作為辨識及純化絲胺酸水解酶的工具。首先，以純化過的poly(3-hydroxybutyrate) depolymerase (PhaZ) 做為測試其反應速度與純化效率的指標。不論是預先加熱(preheating)的步驟或是取代以活性絲胺酸點突變後之PhaZ mutant (S102A)皆會喪失PhaZ原有與探針迅速反應之能力，顯示FP類探針只會與具有活性之絲胺酸水解酶反應，此外以質譜儀分析探針反應後之PhaZ可確認反應位置在99到106之間的片段。更進一步地，利用β-elimination/Michael addition的化學處理，可以使接上活性絲胺酸位置之FP探針脫離，並得以送入另外新的標記分子作為鑑定活性絲胺酸位置之依據。最後利用FP-edo-fluorescein的螢光訊號來建立不同小鼠組織間絲胺酸水解酶之活性圖譜，證明此探針能夠有效地偵測細胞內蛋白質體中酵素的活性狀態，未來將進一步使用帶有biotin的探針來純化並鑑定出這些圖譜中的絲胺酸水解酶成份。

Serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in human genome. However, up to 50% of these enzymes remain unannotated and their substrates are still unknown. In addition, activities of increasing serine hydrolases were shown associated with diseases like cancer neoplasia and invasiveness etc. Therefore, the systematic identification of serine hydrolases which are associated with diseases is of great importance. In this study, two novel hydrophilic fluorophosphonate (FP)-based chemical probes, including FP-edo-biotin and FP-edo-fluorescein, were synthesized and examined for their abilities to recognize as well as to pull down the serine hydrolases via covalent linkage. A recombinantly purified poly(3-hydroxybutyrate) depolymerase (PhaZ) was first used to determine the labeling kinetics and pull-down efficacy. Subsequent experiments using either site-directed mutagenesis of catalytic serine at 102 (S102A) or pre-heating treatment of PhaZ has shown an abolished labeling, indicating the activity-based labeling feature of these FP probes when targeting the active site serine. Mass spectrometric analysis of tryptic peptides from the FP-edo-biotin-labeled PhaZ has confirmed the existence of biotin-labeled peptide (99-106). In addition, I observed that in vitro β-elimination and Michael addition with 2-aminoethanethiol of probe-labeled PhaZ could detach the labeling probe and facilitate future identification of active site serine residues. Finally, the FP-edo-fluorescein has been applied to establish the serine hydrolase profiles of diverse mouse tissues and the results showed that such FP probe is capable to display cellular serine hydrolases efficiently. Further biotin pull-down and protein identification experiments are required in the future.

Contents.............................i
List of abbreviation................ii
中文摘要.............................1
Abstract.............................2
Materials and methods................9
Materials............................9
Instrument...........................9
Methods..............................9
Results..............................15
Discussion ..........................20
Future applications and conclusion...24
Table................................26
Figures..............................35
References...........................57

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