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博碩士論文 etd-0218105-170416 詳細資訊
Title page for etd-0218105-170416
論文名稱
Title
TSG101功能區之定點突變
Site-directed mutagenesis of TSG101 function domain
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
44
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2005-01-31
繳交日期
Date of Submission
2005-02-18
關鍵字
Keywords
定點突變、腫瘤易感基因
TSG101, PTAP, sumoylation, site-directed mutagenesis, ub
統計
Statistics
本論文已被瀏覽 5684 次,被下載 3453
The thesis/dissertation has been browsed 5684 times, has been downloaded 3453 times.
中文摘要
中文摘要:
腫瘤易感基因TSG101具有許多功能,細胞週期之調控,蛋白質分選細胞膜傳輸及核內受體之轉錄活性之調控等。TSG101蛋白具有泛素複合酵素E2類似UBC之功能區,但卻缺乏催化活性所需具備的半胱氨酸(cysteine),而蛋白質泛素化作用(ubiquitination)是細胞內許多蛋白質分解或分選進入多囊泡體(multivesicular body)的一種信號,文獻指出TSG101蛋白質之N端UBC功能區的氨基酸,具備有與PTAP序列及泛素有交互作用的必要氨基酸位點。其中Y63、M95及V141的位置,可與HIV late domain 蛋白之PTAP序列結合,而胺基酸序列的V43、N45及D46,則為與Ubiquitin交互作用的位點。SUMO是一種類泛素修飾蛋白(ubiquitin-like modifier),可對細胞內具有ΨKXE氨基酸序列蛋白進行修飾,Ψ是代表大分子疏水性氨基酸(large hydrophobic amino acid),K是表示與SUMO結合的lysine,X代表任何氨基酸皆可,E則是glutamic acid,將TSG101蛋白氨基酸序列進行分析結果發現其K98、K243、K269位點序列極為可能為SUMO修飾位點,由於實驗室之結果顯示TSG101與SUMO在細胞核內的分布位置有重疊的現象,因此本研究針對TSG101這些功能相關區域進行一系列之定點突變,並以DNA自動定序方法加以確認,並構築TSG101突變蛋白表現載體,以供日後進行TSG101功能測試之使用,以釐清這些位點突變後對TSG101蛋白功能之影響。
Abstract
Abstract:
TSG101 is a tumor susceptibility gene exhibits multiple biological function, including the regulation of cell progression, intracellular protein sorting and membrane trafficking, and transcription activity of nuclear recptor such as estrogen recptor. TSG101 contains an UBC domain which is homologous to that in ubiquitin conjugating E2 enzyme. However, it lacks an essential cysteine residue, which is essential for catalytic activity. Cellular protein ubiquitination serves as a signal for protein degradation or sorting into multivesicular body. UBC domain of TSG101 was proved to contain amino acid residues that are important for its interaction with ubquitin (residues V43, N46, D46 and F88) and PTAP sequence found in the late domain of HIV gag protein (residues Y63, M95, V141). SUMO is an ubquitin-like modifier which can modify cellular protein harbors ΨKXE amino acid sequence, thereby change its subcellular localization and biological activities. TSG101 protein contains K98, K243, K264 and K269 residues that localize in potential SUMO modification site. Our preliminary data indicated that TSG101 colocalize with SUMO in nucleus. It is interesting to know whether TSG101 is sumoylated, and its functional significance. In this thesis, a series of site-directed mutageneic mutant HA and GFP-tagged expression plasmids which contain mutation of the above mentioned functional related amino acid residues were constructed for future TSG101 functional studies.
目次 Table of Contents
目錄
中文摘要 2
英文摘要 4
背景介紹 5
材料與方法 12
結果 18
討論 21
參考文獻 25
圖表 30
參考文獻 References
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