中文摘要

腫瘤易感基因TSG101具有參與胞內蛋白質分選、囊泡運輸和轉錄調控等重要功能。本實驗室以酵母菌雙雜交技術選殖出與TSG101有交互作用的蛋白，其中KLIP1是2003年被發現的一個轉錄抑制因子，被指出具有抑制卡波西氏肉瘤相關疱疹病毒(Kaposi’s sarcoma-associated KSHV)的胸腺嘧啶激酶(TK)基因啟動子的活性。在本研究中，我們首先利用免疫共沉澱法證實TSG101與KLIP1在293細胞有交互作用，並以共軛焦顯微鏡觀察到此兩蛋白在細胞核中有共位現象。接著，我們將表現His-tagged的野生型泛素或無法形成多泛素鏈的突變型K０泛素的表達質體分別與表現HA-KLIP1蛋白之pHA-KLIP1質體共同轉染至293細胞內，並利用Ni-NTA sepharose進行轉染細胞內泛素化蛋白之純化，再利用HA抗體進行泛素化HA-KLIP1蛋白之偵測，結果發現HA-KLIP1蛋白可被多泛素化及單泛素化修飾。進一步實驗並發現隨著細胞內TSG101表現量增加， 71 KDa-KLIP1泛素化蛋白表現量會隨之減少，而60 KDa-KLIP1泛素化蛋白表現量會隨之增加，顯示TSG101會調控KLIP1蛋白泛素化的型態，此外，在其UEV功能區上與泛素交互作用之四個胺基酸位點突變的突變型TSG101會失去影響KLIP1泛素化型態的能力。而在KLIP1蛋白上6個可能被泛素化之賴胺酸位點突變的突變型HA-KLIP1蛋白的泛素化修飾型態(HA-KLIP1-M6)與野生型HA-KLIP1蛋白的型態明顯不同，顯示這六個賴胺酸位點可能包含KLIP1蛋白被泛素化修飾的位點。最後，我們藉由TK啟動子luciferase報導活性分析，確認KLIP1具有抑制TK啟動子之轉錄活性，並發現HA-TSG101扮演共同抑制因子的角色，此外，也發現其60及71 kDa-KLIP1-M6泛素化型式蛋白之缺乏會伴隨著HA-KLIP1- M6之轉錄抑制活性之減弱，顯示，TSG101會調控KLIP1的單泛素化修飾型式，使其以60 kDa-KLIP1單泛素化型態存在細胞核內，以執行其TK啟動子轉錄抑制活性，而KLIP1蛋白質上六個可能被泛素化修飾K位點同時突變，確能使其喪失對TK啟動子轉錄抑制活性，也顯示這六個K位點可能包含形成60 kDa-KLIP1及71 kDa-KLIP1單泛素化蛋白型態的K胺基酸位點，進一步針對這六個K胺基酸位點單一突變之HA- KLIP1蛋白表現質體之研究，將可釐清其確切之泛素化K胺基酸位點之所在。

Abstract

Tumor susceptibility gene TSG101 plays an important role in cellular functions including intracellular protein sorting, vesicular trafficking, and transcription regulation. Our previous results from yeast two-hybrid screening show that TSG101 interacts with a novel transcriptional repressor protein, KLIP1. In this study, we demonstrated in vivo interaction between TSG101 and KLIP1 in nucleus of 293 cells using co-immunoprecipitation assay and confocal imaging. In addition, we found KLIP1 could be modified in a modality of either poly- and mono-ubiquitination when exogenously expressed in 293 cells in conjunction with either wild type His-tagged ubiquitin or a mutant His-tagged ubiquitin (K0-Ub) which has no capability of forming polyubiquitin chain. Furthermore, we found that TSG101 could increase 60 kDa-KLIP1, but decrease 71 kDa-KLIP1 levels of monoubiquitinated KLIP1 protein species in a dose dependent manner. These results indicate that TSG101 might regulate KLIP1 protein function through affecting its monoubiquitin modification status. Further investigation using wildtype pHA-KLIP1 and mutant pHA-KLIP1-M6 containing mutation in its 6 lysine residues for possible ubiquitin modification revealed that wildtype HA-KLIP1, but not HA-KLIP1-M6, could inhibit transcription activity of thymidine kinase (TK) promoter. In conclusion, our results support that TSG101 interacts and acts as a transcriptional co-repressor of KLIP1 by keeping it in 60 kDa-monoubiquitinated status in the nucleus, where KLIP1 functions as a transcription repressor for TK promoter. Further experiment using mutant HA-KLIP1 expression plasmid containing single mutation in the 6 lysine sites should reveal the exact location of ubiquitin-modified lysine site for monoubiquitinated species of KLIP1 protein.

目錄

摘要 2
Abstract 4
前言
TSG101的發現與背景 6
TSG101的蛋白質結構與特性 7
蛋白質泛素化 8
TSG101與蛋白泛素化的相關性 11
KLIP1、CENP-50、MLF1IP的發現與背景 12
實驗目的 17
材料與方法
真核細胞表現載體的製備 18
In vivo Co-IP Assay 22
In VivoTSG101與KLIP1共位分析 28
KLIP1轉錄活性分析--報導基因活性分析 29
RT-PCR分析基因默化之效率 32
實驗結果
TSG101與KLIP1於細胞內產生交互作用 34
TSG101與KLIP1兩者在細胞核有共位(Co-localization)現象 35
KLIP1會進行泛素化作用之轉譯後修飾 36
TSG101與KLIP1蛋白單泛素化修飾之相關性探討 37
TSG101的UEV功能區若喪失與泛素結合能力，將失去調節KLIP1單泛素化的能力 38
確認KLIP1泛素修飾化位點 39
TSG101會影響KLIP1對於TK啟動子轉錄抑制的活性 40
討論 44
參考文獻 53
圖表 57

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