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博碩士論文 etd-0905111-105014 詳細資訊
Title page for etd-0905111-105014
論文名稱
Title
KLIP1蛋白與SUMO-1的交互作用
Interaction between KLIP1 and SUMO-1
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
64
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
2011-07-14
繳交日期
Date of Submission
2011-09-05
關鍵字
Keywords
交互作用、定點突變
KLIP1, SUMO-1, site-directed mutagenesis
統計
Statistics
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The thesis/dissertation has been browsed 5659 times, has been downloaded 480 times.
中文摘要
細胞核內轉錄抑制蛋白KLIP1,又名為MLF1IP,會與myleoid leukemia factor 1(MLF1)共同作用而抑制細胞程式性死亡,而導致腫瘤之形成,其具有抑制卡波西氏肉瘤相關疱疹病毒(Kaposi’s sarcoma-assoicated Hepres Virus)之胸腺嘧啶激酶基因啟動子(TK promoter)的活性, KLIP1亦被報導為一種中節結合蛋白,因而又被稱為CENP-U或CENP-50,其會參與細胞染色體有絲分裂期的姊妹染色體分離之調節。這些證據顯示KLIP1為一種調節細胞轉錄活性及細胞生長週期之重要蛋白。本研究以生物資訊學分析KLIP1蛋白胺基酸的序列發現其至少具有六個SUMO修飾位點的保守序列分別位於K33、K63、K126、K127、K185以及K210。文獻顯示蛋白被SUMO修飾後會影響其與其他蛋白間之交互作用能力、改變其胞內分布狀態、蛋白其穩定性以及轉錄調控活性等,文獻指出染色體中節結合蛋白的SUMO修飾為維持中節與紡錘絲接合之著絲點(kinetochore)生理功能所必須,且前人研究亦指出,KLIP1蛋白中具有與SUMO分子進行交互作用的功能區。為了探討細胞中KLIP1蛋白與SUMO-1間之交互作用,首先將表現GFP-KLIP1以及His-tagged SUMO-1蛋白的表現載體共同轉染到HEK 293細胞中,再進行免疫共沉澱分析,發現可檢測到KLIP1與SUMO-1兩者間有交互作用的現象。且利用雷射共軛焦顯微鏡觀察其螢光影像,也發現在同時表現GFP-KLIP1以及RFP-SUMO-1的細胞中,兩種蛋白在細胞核內有共位現象。因此本研究證實KLIP1確為SUMO-1之交互作用蛋白。本研究中也利用定點突變方法,將以生物資訊預測出的KLIP1蛋白序列中六個可能為SUMO-1修飾位點的Lysine胺基酸位點突變為Arginine,建構成表現突變型KLIP1-M6表現載體pEGFP-KLIP1-M6。將GFP-KLIP1-M6以及His-SUMO-1共同轉染到細胞內,並進行免疫共沉澱,則發現突變型KLIP1-M6與SUMO-1之交互作用現象消失。經影像觀察後發現突變型GFP-KLIP1-M6與RFP-SUMO-1之核共位現象與GFP-KLIP1比較,發現兩者的分布雖然都在細胞核內,但兩者與SUMO-1之核共位現象有顯著的差異,顯示六個K位點突變KLIP1-M6較少與SUMO-1有核共位現象。綜合以上結果顯示KLIP1蛋白的這六個K位點可能與SUMO-1產生交互作用有關,且該K位點突變會影響KLIP1-M6在核內與SUMO-1共位現象,至於其對KLIP1蛋白之生理功能之影響有待進一步實驗加以釐清。
Abstract
Nuclear protein KLIP1 cooperates with myeloid leukemia factor 1 (MLF1) to inhibit the programmed cell death resulting in tumor formation. It also inhibits the activity of thymidine kinase promoter of Kaposi’s sarcoma-associated Herpes Virus. KLIP1 functions as a centromere protein, hence acquires its name as CENP-U or CENP-50, to regulate the separation of sister-chromatids during mitosis. These results indicate that KLIP1 plays important roles in regulation of transcription and cell cycle. In this study, six potential SUMO modification sites, K33, K63, K126, K127, K185 and K210, were identified bioinformatically using SUMOplot. Many reports address that SUMO modification alters the transcriptional activity, protein-protein interaction, the subcellular localization and stability of its target protein. Recent data suggest that SUMO is required for centromere binding protein to mediate proper mitotic spindle attachment to the kinetochore, and previous research suggest that there has a SUMO-interaction motif (SIM) in KLIP1 protein sequence. To reveal the interaction between KLIP1 and SUMO-1, and study its effects on KLIP1 function, we co-express GFP-KLIP1 and His-tagged SUMO-1 in HEK 293 cells. After affinity purification of SUMOylated proteins from transfected cells using nickel conjugated beads and subsequent western blotted with anti-GFP. The results indicated the interaction between KLIP1 and SUMO-1 in co-transfected cells. Our confocal microscopy imaging also found colocalization of GFP-KLIP1 with RFP-SUMO-1 nuclear foci. In addition, we failed to detect the interaction between SUMO-1 and mutant KLIP1-M6 ,whose six potential SUMO modified lysine residues were mutated to arginine. Furthermore, we found a distinct nuclear localization of GFP-KLIP1-M6 as compared to the image of wildtype GFP-KLIP1, which show a significant higher frequency of colocalization with RFP-SUMO-1 foci. Taken together, our data suggest the interaction between KLIP1 and SUMO-1 may be related to these six potential lysine residues, which upon mutation blocks its colocalization with SUMO-1 in nuclear foci. The biological significance of their interaction are awaits for further investigation.
目次 Table of Contents
中文摘要-------------------------------------------------------------------------------------- 3
Abstract-- ------------------------------------------------------------------------------------- 5
前言
KLIP1的發現與背景 -------------------------------------------------------------------- 9
KLIP1蛋白之結構與特性 -------------------------------------------------------------- 9
SUMO修飾作用---------------------------------------------------------------------------10
SUMO與KLIP1---------------------------------------------------------------------------12
材料與方法
大量表現GST-KLIP1(161-280)融合蛋白 -------------------------------------------14
真核表現載體製備------------------------------------------------------------------------17
免疫共沉澱---------------------------------------------------------------------------------20
KLIP1之SUMO修飾位點突變質體之構築-----------------------------------------25
螢光表現載體GFP-KLIP1-M6之次選殖---------------------------------------------26
In vivo KLIP1及RFP-SUMO之共位分析--- -------------------------------------28
實驗結果
KLIP1之特異性抗體製備-------------------------------------------------------------- 30
KLIP1與SUMO-1兩者在細胞內有交互作用的現象 --------------------------- 31
KLIP1蛋白序列中SUMO可能修飾位點之定點突變表現載體之分析 - 34
KLIP1-M6與His-SUMO-1之免疫共沉澱之分析 -------------------------------- 35
GFP-KLIP1-M6與RFP-SUMO-1之共位現象分析 ------------------------------ 36
討論 ------------------------------------------------------------------------------------------- 37
參考文獻----------------------------------------------------------------------------------------41
圖表--------------------------------------------------------------------------------------------- 45
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