蛋白質是生物體內實際執行生理功能的分子，在細胞中蛋白質常進行多種後轉譯修飾作用 (Posttranslational modification)，以賦予蛋白質更多的功能。例如後轉譯修飾可以調節蛋白質在細胞內的位置、蛋白質與蛋白質間的交互作用、蛋白質的活性與穩定度等。在眾多蛋白修飾中，甲基化的研究起步較晚，但其牽涉的生化反應十分廣泛，因此蛋白質甲基化的細胞功能近年來陸續受到重視。
我們實驗室的研究與相關文獻指出，PRMT5 (Protein aRginine MethylTransferase 5) 為一高度表現在數種癌症組織的蛋白質甲基轉移酶，具有促進細胞轉型的活性；PRMT5會甲基化同樣具致細胞轉型活性的HURP (Hepatoma UpRegulated Protein)，此發現促使我們想要了解PRMT5甲基化HURP的機轉及甲基化HURP在細胞中的生理意義。在機轉方面，PRMT5透過支架蛋白 (scaffold protein) Ajuba與蛋白質激酶 (protein kinase) Aurora-A交互作用，進而使PRMT5與Aurora-A可以相互調節彼此的活性；之後， Ajuba與PRMT5協助因子RioK1會幫助PRMT5辨識下游受質HURP，並甲基化HURP於Arg 122的位置。另一方面，本論文也發現去蛋白質甲基酶JMJD6可以去甲基化HURP。細胞的生理意義方面，HURP 122甲基化會促進由Aurora-A所催化於HURP Ser 627的磷酸化，繼而使得HURP蛋白質穩定度提升。此外，HURP 122甲基化會決定HURP在細胞內的分布；例如在細胞分裂期，Arg 122的甲基化會讓HURP遠離紡錘絲與著絲點的交界，HURP因之無法穩定此區域的紡錘絲，使得染色體排列異常。另一方面，HURP 122的甲基化促使高基氏體過於穩定，使高基氏體不易進行碎裂移位重組(polarized assembly)，因而阻礙細胞的爬行；反之，無法甲基化的HURP則能大幅度促進細胞移行。總之，本論文首度揭露PRMT5甲基化HURP的機制與後續的細胞反應，對於解開此PRMT5/HURP訊息途徑的細胞功能做了重要的初步貢獻。

Proteins are molecules of practical implementation of physiological functions , the protein is usually carried out in a variety of cells, post-translational modification (Posttranslational modification), to give the protein more function, E.g., post-translational modification may adjust the position of the protein in the cell, protein-protein interactions, protein activity and stability, etc. Among the many protein modification, the methylation of a late start, but the biochemical reaction which involves very extensive, and therefore protein methylation cell function in recent years have taken seriously.
Our laboratory research and related literature, PRMT5 (Protein aRginine MethylTransferase 5) is a highly expression in several types of cancer tissue protein methyl transferase, promote cell transformation activity. PRMT5 be the same with the methylation-induced cell transformation activity HURP (Hepatoma UpRegulated Protein), this finding promoted us want to know PRMT5-HURP methylation mechanisms and HURP physiological significance in cells. In mechanisms, PRMT5 through scaffold protein-Ajuba with protein kinase-Aurora-A interaction, thereby PRMT5 and Aurora-A can adjust each other's activity. Afterward Ajuba and RioK1 will assist PRMT5 identify downstream HURP, then methylate HURP in Arg 122. On the other hand, this paper also found that protein demethylation enzymes JMJD6 can go demethylation HURP. In cellular significances, HURP 122 promotes methylation catalyzed by Aurora-A in HURP Ser 627 phosphorylation, which in turn makes HURP enhance the stability of the protein. Furthermore, HURP 122 methylation decided HURP distributed in cells. For example, in cell division, Arg 122 methylation make HURP spindle fibers away from the junction with the centromere, HURP consequent spindle can not be stabilized in this region silk, making chromosome misalignment. On the other hand, HURP 122 methylated Golgi is promote to stable, so that the Golgi is not easy to polarized assembly, and thus hinder the cell migration. Conversely, demethylated HURP is promote cell migration. In summary, this study for the first time reveal the mechanisms and cellular significances of PRMT5-induced HURP methylationsubsequent methylation, for the PRMT5/HURP signal pathway made important initial contribution.

目次
誌謝 I
摘要 II
Abstract III
目次 V
圖目次 XII
表目次 XIII
第一章 前言 1
第二章 緒論 2
第一節 蛋白質的後轉譯修飾 (Protein Post-translation Modification) 2
壹 磷酸化 (Phosphorylation) 3
貳 甲基化 (Methylation) 3
第二節 Hepatoma Upregulated Protein (HURP) 4
壹 HURP的序列與結構 4
貳 HURP與細胞週期 4
參 HURP與癌症 5
肆 HURP的蛋白質功能 6
第三節 Aurora-A 9
壹 Aurora-A的蛋白質功能 9
貳 Aurora-A對HURP的修飾及影響 10
第四節 PRMT5 (Protein aRginine MethylTransferase) 12
壹 PRMT5的蛋白質功能 12
貳 PRMT5甲基化HURP 14
第五節 Ajuba 15
壹 Ajuba的蛋白質功能 15
貳 Ajuba分別與Aurora-A及PRMT5交互作用 15
第六節 研究動機與策略 17
第三章 實驗材料與方法 20
第一節 實驗材料與試劑 20
壹 勝任細胞（Competent cells） 20
貳 抗生素（Antibiotics） 20
參 細胞株（Cell lines） 20
肆 定點突變引子(Primer) 21
伍 細胞轉染試劑（Transfection reagents） 23
陸 DNA 染劑（DNA dyes） 23
柒 抗體（Antibodies） 23
捌 細胞週期同步化（Cell cycle synchronization）藥劑 24
第二節 實驗試劑配製 25
壹 Calcium-manganese based 緩衝溶液（CCMB buffer） 25
貳 Luria-Bertain 細菌培養液（LB medium） 25
參 Luria-Bertain 細菌培養基（LB agar plate） 25
肆 10 倍 Tris-borate-EDTA 緩衝溶液（10X TBE buffer） 26
伍 DNA 瓊膠（DNA agarose gel） 26
陸 DME細胞培養液（Dulbecco's modified Eagle's medium, DMEM） 26
柒 DME 細胞凍存培養液（DME cell freezing medium） 27
捌 10 倍磷酸緩衝溶液（10X Phosphate buffer saline buffer, 10X PBS buffer） 28
玖 胰 蛋 白 酶 － 乙 二 胺 四 乙 酸 四 鈉 消 化 液 （ Trypsin- Ethylenediaminetetraacetic acid tetrasodium solution, Trypsin-EDTA.4Na solution） 28
壹拾 免疫螢光染色法－ PLP固定溶液（ Immunofluorescence- periodate-lysine-paraformaldehyde fix solution, IF-PLP fix solution） 28
壹拾壹 免疫螢光染色法細胞透性／蛋白質阻斷溶液 （Immuno- fluorescence cell permeabilization/blocking solution） 29
壹拾貳 免疫螢光染色法清洗溶液 （ Immunofluorescence wash solution） 29
壹拾參 RIPA 細胞溶解緩衝溶液（Radioimmunoprecipitation assay lysis buffer, RIPA lysis buffer） 30
壹拾肆 細胞溶解緩衝溶液（Lysate buffer） 30
壹拾伍 免疫沉澱法清洗溶液（Immunoprecipitation wash buffer, IP wash buffer） 31
壹拾陸 4倍SDS電泳追蹤染劑 （4X SDS electrophoresis sample buffer） 31
壹拾柒 聚丙烯醯胺膠體電泳分離膠體（ Sodium dodecyl sulfate polyacrylamide gel electrophoresis separating gel, SDS-PAGE separating gel） 32
壹拾捌 聚丙烯醯胺膠體電泳焦集膠體（SDS-PAGE stacking gel） 32
壹拾玖 10 倍蛋白質電泳緩衝溶液（10X SDS-PAGE running buffer） 33
貳拾 10 倍全濕式電泳轉漬緩衝溶液（10X immersion transfer buffer） 33
貳拾壹 TBST 緩衝溶液（Tris buffered saline with Tween buffer, TBST buffer） 33
貳拾貳 西方墨點法蛋白質阻斷溶液（Western blot blocking buffer） 33
貳拾參 活體外蛋白質甲基化分析溶液（in vitro protein methylation assay buffer） 33
第三節 實驗儀器 35
壹 高壓蒸氣滅菌釜（Autoclave） 35
貳 桌上型酸鹼度計（pH meter） 35
參 超純水機（Milli-Q） 35
肆 水浴槽（Water bath） 35
伍 －80°C冰箱（－80°C freezer） 35
陸 －20°C冰箱（－20°C freezer） 35
柒 4°C雙門藥品冷藏櫃（4°C two glass door drug freezer） 35
捌 4°C冰箱（4°C freezer） 35
玖 聚合酵素鏈鎖反應器（Thermal cycler） 35
壹拾 回轉式恆溫振盪培養箱（Orbital shaking incubator） 35
壹拾壹 微波爐（Ｍicrowave） 35
壹拾貳 水平式無菌無塵操作台（Laminar flow） 35
壹拾參 二氧化碳培養箱（Direct heat CO2 incubator） 35
壹拾肆 桌上型低溫高速離心機（Refrigerated centrifuge） 36
壹拾伍 分光光度計（Spectrophotometer） 36
壹拾陸 液態氮桶（Liquid nitrogen storage vessels） 36
壹拾柒 試管旋轉混合器（Mixer for rotator） 36
壹拾捌 迴轉式振盪器（Orbital shaker） 36
壹拾玖 倒立式螢光顯微鏡（Inverted fluorescence microscopy） 36
貳拾 直立式螢光顯微鏡（Fluorescent microscopy） 36
貳拾壹 顯微鏡數位彩色照相系統（Microscope digital camera） 36
貳拾貳 震盪器（Vortex） 36
貳拾參 盤式酵素免疫分析儀（ELISA microplate reader） 36
貳拾肆 流式細胞儀（Flow cytometry） 36
貳拾伍 熱風循環烘箱（Hot circulator exact oven） 36
貳拾陸 磁力加熱攪拌器（Magnetic hotplate stirrer） 36
第四節 實驗方法 37
壹 質體量化（Plasmid amplification） 37
壹之一 勝任細胞製備（Competent cell preparation） 37
壹之二 勝任細胞轉形作用（Competent cell transformation） 38
壹之三 質體純化（Miniprep）及定量 38
壹之四 DNA 瓊膠電泳分析（DNA agarose gel electrophoresis） 40
貳 定點突變質體製備 (Point mutagenesis plasmid) 40
貳之一 引子設計 (Primer design) 40
貳之二 聚合酶鏈反應 (Polymerase Chain Raction, PCR) 41
貳之三 PCR轉型作用 (PCR Transformation) 42
參 細胞培養（Cell culture） 43
参之一 細胞繼代培養（Cell sub-culture） 43
参之二 細胞冷凍及解凍（Cell freezing and thawing） 44
肆 質體表現 （Plasmid expression） 46
細胞轉染（Transfection） 46
伍 蛋白質分析（Proteinic analysis） 46
伍之一 免疫螢光染色法（Immunofluoresence） 46
伍之二 細胞蛋白質萃取（Protein extraction） 47
伍之三 蛋白質濃度測定 48
伍之四 免疫沉澱法（Immunoprecipitation） 49
伍之五 聚丙烯醯胺膠體電泳（ Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE） 49
伍之六 西方墨點法（Western blot） 51
陸 細胞增生分析（Cell proliferation assay） 52
細胞存活率分析（3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, MTT assay） 52
柒 細胞週期分析（Cell cycle analysis） 53
有絲分裂指數（Mitotic index） 53
捌 類傷口癒合分析（Wound healing assay） 53
玖 活體外蛋白質甲基化分析（in vitro protein methylation assay） 53
壹拾 HURP 進出細胞核觀察（Nuclear targeting of HURP） 54
壹拾壹 統計分析 54
第四章 實驗結果 55
第一節 確認PRMT5甲基化HURP的位點 55
壹 建立甲基化HURP的突變質體 55
貳 PRMT5甲基化HURP在HURP R122和R496位置 55
第二節 PRMT5甲基化HURP的機轉 56
壹 PRMT5和Aurora-A會透過Ajuaba相互調節 56
貳 HURP的甲基化和磷酸化之間的交互作用 58
參 RioK1和Ajuba會協助PRMT5辨識下游受質HURP 58
肆 JMJD6 去甲基化HURP 58
第三節 甲基化HURP在細胞中的分子意義 59
壹 在細胞中表現模擬HURP甲基化突變質體。 59
貳 甲基化HURP影響HURP的蛋白質穩定度 59
第四節 甲基化HURP在細胞中的生理意義 60
壹 甲基化HURP影響細胞的生長速率 60
貳 模擬甲基化HURP突變體增加chromosome misalignment的比例 60
參 去甲基化的HURP會穩定spindle 61
肆 甲基化HURP對migration 的調控 61
伍 模擬甲基化HURP突變體促進Golgi reassemble 62
第五章 討論 63
壹 HURP的甲基化和磷酸化之間的交互作用 63
貳 RioK1和Ajuba會協助PRMT5辨識下游受質HURP 63
參 HURP甲基化突變質體在細胞中表現情形 63
第六章 結論 64
參考文獻 82

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