多功能的腫瘤易感基因TSG101(Tumor susceptibility gene 101) 產物為泛素化路徑之無活性E2泛素結合酶，參與細胞內蛋白質的分選、囊泡運輸、轉錄調節及細胞生長分化的調控等功能。過去研究顯示，細胞中的TSG101可被單或多泛素化修飾，蛋白質泛素化修飾與其在細胞內之功能息息相關，但過去之研究並未對TSG101泛素化鍵結狀況進行詳盡探討。泛素上有七個Lysine (K)位點，分別為K6、K11、K27、K29、K33、K48、K63，這些不同位點之多泛素化，賦與被修飾蛋白不同生物功能如蛋白質的降解、DNA 受損修復以及細胞內噬作用、蛋白分選等。另外文獻報導，在細胞內E3泛素連接酶AIP4藉由多種泛素K位點鏈結調節其受質蛋白的多泛素化，而參與多種細胞生理功能，包括紅血球 (erythroid)和淋巴細胞 (lymphoid cell)分化和免疫反應的調節，且其基因突變與多系統器官自體免疫性疾病形成原因之一。為了探討細胞內TSG101蛋白修飾態狀況及其是否為AIP4之泛素化活性之受質，以增進對其參與生理功能調節訊息路徑之了解，本研究首先探討TSG101蛋白之泛素修飾狀況，實驗中將pHA-TSG101分別與各種單一K位點泛素表達質體p6His-Ub-K共同轉染到HeLa細胞中，以進行泛素化修飾蛋白之分析。並觀察proteasome抑制劑MG132之影響。結果發現不同K位點單泛素化修飾會影響HA-TSG101在轉染細胞中之steady-state含量，以MG132抑制proteasome活性，可以減緩其降解，尤其在含單一K63位點泛素表達組之影響最顯著，顯示該K63位點多泛素化E3泛素連接酶在HA-TSG101細胞中含量之維持相當重要。將pHA-TSG101和pFlag-AIP4表達質體共同轉染後進行免疫共沉澱分析發現兩者有交互作用，而HA-TSG101可能為AIP4之受質。將pHA-TSG101分別和野生型Flag-AIP4或顯性負向突變型Flag-AIP4DN表達質體共同轉染至His-Ub-HeLa細胞(為6His-Ub-WT質體之永久轉染HeLa細胞株)內，發現AIP4之表現會使HA-TSG101之表現量顯著增加，而Flag-AIP4DN則造成HA-TSG101含量下降到比控制組還低，顯示此穩定HA-TSG101蛋白之作用具有AIP4 E3泛素連接酶依賴性，其是否暗指AIP4活性訊息路徑介導之K63位點泛素化在TSG101 steady-state含量之維持扮演相當重要之角色則有待進一步實驗加以證實。

Tumor susceptibility gene 101（TSG101）encodes an inactive ubiquitin conjugating E2 enzyme implicated in regulation of protein sorting, vesicular trafficking, transcription activation of nuclear receptor, cell growth and differentiation. Previous studies showed that TSG101 can be mono- or poly- ubiquitinated, which is relevant to its functional status. There are seven Lysine (K) sites, K6, K11, K27, K29, K33, K48 and K63, on ubiquitin (Ub). Polyubiquitination using different Ub K sites confers differential function for protein degradation, DNA damage repair, endocytosis and protein sorting. AIP4 E3 ubiquitin ligase modifies its substrates involved in erythroid and lymphoid lineage differentiation and the associated immune responses. Mutation in AIP4 gene resolves in multisystemic autoimmune disease. TSG101 was recently shown to be a molecular checkpoint for T cell receptor downregulation. Here we investigate the ubiqutination status of TSG101. The ubiquitin-conjugated protein in lysate of cells co-transfected with pHA-TSG101 and His-tagged wild type Ub or each K site mutant ubiquitin expression plasmids was purified on nickel beads and then subjected to western blotting using antibodies against HA-TSG101 or His-tag. The results showed that K series mutant had differential effect on the steady-state of HA-TSG101. Proteasome inhibitor could alleviate its degradation especially in the K63 ubiquitin expression group, implying K63 ubiquitination E3 ligase is critical in maintaining HA-TSG101 level. Our coimmunoprecipitation result demonstrated the interaction between AIP4 and HA-TSG101, implying that TSG101 might be a substrate for AIP4. The ectopic overexpression of AIP4 increased the amount of HA-TSG101 in an E3 ligase activity depended manner. Taken together, these results indicated that AIP4 activity mediating Ub K63 modification might be critical for regulating cellular TSG101 protein level. Further experiment should clarify this issue.

中文摘要 3
Abstract 5
第一章 背景介紹
第一節 前言 7
第二節 研究目的 18
第二章 材料與方法
第一節 質體製備 19
第二節 細胞的培養與轉染 23
第三節 建立pCB6-6His-Ub-WT表達質體之永久轉染細胞株 24
第四節 TSG101蛋白泛素化修飾分析和AIP4與TSG101蛋白穩定度之關係 25
第三章 結果
第一節 細胞內TSG101蛋白質之泛素修飾狀況 31
第二節 MG132蛋白質分解體抑制劑的處理會影響細胞內TSG101蛋白表現量 33
第三節 TSG101 蛋白可與AIP4蛋白交互作用 33
第四節 AIP4與TSG101蛋白穩定度之關係 34
第四章 討論 36
第五章 參考文獻 40
第六章 圖表 51

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