細胞內噬作用參與調控內吞營養物質、細胞表面接受器再循環利用和分解等過程，其中內含體分選傳輸複合體ESCRTs會協助多囊泡體(MVBs)之形成、蛋白質分選及囊泡運輸作用。腫瘤易感基因101 (TSG101)是組成ESCRT-I成員之一，參與內含體蛋白傳送至多囊泡體之步驟。細胞的內噬路徑囊泡與細胞自噬路徑囊泡皆會直接與溶酶體融合，藉由溶酶體內的酵素將其內涵之蛋白分解回收再利用。在有些情況下，細胞自噬小體與多囊泡體會融合形成amphisome，進一步分解細胞自噬體與多囊泡體中的分子，其主要功能是清除細胞內錯誤摺疊、異常堆積的泛素化蛋白及老化的胞器，以維持細胞的生理平衡。文獻指出細胞內噬路徑異常會影響細胞自噬作用的進行。細胞藉由自噬作用清除無用蛋白或胞器的過程中需要ESCRTs的幫助，避免造成與蛋白質沉積相關的退化性疾病如神經退化或癌症之發生。而SQSTM1/p62具有與泛素結合功能區，可標記細胞中異常聚積之泛素化蛋白，在自噬作用路徑受阻無法順利將之清除時，會有SQSTM1/p62累積現象。我們結果顯示當細胞處於飢餓狀態或是處理細胞自噬誘導劑時，會增加TSG101及細胞自噬作用蛋白MAP1LC3-II的表現量，顯示TSG101可能參與細胞自噬作用的調控。本研究中以神經母細胞瘤SHSY5Y細胞模式及建立自噬標幟螢光蛋白GFP-MAP1LC3報導細胞株，用以探討TSG101在細胞自噬作用之角色，實驗中以siRNA抑制TSG101蛋白表現後，細胞再處理細胞自噬誘導劑rapamycin或是細胞自噬抑制劑chloroquine後，來觀察TS101表現降低對自噬作用相關標幟蛋白MAP1LC3-II、SQSTM1/p62及細胞中泛素修飾蛋白的表現量之影響，以釐清TSG101在細胞自噬作用之角色。實驗結果發現以siRNA抑制TSG101表現會造成MAP1LC3-II、SQSTM1/p62及泛素修飾蛋白質之累積。另一方面，當內源性TSG101表現被抑制時也會增加報導細胞株中的GFP-LC3 綠瑩光亮點數目，且被DsRed-Rab7標定的多囊泡體與細胞自噬體的共位現象也會減少。此結果表示TSG101可能會幫助多囊泡體與細胞自噬小體融合步驟以促進細胞自噬作用進行。本研究結果更增進我們對TSG101參與細胞自噬作用分子機制之瞭解。

The endocytosis involves in a variety of cellular activities including nutrient intake, and receptor recycling or degradation. The endosomal sorting complexes required for transport (ESCRTs) plays an important role that orchestrate the formation of multivesicular bodies (MVBs), which regulates protein sorting and vesicular trafficking. Tumor Susceptibility Gene 101(TSG101) is a member of ESCRT-I responsible for sorting ubiquitin-modified protein into MVBs. The endocytic and autophagic pathways merge at the lysosomal compartment. Proteins sorting into the MVBs are processed upon fusion of autophagosome and lysosome to form autophagolysosome by lysosomal enzymatic digestion. The autophagosome can fuse with endosomes/multivesicular bodies to generate the amphisome for degradation cargo containing both autophagic and endocytic materials. This autophagic pathway plays a critical role for cellular physiological homeostasis by clearing misfolded protein, aberrant accumulation of ubiquitin modified proteins, and damaged organelles. Recent reports confirmed that abnormal endocytic pathway may affect autophagic process, which is critical for the prevention of cancer or neuronal degenerative diseases. In addition, SQSTM1/p62 contains an ubiquitin binding domain that can bind to the ubiquitylated proteins destined for selective degradation. The accumulation of SQSTM1/p62 serves as a marker for perturbation of autophagic process. Our previous data indicated that TSG101 and MAP1LC3-II are upregulated simultaneously upon starvation or autophagic induction with rapamycin. In this study, the neuroblastoma SH-SY5Y and GFP-LC3 autophagic reporter cell lines were used to investigate the effect of TSG101 on the LC3-II, p62 and ubiquitinated protein levels upon deprivation of TSG101 using siRNA in conjunction with the treatment of autophagic modulators, rapamycin and chloroquine. We found that silencing of TSG101 resulted in the accumulation of MAP1LC3-II, SQSTM1/p62, and ubiqutinated proteins. The number of GFP-LC3 puncta was increased in TSG101 knockdown SHSY5Y cells, while co-localization of DsRed-Rab7-associated MVB and autophagosome were diminished, suggesting TSG101 may facilitate fusion steps of autophagosome with MVB to promote autophagic activity. These results provide the new role and molecular mechanism of TSG101 in the autophagic pathway.

論文審定書 ..................................................................................................................... i
致謝 ................................................................................................................................ ii
摘要 ............................................................................................................................... iii
Abstract ........................................................................................................................... v
前 言 .............................................................................................................................. 1
腫瘤易感基因 TSG101 的功能與背景 ................................................................. 1
細胞內噬作用(endocytosis)的功能 ....................................................................... 2
ESCRT 的功能 ....................................................................................................... 2
細胞自噬作用的功能與背景 ................................................................................ 3
蛋白質的泛素化作用 ............................................................................................ 5
細胞內噬作用與細胞自噬作用的關係 ................................................................ 6
神經退化性疾病與細胞內噬作用及細胞自噬作用的關聯性 ............................ 7
材料與方法 .................................................................................................................. 10
一、細胞培養 ...................................................................................................... 10
二、質體的製備 .................................................................................................. 10
三、真核細胞轉染(transfection) ......................................................................... 12
四、蛋白質萃取 .................................................................................................. 13
五、西方墨點法(Western blotting) ..................................................................... 14
六、RNA 干擾技術(RNA interference) .............................................................. 15
七、免疫螢光染色法（immunofluorescence staining） ................................... 16
八、永久轉染細胞株 (stable transfection) ........................................................ 16
結果 .............................................................................................................................. 18
誘發細胞自噬作用會增加 TSG101 的蛋白質表達 ........................................... 18
誘發細胞自噬作用會增加 GFP-MAP1LC3 的螢光蛋白質表達 ...................... 19
抑制 TSG101 的表達會影響細胞自噬標誌蛋白 MAP1LC3 的表現 ............... 20
抑制 TSG101 蛋白質表現會導致 GFP-MAP1LC3 螢光標定亮點累積 ......... 21
抑制 TSG101 蛋白質表達會影響泛素化修飾蛋白之分解路徑 ....................... 22
TSG101 與細胞自噬作用路徑之關係 ................................................................ 24
進一步探討 TSG101 對 amphisome 形成之影響 .............................................. 26
討論 .............................................................................................................................. 28
細胞可能藉由增加 TSG101 的蛋白質表達協助細胞自噬作用進行 ............... 28
抑制 TSG101 蛋白質會影響細胞自噬作用的進行造成細胞自噬體累積 ....... 31
探討泛素修飾蛋白質與 TSG101 及細胞自噬作用之關係 ............................... 34
TSG101 對於細胞自噬作用路徑之調控角色 .................................................... 36
總結與展望 .................................................................................................................. 40
圖表 .............................................................................................................................. 42
參考文獻 ...................................................................................................................... 62
補充結果圖表 .............................................................................................................. 69
附錄 .............................................................................................................................. 72

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