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研究生:陳克偉
研究生(外文):Ke-Wei Chen
論文名稱:PLEKHF1參與細胞吞噬作用之研究
論文名稱(外文):Roles of PLEKHF1 in endocytosis-related processes.
指導教授:林奇宏林奇宏引用關係
指導教授(外文):Chi-Hung Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
中文關鍵詞:細胞吞噬
外文關鍵詞:endocytosisearly endosomePLEKHF1Rab5Rab7phafinlysosomeFYVE
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在實驗室之前的研究中,利用基因體研究及生物資訊的方法尋找肝癌細胞中表現量改變的基因,並且在配合visual screening下發現兩個分布於內膜體的功能未知新穎基因:PLEKHF1及PLEKHF2 (pleckstrin homology domain containing, family F (with FYVE domain) member 1/2, or PF1/2)。在細胞中大量表現PF1和PF2時可造成早期內膜體的增大;本研究主要針對PF1造成此現象的原因以及在細胞吞噬中扮演的角色。PF1與P2同屬一個新發現的蛋白家族:Phafin(protein containing both PH and FYVE domains),其成員保留在不同物種之中,目前發現至少共有十四個基因。以綠色螢光蛋白作為融合標籤發現PF1可同時分布在早期內膜體和溶酶體;PF2則無溶酶體的分布。大量表現時造成早期內膜體增大的能力需仰賴Rab5的活性,顯示PF1可能作用於Rab5上游;而PF1進入晚期內膜體/溶酶體的現象則在細胞同時表現顯性抑制型Rab7時消失,可推測PF1與Rab7的功能相關。如同其他已知的FYVE蛋白,PF1的FYVE區域即可進入帶有Rab5或運鐵蛋白的早期內膜體,同時in vitro實驗也證實PF1的確有結合PI(3)P的能力。為了進一步研究在早期內膜體的功能,觀察活體細胞攝取運鐵蛋白發現PF1在吞噬早期即有所參與,但並不影響其速率。另一方面,PF1的C端序列可能與進入溶酶體的能力相關,將其尾端三十個氨基酸融合到PF2後,分布到LysoTracker Red的能力明顯增加;但尾端序列本身卻無法有效地進入晚期內膜體/溶酶體,因此推測此尾端序列可能與其上游共同組成一蛋白結合區域,控制PF1在溶酶體的分布。綜合以上發現,本研究初步地探討了PF1在細胞吞噬過程可能扮演的角色,同時也發現其同源蛋白PF2在細胞中參與不同的功能,兩者的差異可能有部分與C端序列相關。
Based on a series of genomics analysis that targets differentially expressed genes in liver cancers, we have identified, from their distinctive endosomal localization patterns, two novel genes named PLEKHF1 (pleckstrin homology domain containing, family F (with FYVE domain) member 1, or PF1) and PLEKHF2 (PF2). Overexpression of either PF1 or PF2 resulted in the enlargement of the endosome. This study focuses on deciphering the signalling pathway underlying this intriguing phenotype of PF1. PF1 and PF2 represent two members of a new protein family called Phafins (protein containing both PH and FYVE domains), which consists of 14 genes from different species. GFP-tagged PF1 proteins were found in early endosomes and lysosomes, where they colocalized with EEA1 and LysoTracker Red, respectively, and this early endosomal localization is dependent on its FYVE domain. Increased expression of PF1 caused an enlargement of early endosome, as had been observed in cells expressing the constitutively active form of Rab5 gene. Endosomal enlargement caused by PF1 overespression was abolished by the simultaneous presence of the dominant negative Rab5, indicating that PF1 could regulate the endocytosis process in a Rab5-dependent manner. In addition, the presence of PF1 in the lysosome was reduced in cells containing the dominant negative Rab7. Transferrin uptake assay further showed that PF1 is involved in the early stages of endocytosis, but its overexpression had no effect on internalization rate. Furthermore, the C terminal sequence may comprise elements necessary for PF1 to enter lysosomal compartments. While confering PF2 the ability to enter lysosomes, the tail domain of PF1, when expressed alone, showed only minor colocalization with LysoTracker, suggesting tail domain may act in concert with upstream sequences to mediate lysosomal loclization. Taken together, our results suggested that PF1 represents a new player in Rab-mediated endocytosis precesses, as its effects on the formation of early endosomes was mediated by Rab5 signalling, whereas its continuous presence and metabolisms in the lysosome was modulated by the Rab7 signalling.We propose that PF1 may act as an adaptor functioning along the endocytic pathway through protein-lipid and possibly protein-protein interactions.
壹、中文摘要.........4
貳、英文摘要.........5
叁、前言.............7
肆、實驗材料與方法..20
陸、結果............34
柒、討論............42
捌、參考文獻........47
玖、圖目錄..........52
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