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研究生(外文):Jian-Fa Hsu
論文名稱(外文):Studies of a novel protein delivery system and interacting proteins of the human coactivator SRC-1
指導教授(外文):Han-Jung Lee
外文關鍵詞:nuclear receptorprotein transduction domains
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細胞核受體 (nuclear receptor) 是一個受ligand調控的轉錄因子,他可以直接結合到標的基因的hormone response elements (HREs)或者形成同二元體甚至與retinoid X receptor (RXR) 形成異二元體結合到HREs上。共調節者的參與也是影響細胞核受體轉錄反應的重要元素,其中steroid receptor coactivator-1 (SRC-1) 是最早被選殖出來的的共活化者,我們欲瞭解此共調節者如何在這複雜的轉錄程序上扮演他的角色。在本研究中涵蓋了兩個目標,其一為探索SRC-1在細胞中所處的位置,其二為選殖SRC-1交互作用的蛋白質。因為細胞膜生物特性的限制,將蛋白質送進細胞仍是一個障礙。最近幾年,一些具有蛋白質傳導區域 (protein transduction domains ; PTDs) 的胜�L相繼被發現能夠有效的穿透哺乳動物細胞膜,此作用稱為蛋白質傳導 (protein transduction)。Human immunodeficiency virus (HIV)-1 transactivator protein (TAT)的蛋白質傳導區域 ( 47-57個胺基酸)已經被證實能夠穿透細胞膜。我們以TAT-GFP這個融合蛋白來探討TAT蛋白質在HepG2細胞內的輸送情形。結果顯示TAT-GFP融合蛋白不僅能夠穿透哺乳動物細胞的細胞膜,更能夠穿透細胞核膜聚集在細胞核中,可是單單以green fluorescent protein (GFP) 卻無法通過哺乳動物細胞的細胞膜。我們進一步想以TAT-SRC-GFP融合蛋白來探討SRC-1在經由與TAT形成融合蛋白進入細胞內時,其所在位置及是否仍具有生物活性。但是目前TAT-SRC-GFP在大腸桿菌中的蛋白質表現表現遇到些許困難。為了選殖SRC-1可能的交互作用蛋白質,我們利用酵母菌二合法,以SRC-1 118-780的片段基因 (包括PAS區域和LXXLL區域) 為餌,來篩選人類cDNA基因庫中是否還有與SRC-1有交互作用的蛋白質。截至目前為止,我們尚未從人類HeLa、kidney和brain的cDNA基因庫中選殖出與SRC-1有交互作用的蛋白質。原因可能為mating效率太低,基因庫品質欠佳,或與SRC-1有
Nuclear receptors act as ligand-dependent transcription factors by directly interacting as monomers, homodimers, or heterodimers with the retinoid X receptor (RXR) with hormone response elements (HREs) of target genes. The effects of nuclear receptors on transcription are mediated through recruitment of coregulators. Steroid receptor coactivator-1 (SRC-1) was the first identified coactivator. We want to know that how assembly of transcriptional complexs activates nuclear hormone receptors by recruitment of distinct subclass of coregulators. We have two goals in this approach, one is to observe locolization of SRC-1, and another is to isolate interacting proteins of SRC-1. Intracellular delivery of proteins remains a problem because of the bioavailability restriction imposed by the cell membrane. Recently, several peptides, called protein transduction domains (PTDs) have been identified that possess the ability to tranverse cell membranes efficiently in a process termed protein transduction. The PTD in the human immunodeficiency virus (HIV-1) transactivator (TAT) protein (amino acid 47-57) has been shown to successfully mediate the introduction of proteins into mammalian cells. TAT-GFP fusion protein was used to study the intracellular localization and traffick in the human HepG2 cells. The results showed that TAT-GFP fusion protein can enter into cytoplasm and accumulate in the nucleus.However, green fluorescent protein (GFP) itself fails to do so. Furthermore, TAT-SRC-GFP was used to monitor SRC-1 biological activity and localization. Unfortunately, we currently have difficulty in over-expression of TAT-SRC-GFP in E.coli strains. Using yeast two-hybrid system, we want to isolate SRC-1 interacting protein from human cDNA library using SRC-1 118-780 fragments with PAS domain and LXXLL motifs as bait. So far, We did not find any SRC-1 interacting protein from screening human HeLa, kidney, and brain cDNA libraries. We assume that low efficiency of yeast mating, low quality of cDNA libraries, or SRC-1 interacting proteins have tissue specificity, or even don’t exit.
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