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標題: | 基質金屬蛋白酶-7最小功能片段(6 kDa)參與調控αvβ3整合蛋白在膜上富含膽固醇硫酸鹽微域的胞吞作用之研究 A novel cryptic disintegrin-like molecule regulates αvβ3 integrin endocytosis in cholesterol sulfate-rich cell surface microdomain: 6 kDa mini-MMP-7 |
作者: | Ya-Wen Kao 高雅雯 |
指導教授: | 游偉絢 |
關鍵字: | 6 kDa基質金屬蛋白酶,-7,αvβ3整合蛋白,胞吞作用,膽固醇硫酸鹽,網格蛋白, 6 kDa mini-MMP-7,αvβ3 integrin,endocytosis,CS,clathrin, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 基質金屬蛋白酶 (MMP)屬於一種依賴鋅離子的胜肽內切酶,其在細胞外基質的重塑、表面受體的更迭、細胞膜上配體的活化扮演重要的角色。基質金屬蛋白酶-7 (MMP-7)是MMP家族中的最小成員,其C端的63個胺基酸為組成鋅離子結合模體的最小功能片段,結構為螺旋-彎-螺旋模體,所有的MMP成員皆具有此分子量為6 kDa的片段,在本研究稱作「6 kDa mini-MMP-7」。首先,在HEK293T以及CL1-1細胞中表現6 kDa mini-MMP-7,發現細胞型態呈現圓型,並且失去附著生長的能力,由TUNEL檢測細胞凋亡以及流式細胞儀分析細胞週期的結果,顯示這些細胞沒有走向細胞凋亡。接著在細胞內表現6 kDa mini-MMP-7與αv整合蛋白,經由螢光顯微鏡觀察,發現兩者共同位於細胞表面的位置。此外,由免疫細胞染色以及免疫沉澱分析法顯示6 kDa mini-MMP-7和MMP-7皆會與αvβ3整合蛋白交互作用,且發現MMP-7是透過位在細胞膜穴樣內陷的膽固醇硫酸鹽與αvβ3整合蛋白形成複合體。因此,我們構建一個含有RGD序列的MMP-7的片段「TAD」,進而由免疫沉澱分析法證實MMP-7與αvβ3整合蛋白的交互作用非關RGD序列。另外,經由共軛焦顯微鏡觀察,發現αv整合蛋白聚集的現象,而6 kDa mini-MMP-7則位於由αv整合蛋白形成的胞吞小泡之中,進而利用免疫沉澱分析法證實,6 kDa mini-MMP-7所誘導的αvβ3整合蛋白胞吞作用不是透過網格蛋白所調控。綜合以上結果,我們發現6 kDa mini-MMP-7與αvβ3整合蛋白結合在細胞膜穴樣內陷,促使αvβ3整合蛋白進行胞吞作用,由於細胞膜上αvβ3整合蛋白的降低,進而導致細胞失去附著生長的能力。由此可知,6 kDa mini-MMP-7對於αvβ3整合蛋白的更迭佔有重要的調控地位。 Matrix metalloproteinase-7 (MMP-7, matrilysin, pump-1) is the smallest member in the MMPs family which is a zinc-dependent endopeptidases that regulate extracellular matrix (ECM) remodeling, surface receptor turnover and membrane-bound ligand activation. The 6 kDa mini-MMP-7 is a 63 residues truncated enzyme with the minimal zinc-binding motif (helix-Met turn-helix) at the C-terminal in all members of MMPs. First, both HEK293T and CL1-1 cells that expressed 6 kDa mini-MMP-7 were rounding and detached from culture dishes and the results of TUNEL assay and flow cytometry showed that these cells were still alive. Then, the well-colocalization of 6 kDa mini-MMP-7 and αv integrin on the cell surface was observed by fluorescence microscopy. From the immunocytochemistry and immunoprecipitation (IP) assay, we found that both 6 kDa mini-MMP-7 and endogenous MMP-7 could interact with αvβ3 integrin, moreover, MMP-7 formed a complex with αvβ3 integrin through cholesterol sulfate (CS) in cell surface microdomain. Furthermore, MMP-7 fragment ‘TAD’ containing RGD-motif was constructed and we found that the binding of MMP-7 and αvβ3 integrin was RGD-independent manner. Besides, confocal microscopy images indicated that αv integrin clustered and 6 kDa mini-MMP-7 was colocalized in the endocytotic vesicle-like intracellular compartments. Thus, endocytosis of αvβ3 integrin via clathrin-independent pathway was also established by IP. Taken together, these results provide evidences for a novel regulatory role of the 6kDa mini-MMP-7 in αvβ3 integrin turnover, and it serves as a putative disintegrin-like molecule and induces clathrin-independent endocytosis of αvβ3 integrin. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23387 |
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顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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