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標題: | 以定量蛋白質體學分析小鼠集尿管細胞株(mpkCCD)頂尖膜蛋白體受抗利尿激素刺激之變化 Quantitative Analysis of Apical Membrane Proteome of mpkCCD Cells in Response to Vasopressin |
作者: | Chin-San Loo 呂振山 |
指導教授: | 余明俊(Ming-Jiun Yu) |
關鍵字: | aquaporin-2,apical trafficking,apical proteome,SILAC quantitative, 第二型水通道蛋白,頂膜轉運,頂膜蛋白質體,SILAC定量, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 在抗利尿激素(antidiuretic hormone vasopressin)的刺激之下,腎臟集尿管細胞中的水通道蛋白(aquaporin-2, AQP2)會從細胞內液胞轉運到細胞頂膜(apical plasma membrane)上,藉此增加腎臟集尿管細胞對水分的通透性以及再吸收。 上述調控機制對於身體水分平衡扮演重要的角色,然而AQP2頂膜轉運的分子機制尚未被完全瞭解。 我們以生物素標定術(surface biotinylation)配合上卵白素純化術(streptavidin affinity chromatography)收集小鼠集尿管細胞株(mpkCCD)頂膜上之蛋白質,再藉由SILAC為基礎的定量蛋白質體學方法分析受抗利尿激素調控並參與頂膜轉運的蛋白質。 在三重複實驗之下,我們一共鑑定出1,028個蛋白質,其中100個蛋白質會因在抗利尿激素類似物(dDAVP)的刺激而在頂膜區域上有量的變化,其中有45個蛋白質是增加,而有55個蛋白質是減少。 進一步利用DAVID Gene Ontology分析得知有變化之蛋白質主要扮演著蛋白質分佈(protein localization)以及肌動蛋白骨架重組(actin cytoskeletal reorganization)之角色。 在這100個受dDAVP調控之蛋白質中,有4個蛋白質在dDAVP刺激下增加至1.4倍(log2[dDAVP/vehicle]>0.5)以上的量,分別為Aquaporin-2,Protein Mal2,Clamin isoform b與MAGUK p55 subfamily member 5;另外Tumor necrosis factor receptor superfamily member 10B precursor則是減少至1.4倍(log2[dDAVP/vehicle]<-0.5)。 我們利用細胞免疫螢光染色(immunofluorescence staining)與免疫墨點法(immunoblotting)證實Protein Mal2 (MAL2) 會在dDAVP刺激之下轉運至細胞頂膜上。 先前研究指出MAL2為一種脂筏(lipid raft)蛋白質且與細胞頂膜蛋白質之胞轉作用(transcytosis)有關。 我們的結果顯示部分AQP2在dDAVP刺激下會移動到被認為是脂筏的detergent-resistant membrane fraction中。 因此,我們的研究顯示經由抗利尿激素調控的AQP2頂膜轉運可能需要皮質肌動蛋白細絲的動態變化與MAL2脂筏蛋白質的參與。 In response to the antidiuretic hormone vasopressin, the molecular water channel protein aquaporin-2 (AQP2) redistributes from the intracellular vesicles to the apical plasma membrane to increase water permeability of the renal collecting ducts. This vasopressin-regulated water permeability is critical to whole body water homeostasis; however, the molecular details of the AQP2 trafficking mechanism are largely unknown. Here, we used surface biotinylation and streptavidin affinity chromatography to enrich for apical membrane proteins of the mouse cortical collecting duct (mpkCCD) cells followed by SILAC based quantitative proteomics to identify potential apical trafficking machinery regulated by vasopressin. A total of 1,028 proteins were quantified in three replicates. Among the quantified proteins, 100 showed significant changes in abundance in response to the vasopressin analog dDAVP (p<0.05) including 45 increased and 55 decreased proteins. DAVID Gene Ontology term analysis of these changed proteins revealed predominant roles of these proteins in protein localization and actin cytoskeletal reorganization. Among the dDAVP-regulated proteins, 4 proteins (Aquaporin-2, Protein Mal2, Clamin isoform b, and MAGUK p55 subfamily member 5) showed significant increases greater than 1.4 fold (log2[dDAVP/vehicle]>0.5) in response to dDAVP versus vehicle; 1 protein (Tumor necrosis factor receptor superfamily member 10B precursor) showed a significant decrease greater than 1.4 fold (log2[dDAVP/vehicle]<-0.5). Immunoblotting and immunofluorescence staining of Protein Mal2 (MAL2) confirmed apical translocation of MAL2 in response to dDAVP stimulation. MAL2 is a lipid raft protein thought to mediate trafficking of apical membrane proteins via transcytosis. In response to dDAVP, a portion of AQP2 moves into detergent-resistant membrane fraction (i.e. lipid raft). Our data suggests that vasopressin-mediated apical AQP2 trafficking involves dynamic changes in cortical actin filaments and potentially involves the lipid raft protein MAL2. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15881 |
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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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