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標題: | 人類腺嘌呤核苷二磷酸核醣化相似因子四與其結合蛋白特性探討 Functional Characterization of human ARL4s and their interacting partners |
作者: | Meng-Chen Tsai 蔡孟真 |
指導教授: | 李芳仁(Fang-Jen Lee) |
關鍵字: | 腺嘌呤核?二磷酸核醣化相似因子,細胞移動,磷酸肌醇, Arf,Arl,Robo,Cdc42,Erk2,phosphatidylinositide,liposome, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 人類四腺嘌呤核苷二磷酸核醣化相似因子(ARL4A)可以幫助維持高基氏體結構以及調控肌動蛋白的組裝,我們先前的研究發現在發育的過程當中ARL4A主要表現在神經系統中。我們發現Robo1為一個新的ARL4A作用蛋白,Robo1為一個穿膜受器,其受質為Slit,兩者的作用可以調控神經軸突的生長方向。ARL4A藉由幫助Robo1和srGAP之間的作用來達到增加細胞移動力的目的,降低ARL4A或Robo1的表現會抑制細胞的移動力,而表現野生型的Robo1可以恢復細胞的移動力,但是不能和ARL4A作用的Robo1-A1及Robo1-A2兩個突變則不能恢復恢復細胞的移動力。因此,我們的實驗結果指出,ARL4A促進Robo1和srGAP的作用,以達到促進細胞移動的功用。 磷脂醯肌醇(phosphatidylinositide)在細胞中扮演重要的角色,磷脂醯肌醇可以作為生物膜上讓蛋白質結合的脂質,並可進一步促進訊息傳遞、細胞移動、胞吞作用等等的細胞反應。我們發現到ARL4A、ARL4C及ALR4D (ARL4s)可以幫助膜上磷脂醯肌醇的累積,為了找到ARL4s達成此目的下游蛋白,我們利用脂質囊浮力試驗(Liposome floatation assay)找到了一群會與ARL4s作用的蛋白質,並利用酵母雙雜交試驗證明我們找到的其中一個蛋白質Erk2會與ARL4s有作用。 ADP-ribosylation factor-like protein 4A (ARL4A) is a member of Arf family small G proteins. ARL4A functions in maintenance of Golgi structure and regulation of actin dynamics. ARL4 is developmentally regulated and expresses in neuron system. We identify a novel interacting protein of ARL4A, Robo1. Robo1 is a transmembrane receptor and, alone with its ligand Slit, involves in the axon neuron guidance. Both in vitro and in vivo assays are used to demonstrate direct interaction between ARL4A and Robo1. Moreover, ARL4A interacts with Robo1 to promote the recruitment of Slit-Robo GTPase activating proteins 1 (srGAP1), a downstream effector of Robo1. ARL4A-Robo signaling pathway facilitates cell migration. Knockdown of ARL4A or Robo1 attenuates cell motility, which can be rescued by expressing wildtype Robo1. However, expressing ARL4A-interaction defective Robo mutants does not rescue defect in cell motility. Our observation indicates ARL4A interacts with Robo1 to facilitate recruitment of srGAP1, which leads to inactivation of Cdc42 and promotion of cell migration. Phosphatidylinositol plays various roles to cell physiology. Phosphatidylinositol acts as dock site for proteins binding to facilitate cell signaling, migration, phagocytosis, and various cellular functions. We observe that ARL4A, ARL4C, and ARL4D (ARL4s) promote accumulation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), phosphatidylinositol (3,4,5)-trisphosphate (PI(3, 4, 5)P3) and phosphatidylserine (PS). To identify the downstream effectors by which ARL4s modulates membrane lipid composition. We develop liposome floatation assay to seek for ARL4s effectors in the presence of lipid bilayer. We identify several proteins involve in lipid biogenesis, especially phosphatidylinositol 5-phosphate 4-kinase (PIP4K). We examine one of the obtained proteins, Erk2, for its interaction with ARL4s by yeast-two hybrid. Our results show the liposome floatation assay is capable of identify novel interacting proteins of ARL4s. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52126 |
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