請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70739
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IRSp53/BAIAP2特性之探討 Functional characterization of human Arl4D and its interacting protein, IRSp53/BAIAP2 |
作者: | Tai-Chen Tsai 蔡岱蓁 |
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4D,IRSp53/BAIAP2,Cdc42,肌動蛋白細胞骨架重塑,絲狀偽足,板狀偽足, Arl4D,IRSp53/BAIAP2,Cdc42,actin cytoskeleton remodeling,filopodia,lamellipodia, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 二磷酸線苷核醣基化因子 (Arfs) 是一群調控膜運輸、胞器完整性及細胞骨架動態性的小分子鳥嘌呤核苷酸結合蛋白 (Small GTP binding protein),其中與其結構相似的腺嘌呤核苷二磷酸核醣化相似因子4 (Arl4s) 家族包含了三個成員,Arl4A,Arl4C及Arl4D。在這篇論文中,我們發現一個新的Arl4D的作用蛋白-IRSp53。IRSp53受到兩個小分子鳥嘌呤核苷酸結合蛋白,Cdc42及Rac1的調控,分別決定其促進絲狀偽足 (filopodia) 或是板狀偽足 (lamellipodia) 的形成。透過酵母菌雙雜交系統 (yeast two hybrid) 的檢測,只有Arl4D會專一和IRSp53結合,Arl4A及Arl4C不會。進一步由試管結合實驗 (in vitro binding assay) 及免疫共沉降 (co-immunoprecipitation) 實驗驗證,我們發現持續活化及不活化的Arl4D都能和IRSp53結合。由酵母菌雙雜交系統及試管結合實驗,我們發現IRSp53 N端的229個胺基酸殘基對於兩者的結合是重要的,且和過去被報導的Rac1結合位相同。靜默IRSp53不會改變Arl4D在膜上的分布,相反地,剔除Arl4D會讓IRSp53誘使的突出數目減少,此外,只有持續活化的Arl4D會降低IRSp53誘使的絲狀偽足的長度。透過免疫共沉降實驗,我們發現Arl4D能夠和Cdc42及IRSp53存在於同一個複合體當中。總而言之,我們發現一個新的小分子鳥嘌呤核苷酸結合蛋白Arl4D能夠和IRSp53交互作用,Arl4D可能透過未知的調控方式參與在絲狀偽足的形成當中。 ADP-ribosylation factors (Arfs) are small GTP binding proteins involved in membrane transport, the maintenance of organelle integrity and cytoskeletal dynamics. The Arf-like 4 (Arl4) family is composed of three isoforms, Arl4A, Arl4C and Arl4D. Here, we identified a novel interacting protein of Arl4D, called insulin substrate receptor p53 (IRSp53). IRSp53 is a main actin modulator that regulates filopodia and lamellipodia formation, mediated by two small GTPases, Cdc42 and Rac1, respectively. Through yeast two hybrid, we found that only Arl4D, but not Arl4A or Arl4C, interacted with IRSp53. Confirmed by in vitro binding assay and co-immunoprecipitation, we demonstrated that both constitutively active and inactive mutants of Arl4D interacted with IRSp53. Revealed from yeast two hybrid and in vitro binding assay, the shortest region important for their interaction resided in the N terminal 229 residues of IRSp53, which is the same as reported Rac1 binding region. Knockdown of IRSp53 did not affect the membrane localization of Arl4D. By contrast, knockout of Arl4D decreased the number of IRSp53-induced filopodium-like protrusions. In addition, coexpression of WT and constitutively active but not inactive mutant of Arl4D reduced the average length of IRSp53-induced protrusions. Last but not least, Arl4D was found within the same complex with Cdc42 and IRSp53 from co-immunoprecipitation. In conclusion, we found that Arl4D is a novel small GTPase that interacts with IRSp53, which might regulate filopodia formation through unknown mechanism via IRSp53. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70739 |
DOI: | 10.6342/NTU201802758 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子醫學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-107-1.pdf 目前未授權公開取用 | 3.39 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。