以一個大規模的Pull-Down實驗來尋找與果蠅Echinoid有直接交互作用的蛋白質

A Large-Scale Pull-Down Assay to Screen for Direct-Interacting Proteins of Echinoid

陳惠民(Hui-Min Chen)

指導教授 ： 徐瑞洲

國立清華大學/生命科學院/分子醫學研究所/碩士(2006年)

https://doi.org/10.6843/NTHU.2006.00499

摘要

本論文的研究主題在於尋找和果蠅基因echinoid有直接交互作用的其他蛋白質分子。echinoid (ed)是一個位在細胞膜上的cell adhesion molecule。它已經被證明參與在果蠅複眼與胸部剛毛的發育過程中，其本身是一個EGFR訊號傳遞的負調控因子並且和Notch共同作用在神經元的生成機制上。

在此，我們進行了一個大規模的Pull-Down實驗，用來尋找和Ed蛋白直接或間接交互作用的其他蛋白質，希望藉此能讓我們對ed這個基因的功能有更進一步的了解。在我們的實驗中總共找到了53個蛋白質，再經過質譜儀的分析，我們將它們每個胜肰的身份鑑定出來。在它們之中，我們再依偏好挑選出12個基因，成為接下來研究的目標。其中，有6個於驗證用的一對一in vitro Pull-Down實驗，被我們證明與Ed有直接的交互作用。若依照與Ed蛋白互相黏著的強度順序來排列，這6個基因分別是CG6459，jaguar，coronin，RACK1，CG3957和ran1（由強至弱）。這些基因在之前的文獻上，已經被報導參與在各式各樣的生物現象中，諸如：訊號傳遞、細胞移動、細胞骨架運作、細胞內微泡運輸、細胞分裂增殖與細胞核質間傳輸。

然而，本論文所敘述之主題，只是二年以來的研究初步結果。我們需要更多更充足的生物本體的實驗證據，來驗證本計畫所篩選出的目標基因蛋白是否確為與Ed蛋白質實際上有反應的分子。希望未來這些侯選標的基因，能夠幫助我們對Echinoid這個多功能的蛋白分子，能有更宏觀概括的了解。

關鍵字

果蠅 ； 基因篩選 ； 交互作用

並列摘要

The focus of this thesis is centered on the gene echinoid (ed), which is a cell adhesion molecule integral to the plasma membrane. ed has been previously shown to be involved in the development of Drosophila compound eyes and notum bristles. It is a negative regulator of EGF receptor signaling pathway and synergizes with Notch in mesothorax bristle patterning.

Here, we performed a large-scale pull-down assay to screen for Ed direct-interacting proteins in order that we could understand how ed executed its multiple biological functions. A total of 53 proteins were identified in this assay, and twelve of them were selected as possible candidates and taken for further studies. Among these genes, six were proved to interact specifically with Ed in vitro. They were CG6459, jaguar, coronin, RACK1, CG3957 and ran, ranked and mentioned following their binding strength to Edintra (from strong to weak). These genes were formerly reported to be involved in diverse cellular processes, such as signal transduction, cell movement, cytoskeleton arrangement, vesicle transport, cell proliferation and even nuclear transport.

Nonetheless, the project presented here is only the initial work of the research on echinoid. We need more biological evidence to prove these plausible candidates as true and meaningful research targets. Hopefully, in the end, several of them will reveal some interesting processes ed has taken a part in and help us gain a whole view of the physiological versatility of this membrane protein, Ed.

並列關鍵字

Drosophila ； echinoid ； Embryo Extracts ； Pull-Down

參考文獻

Ahmed, A., Chandra, S., Magarinos, M. and Vaessin, H. (2003) echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway. Development, Vol. 130, 6295-6304.

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Besson, A., Wilson, T.L. and Yong, V.W. (2002) The anchoring protein RACK1 links Protein Kinase C to Integrin Chains. The Journal of Biological Chemistry, Vol. 277, 22073-22084.

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