建構Securin蛋白質交互作用網絡

Abstract

Securin是一個多功能蛋白質，且在許多癌症中有高量的表現，但是對於securin如何調控癌症細胞的機制目前尚未完全解開，所以希望能藉由建構與securin蛋白質作用的網絡，更進一步的了解securin的作用機制。我們使用免疫沉澱(IP)、酵母菌雙雜交系統(yeast two hybrid)及遠西方墨點法(Far Western)等方法篩選出與securin結合的蛋白質，經由質譜儀(MALDI-TOF Mass)分析其序列後，確定這些蛋白質為PCAF、T-box3、Histone H4、KRT1、BCl2A1、Dopamine receptor D4、及RNA cyclase，藉由這些與securin作用的蛋白質，我們可以知道，securin除了已知功能外，還可能與蛋白質乙醯化的調節、Histone H4、Cytoskeleton及抗細胞凋亡的BCl2A1有關。另外我們使用兩種方式測量蛋白質之間的交互作用：遠西方墨點(Far Western)法及恆溫滴定微卡洛里計(ITC)，實驗中以p53與securin為代表，分別用兩種方式測得其作用力大小，此測量系統能應用於測量不同環境下蛋白質之間的作用力，以及不同修飾狀態下的蛋白質作用力大小，作為探討蛋白質之間分子作用機制的分析方法。

Human securin is a multifunctional protein and highly expressed in many types of tumor cell. The function of securin is regulating separase that control cell division and cell cycle, and DNA repair mechinary. However, the molec1ule mechanism of securin in cell cycle regulation remains unclear. To understand the role of securin in cell cycle, a protein-protein interaction network of securin was constructed try using immunoprecipitation, yeast two hybrid and Far Western methods. We found several proteins may interact with securin such as PCAF, T-box3, Histone H4, KRT1, BCl2A1, Dopamine receptor D4 and RNA cyclase. From the results of the experiments, securin may participate in protein acetylation, mofification of Histone H4 and apoptosis. Meanwhile we measured the protein-protein interaction by using Far Western and ITC. The two systems offered us a useful tool to analyze protein-protein interaction in different environments and different situations of protein modification.