CTEN 於細胞核中的功能探討

Abstract

CTEN (C-terminal tensin-like) 蛋白質位於集中附著點，屬於 tensin family 中的一員，並且在細胞的附著、移動和癌症發生過程中扮演著重要角色。先前的研究顯示 CTEN 在大腸癌中除了有過量表現之情形外，同時在細胞核內也能偵測到高表現量的 CTEN，而這樣的現象促使我們欲研究 CTEN 在細胞核中的功能。其他的研究指出，某些 focal adhesion 蛋白質會進入至細胞核內作為 coregulatory 或 coactivator，因此我們假設 CTEN 是一個能與 DNA 結合之蛋白質，在癌細胞中會進入至細胞核內參與調控促進腫瘤發生的基因表現。本論文先利用線上資料庫分析 CTEN 的胺基酸序列和蛋白質結構，但是並沒有發現可與 DNA 結合之高度保守性序列，因此另一方面我們想以實驗進一步證實 CTEN 是否具有與 DNA 結合之能力。首先本論文成功選殖出可在大腸桿菌中表現 CTEN 的重組質體 pET-28a-CTEN，想要利用純化的 CTEN 蛋白質測試其與 DNA 結合的能力，嘗試不同的表現與純化條件發現部分重組蛋白質 CTEN 會形成不可溶體，無法得到大量的可溶性 CTEN 蛋白質。同時，本論文利用 CTEN 抗體進行 ChIP assay 再經由毛細管電泳分析，實驗結果發現表現 CTEN 蛋白質之組別其 DNA 片段含量有較高之情形，符合 CTEN 是一個能與 DNA 結合的蛋白質之假設，此結果為 CTEN 於癌細胞核中累積之現象提供了一可能解釋。

C-terminal tensin-like (CTEN) protein is localized at focal adhesion complex. It is a member of the tensin protein family, and also plays a crucial role in cell adhesion, migration, and the development of malignancies. Previous studies have indicated that the expression levels of CTEN is elevated in colon cancer, accompanied with the detection of high population of CTEN in the nucleus. It prompts us to study the functions of human CTEN protein in the nucleus. Some of focal adhesion proteins are able to enter the nucleus and function as coregulators or coactivators. Therefore, we hypothesize that CTEN is a DNA binding protein, and could enter the nucleus in cancer cells to regulate the gene expression contributing to tumorigenicity. In this work, we firstly use the online database to analyze the amino acid sequence and the protein secondary structure of CTEN. It turns out that no highly-conserved DNA binding domain is found. On the other hand, we want to determine whether CTEN possesses the ability to bind DNA. First, the recombinant plasmid pET-28a-CTEN, which can express CTEN in E.coli, is successfully constructed. Then we plan to use the purified recombinant CTEN protein to examine its ability to bind DNA. However, high levels of the recombinant protein form inclusion bodies, and the yield of soluble CTEN is low. Moreover, ChIP assay followed by capillary electrophoresis is used to analyze whether CTEN is able to bind DNA. Our results show that the amount of DNA after ChIP is increased in the cells expressing CTEN protein. This result supports the hypothesis that CTEN functions as a DNA binding protein, and provides one potential explanation for the accumulation of CTEN in the nucleus in cancer cells.