探討N端乙醯基轉移酶與H3K9組蛋白甲基轉移酶G9a在癌細胞腫瘤發生中扮演的角色

Evaluation of the role between N-α-acetyltransferase 10 protein (Naa10p) and H3K9 histone methyltransferase G9a in cancer cell tumorigenesis

林才甯(Tsai-Ning Lin)

指導教授 ： 郭明良

國立臺灣大學/醫學院/毒理學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10126

摘要

在癌症發展中，特定的蛋白質轉譯後修飾作用會促進基因表現或靜默，進而調控癌細胞的生長。真核生物中最普遍的共價修飾作用為N端乙醯化修飾，藉由特定N端乙醯基轉移酶來催化轉移百分之八十至九十的人類蛋白質乙醯基。其中，Naa10p為具催化N端乙醯化修飾能力的酵素反應次單元，能進行N端α氮及ε氮上之乙醯基轉移，亦可催化自乙醯化，已知參與許多細胞內功能調控。近年許多研究顯示Naa10p異常對癌細胞的發展有關，但在癌細胞中Naa10p扮演的角色仍不清楚。另一調控因子G9a，主要是催化DNA組蛋白H3上第九個胺基酸離胺酸(Lysine 9)進行甲基化的甲基轉移酶，進而抑制某些腫瘤抑制基因的轉錄而促使癌症發生。先前本實驗室研究發現G9a高度表現在末期惡性癌症病人的腫瘤組織中，認為G9a為一致癌基因。2005年Rual et al利用高通量酵母菌兩蛋白質雜交系統指出Naa10p與G9a之間會交互作用。因此在本篇研究中，我們試圖確認Naa10p與G9a的交互作用與兩者之間結合位置，更進一步想了解此兩酵素結合後會發生甚麼樣的調控作用。有趣的是，我們發現當過度表現Naa10p時，會顯著的抑制G9a甲基轉移能力，同樣的當靜默Naa10p時，G9a催化之H3K9二甲基化會顯著的增加。綜合以上實驗結果，顯示G9a會在N端以及Cys-ANK 重複序列聯結之間與Naa10p之N端58個胺基酸序列之間交互作用，並受到Naa10p調控而失去催化H3K9二甲基化的酵素活性。我們也利用transwell的細胞遷移實驗，指出Naa10p及G9a之間的確會有一條控的角色存在。未來可進一步研究Naa10p對G9a的蛋白質交互作用調控機制，並多加探討G9a是否會藉由調控Naa10p而影響下游轉錄轉譯基因改變，以便於未來有機會能運用在癌症之臨床檢測與治療上。

關鍵字

蛋白質轉譯後修飾 ； N端乙醯基轉移酶組蛋白甲基轉移酶蛋白質交互作用

並列摘要

Specific combinations of post-translational modifications of proteins facilitating gene transcription or silencing and play an important role in regulating tumor progression. One of the most common covalent modifications in eukaryotes is N-terminal acetylation (Nt-acetylation), by specific acetyltransferase catalyzing the transfer of acetyl moieties to the N-termini of 80-90% of all human proteins. Human N-α-acetyltransferas 10 protein (Naa10p), the catalytic subunit of N-acetyltransferase A, catalyzes both N-α-acetylation and ε-acetylation, as well as autoacetylation and is involved in a variety of cellular functions and. Some studies shows that dysregulation of Naa10p is associated with tumorigenesis, however it plays a conflict role and the biological functions remains largely unknown. Another regulated factor, DNA Histone 3 Lysine 9 (H3K9) methyltransferase G9a can promote tumor metastasis by repressing downstream tumor suppressor genes. Our previous studies demonstrated that G9a is highly expressed in late stage aggressive cancer patients and has been identify it as an oncoprotein. In 2005, Rual et al. reported that Naa10p will interact with G9a by a high-throughput yeast two-hybrid system. In this study, we confirm the interaction between Naa10p and G9a and also evaluate the binding domain among each other. Interestingly, we found that enforced expression of Naa10p significantly impaired the G9a enzyme activity, whereas Naa10p silencing increased H3K9 dimethyltransferase activity. We also used transwell migration assay to indicate that there is a regulated role between Naa10p and G9a. Our study indicated that Naa10p functions as a suppressor of the oncogenic protein G9a. We hypothesize that the protein-protein interaction functions and mechanisms between Naa10p and G9a is possible to anticipate novel anticancer drugs in the future.

並列關鍵字

Post-translational modifications ； N-acetyltransferase ； Histone methyltransferase ； Protein-Protein interaction

參考文獻

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