人類粒腺體胸苷酸激酶的分子選殖及分析

Abstract

為了維持粒腺體DNA的完整性，已知粒腺體中有許多的特殊的激酶 (kinase) 負責合成粒腺體DNA複製與修補所需要的dNTPs，時至今日，已經有數個在粒腺體中扮演這類角色的激酶被發現，包括deoxyribonucleoside kinases (TK2 and dGK) 以及nucleoside diphosphate kinases ( nm23-H4 and nm23-H6) ，然而，尚未有研究指出粒腺體中有dNMP kinase的存在。 本論文的目的即在探索粒腺體中dTMP kinase (TMPK2) 存在的可能性，透過Genebank蛋白質序列的比對以及分析，我們發現TMPK2的可能序列，以RT-PCR的方法將之clone出來，並接上綠色螢光蛋白 (GFP) 基因，形成TMPK2-GFP融合基因後，表現於HeLa細胞中，證明此基因的確為粒腺體的蛋白質，而且其表現可造成細胞內dTTP pool的上升，說明了其在細胞內的功能特性。另外、在大腸桿菌中表現並純化TMPK2，經由活性測試分析也證明了TMPK2具有將dTMP轉換為dTDP的活性。 總括上述，我發現了一個新的粒腺體蛋白質，而且這個蛋白質具有TMPK的活性，為粒腺體內調控dTTP pool的新酵素。

To maintain mitochondrial DNA integrity, mitochondria contain specific kinases for salvage of deoxyribonucleotides. To date, several kinases involved in dNTP synthesis within mitochondria have been identified, including ribonucleoside kinases (TK2 and dGK) and dNDP kinases (nm23-H4 and nm23-H6). However, little is known about mitochondrial dNMP kinases. In this study, by blast search based on the criteria of conserved TMPK domain and mitochondria targeting sequences, I found a novel mitochondrial protein, designated as TMPK2. The cDNA of TMPK2 encodes a 49 kDa polypeptide, containing a C-terminal conserved TMPK domain and an N-terminus typical mitochondria targeting sequence. By expression of TMPK2-GFP in HeLa cells, I showed that TMPK2 was localized within mitochondria. In parallel, I found that overexpression of TMPK2 increased dTTP pool as TMPK did, suggesting that functional contribution of TMPK2 to intracellular dTTP pool. The enzymatic function of TMPK2 was assessed with recombinant TMPK2 protein. Through NADH-couple TMPK activity assay, I also demonstrated TMPK activity of the recombinant TMPK2 in vitro. In summary, I identified a novel mitochondrial protein with TMPK activity, providing an opportunity to characterize the regulation of dTTP pool within mitochondria.