蛋白質精胺酸甲基轉移酶之特性分析及精胺酸甲基接受蛋白於細胞內分布之研究

中文摘要 由蛋白質精胺酸甲基轉移酶 (PRMT) 催化的蛋白質精胺酸甲基化是一種轉譯後修飾作用。第一型的甲基轉移酶催化甲基接受蛋白形成單甲基和不對稱型雙甲基化，而第二型的甲基轉移酶能催化其它的甲基接受蛋白形成單甲基和對稱型雙甲基化。一直到最近定出6種不同PRMT基因前，以生化法定出的各種生物來源的第一型和第二型的甲基轉移酶的分子量皆介於275 kDa到500 kDa之間。而這些PRMT單體的分子量卻只在40 kDa到70 kDa間，因此PRMT單體可能是甲基轉移酶複合體中的催化次單元。 本研究中，我們使用生化法來純化、分析豬腦中的精胺酸甲基轉酶。以不同的離心速度將組織蛋白萃取液分fraction，所得之上清液和沉澱物分別命名為S1、S2、S3和P1、P2、P3。而以超高速離心得到沉澱物P3回溶於含有0.5 M KCl的萃取液中再離心一次得到S4和P4。大多數的PRMT活性 (以第一型甲基化的受質 fibrillarin來做測試) 在S3 (cytosolic)，而在S4 fraction卻有最高的比活性。將淋巴母細胞萃取液或 P3 fraction加入0.5 M KCl後PRMT活性亦顯著的增加，而將S4 fraction中的KCl透析後則PRMT活性下降。因此證實0.5 M KCl會影響PRMT活性。然而，在S3中卻含有較S4大量的PRMT1 (在哺乳動物系統中最主要的第一型蛋白質精胺酸甲基轉移酶)，且重組的PRMT1活性不受0.5 M KCl影響。因此有可能高濃度 (0.5 M) 的KCl會讓P3中與甲基轉移酶結合的抑制者分開而使得S4中的甲基轉移酶有較高的比活性。經陰離子交換樹脂 (Mono Q) 分離後兩個fractions中的第一型甲基化活性幾乎出現在同樣的位置。因此現在我們試著去純化這兩個fractions中的酵素去瞭解它們的成分。 在細胞中含有RGG motifs (arginin- and glycine-rich) 的RNA結合蛋白會被細胞中具專一性的蛋白質精胺酸甲基轉移酶催化形成精胺酸甲基化。我們先前的資料顯示將淋巴母細胞養在含AdOx (一種間接的甲基轉移酶抑制劑) 的培養基中，可獲得大量低甲基化的受質。我們的資料亦顯示蛋白質精胺酸甲基轉移酶及其受質皆廣泛的分布於淋巴母細胞中的不同fractions中。最近的資料亦顯示精胺酸甲基化與蛋白間的交互作用及某些甲基接受蛋白於細胞內的分佈有關。在此實驗中我們將淋巴母細胞分為不同的fractions來研究含有 RGG的蛋白 (例如:FMRP, hnRNP-A1, 和hnRNP-A2/B1) 分布情形是否會受甲基轉移酶抑制劑Adox影響。運用西方點墨法來分析這些蛋白，我們看到FMRP、hnRNP-A2/B1及一些甲基接受蛋白在以AdOx處理和正常細胞內的分佈情形有所差異。未來我們將進一步以細胞免疫螢光染色和二維電泳來研究精胺酸甲基接受蛋白的甲基化狀態與其在細胞內分佈情形的相關性，以提出更多關於這方面的資料。

Abstract Protein arginine methylation is a posttranslation modification catalyzed by protein arginine methyltransferase (PRMT). The type I methyltransferase catalyzes the formation of mono- and asymmetric di-methylarginine and another type II activity can catalyze the formation of mono- and symmetric di-methylarginine on other methylaccepting proteins. Before the identification of five different PRMT genes recently, the type I and II PRMT from various biological resources were biochemically characterized to be of molecular weight from 275 kDa to 500 kDa. All these PRMT monomers are of molecular weight from 40 kDa to 70 kDa, thus are likely to be the catalytic subunit in a methyltransferase complex. In this study, we used the biochemical approach to isolate and analyze the arginine methyltransferase from a natural source porcine brain. We fractionated the lysates by differential centrifugation to obtain the supernatant S1, S2 and S3, as well as the pellet fractions P1, P2 and P3. Part of the P3 was resuspended in lysis buffer with 0.5M KCl and subjected to ultracentrifuge again to obtain S4 and P4. The majority of the PRMT activity (assayed by a type I substrate fibrillarin) was in the S3 (cytosolic) fraction while the highest specific activity was in the S4 fraction. Addition of KCl increase the arginine methyltransferase activity in the P3 fraction as well as that in the lymphoblastoid cell. Removal of the KCl in the S4 fraction decrease the PRMT activity. It is possible that high KCl concentrate can help to release the PRMT in P4 from associated inhibiting factors, thus increase the methyltransferase activity. However, PRMT1, the predominant type I PRMT in the mammalian system is much more abundant in S3 than S4, and the PRMT activities in S3 and recombinant PRMT1 were not affect by 0.5M KCl. Whether the methyltransferase in S3 and S4 were the same or different enzyme is under investigation. The type I activity in both fractions almost eluted at the same position by anion-exchange (Mono Q) column. We are now trying to purify the enzyme in both fractions to understand their identities. Arginine methylation in RNA-binding proteins containing arginine- and glycine-rich RGG motifs is catalyzed by specific protein arginine N-methyltransferase in cells. We previously showed that lymphoblastoid cells grown in the presence of an indirect methyltransferase inhibitor, adenosine dialdehyde (AdOx), accumulated high level of hypomethylated protein substrates. We also demonstrated that protein-arginine methyltransferases as well as their methyl-accepting substrates were widely distributed in different subcellular fractions of lymphoblastoid cells. Recently arginine methylation has been suggested to be related with protein-protein interaction and subcellular localization of some methyl-accepting proteins. Therefore, in this study we fractioned lymphoblastoid cells into different subcellular fractions to study if localization of certain RGG containing proteins such as FMRP, hnRNP-A1, hnRNP-A2/B1, and nucleolin would be interfered upon Adox treatment. By western blot analyses using antibodies against these proteins, we demonstrated that subcellular localization of FMRP and hnRNP-A2/B1 appeared to be different in AdOx-treated and normal cells. Further studies utilizing immunofluorescent staining and 2-dimensional gel electrophoresis will provide more information of the relationship between the methylation status of arginine methyl-accepting proteins and their subcellular localization.