甲基轉移酶複合體於細胞轉型的角色

The Role of Methylosome in Cell Transformation

魏同佑(Tong-You Wade Wei)

指導教授 ： 余長澤

國立暨南國際大學/科技學院/生物醫學科技研究所/碩士(2010年)

摘要

至今為止有許多的蛋白質後修飾作用已經被發現，而這些修飾作用對於維持許多細胞生理都屬於不可或缺的重要條件，諸如蛋白質在細胞內的位置與功能、蛋白質－蛋白質間的交互作用以及蛋白質的活性與穩定度…等，都扮演著極為重要的角色。其中，蛋白質的甲基化修飾牽涉到十分廣泛的生化反應，例如：基因的表現與否、RNA的剪切與加工、轉譯作用的調節、DNA的修復以及細胞增生…等，雖然辨識蛋白質上被甲基化的胺基酸有其難度，蛋白質的甲基化仍然吸引了越來越多的重視。近來一個被定義為甲基轉移酶複合體（Methylosome）的蛋白質複合體是由PRMT5、MEP50及CLNS1A所組成。這個20S的Methylosome會將兩個甲基團轉移至受質精胺酸上的兩個N端，形成對稱性二甲基精氨酸。在許多人類的疾病中都發現表現及活性改變的Methylosome；然而，極少文獻指出Methylosome與癌症形成的關係。本篇論文指出，穩定表現PRMT5、MEP50與Methylosome的細胞株能夠在一般及低血清濃度的培養液中具備快速細胞增生速率與低生長因子依賴性的能力，而且在不易貼附的環境下具有非固著依賴性生長的能力；另一方面，穩定表現CLNS1A、MEP50與Methylosome的細胞株則具有細胞移行的能力。本論文也發現Methylosome複合體於細胞中所造成的細胞轉型能力明顯高於各單元於細胞中的效應，並且在短暫表現的單一細胞增生與穩定表現的非固著依賴性生長中具有協同作用；最後，這三個構成Methylosome的蛋白質皆高度表現於病患的肺癌組織且具有相關性。上述結果顯示，表現Methylosome各單元或複合體的細胞展現了細胞轉型的型態，指出Methylosome具有使細胞轉型的能力，為一潛在的致癌性蛋白質複合體。為了了解Methylosome導致細胞轉型的相關分子機制，本篇論文也探討了Methylosome的受質，以及鮮少被探討的蛋白質甲基化與細胞轉型之間的關係；本篇發現PRMT5、MEP50和CLNS1A都與HURP（Hepatoma Upregulated Protein）具有交互作用且HURP為Methylosome的受質。HURP是一個高度表現在數種癌症組織中、潛在的致癌性蛋白質，將外緣性的HURP高量表現於細胞中，可以使細胞擁有低生長因子依賴性及非固著依賴性生長的能力，顯示HURP具有造成細胞轉型的能力。本篇論文探討了Methylosome對HURP的甲基化修飾以及Methylosome與HURP造成細胞轉型所具有的協同作用。本篇研究提出許多證據顯示Methylosome與癌症的關係，以及這個蛋白質複合體所造成快速細胞增生速率、低生長因子依賴性、非固著依賴性生長與細胞移行的能力，這些使細胞轉型的特性都與癌症的發生及惡化具有高度相關。最後，也探討了其受質HURP的甲基化及Methylosome與HURP協同地促進細胞轉型的關係。

關鍵字

甲基轉移酶複合體 ； 細胞轉型 ； 致癌基因 ； 甲基化 ； HURP

並列摘要

There are a huge number of posttranslational modifications of protein found so far. Many of them play important roles in protein subcellular localization, protein-protein interaction, protein stability or protein activity. Although the difficulties of identifying the methylated amino acids, protein methylation attracts more and more attention because of its involvement in a wide range of biological processes such as gene expression, RNA splicing, transcriptional regulation, DNA repair and cell proliferation. A newly indentified protein complex termed Methylosome is composed of PRMT5, MEP50 and CLNS1A. The 20S Methylosome performs their cellular function by methylating proteins, which catalyzing the formation of symmetrical methylated arginines of a protein. Altered expression or activities of Methylosome is associated with a number of human diseases. However, limited documents have mentioned the involvement of Methylosome in tumor formation. In this thesis, we point out that the cell lines stably overexpressing PRMT5, MEP50 and Methylosome stimulated fast cell proliferation rate and low growth factor demanding in normal and low serum. This cell lines also obtained anchorage-independent growth ability. On the other side, cell lines stably overexpressing CLNS1A, MEP50 and Methylosome promoted cell migration. This thesis also found that the stable clone overexpressing Methylosome complex display higher cell transformation ability than the stable clones overexpressing Methylosome components, and furthermore Methylosome shows synergistic effect on short term expressing of single cell proliferation and long term expressing of anchorage-independent growth. Finally, three Methylosome components are overexpressed in a collected panel of lung cancer tissues. The results above showed that Methylosome components or complex display higher transformed phenotype; implicating Methylosome may possess cell transforming activity and work as a protential oncoprotein complex. To understand how Methylosome transforms cells, the thesis also explored the substrate of Methylosome and discussed the relationship between protein methylation and cell transformation. We have identified HURP (Hepatoma upregulated protein) existing protein-protein interaction with Methylosome components and acting as a substrate of the Methylosome. HURP is a potential oncoprotein, is originally isolated form screening for tumor-associated genes. Overexpression of exogenous HURP stimulates low growth factor demanding and anchorage-independent growth ability, indicating HURP possesses cell transforming activity. In this study, we survey the effect of HURP methylation and the synergistic effect of Methylosome and HURP on cell transformation status. The studies present a number of evidence showing the association of Methylosome with cancer incidences and displaying the fast cell proliferate rate, low growth factor demanding, anchorage-independent growth and cell migration activities of the protein complex. Eventually, the the substrate HURP methylation and significance of Methylosome and HURP on cell transformation is explored and discussed.

並列關鍵字

Methylosome ； Cell transformation ； Oncogene ； Methylation ； HURP

參考文獻

Anne, J., Ollo, R., Ephrussi, A., and Mechler, B.M. (2007). Arginine methyltransferase Capsuleen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila. Development 134, 137-146.

Azzouz, T.N., Pillai, R.S., Dapp, C., Chari, A., Meister, G., Kambach, C., Fischer, U., and Schumperli, D. (2005). Toward an assembly line for U7 snRNPs: interactions of U7-specific Lsm proteins with PRMT5 and SMN complexes. J Biol Chem 280, 34435-34440.

Bassal, S., Nomura, N., Venter, D., Brand, K., McKay, M.J., and van der Spek, P.J. (2001). Characterization of a novel human cell-cycle-regulated homologue of Drosophila dlg1. Genomics 77, 5-7.

Bedford, M.T. (2007). Arginine methylation at a glance. J Cell Sci 120, 4243-4246.

Bedford, M.T., and Clarke, S.G. (2009). Protein arginine methylation in mammals: who, what, and why. Mol Cell 33, 1-13.