臺灣博碩士論文加值系統

RhoGDIα (Rho GDP-dissociation inhibitor) 透過負向或正向調節Rho family GTPases的活性，來調控多種重要的細胞運作，包括細胞分化以及惡性疾病生成。血小板在人體中執行凝血功能，而巨核球 (MKs) 是循環血小板的唯一來源。我們之前的研究揭露，當 PMA (phorbol 12-myristate 13-acetate) 處理K562細胞時，RhoGDIα會促進巨核球分化。我們還發現Rac1能介導RhoGDIα對巨核球分化的影響，並且為RhoGDIα下游GTPase。目前已經在RhoGDIα上發現了許多轉譯後修飾 (PTM)，然而這些轉譯後修飾如何影響RhoGDIα的功能仍然有很大程度上的未知。我們最近透過液相層析串連式質譜儀鑑定出K562細胞中RhoGDIα的R111和R172具有雙甲基化修飾。我的研究發現，R111突變為K111顯著降低了PMA誘導巨核球分化時RhoGDIα的促進作用。R172的突變雖然影響了分化，但程度較小。另外，過度表現這些非甲基化RhoGDIα突變蛋白會對K562細胞走向巨核球分化有dominant negative的影響。我的研究進一步揭露，R111K突變大幅降低了RhoGDIα與Rac1 GTPase的結合，這可能導致Rac1的活化和巨核球分化的抑制。在努力找尋RhoGDIα上游潛在的甲基轉移酶之下，我以體外甲基化的方式進行測定並且結果顯示蛋白質精氨酸甲基轉移酶6 (PRMT6) 可以甲基化RhoGDIα。由於我們先前實驗室研究發現PRMT6可以促進K562細胞的巨核球分化，而RhoGDIα R111能反過來透過結合更多的Rac1 GTPase促進巨核球分化，因此PRMT6可能是甲基化RhoGDIα R111的潛在調節因子。未來，我們希望透過瞭解巨核球分化的機制，為血球分化異常相關疾病提供新的治療策略。

RhoGDIα (Rho GDP-dissociation inhibitor) plays important roles in regulating a variety of cellular processes, including cell differentiation and malignancy, by either negatively or positively modulating the activity of Rho family GTPases. Megakaryocytes (MKs) are the sole source of circulating platelets which perform coagulation in human. Our previous research showed that RhoGDIα up-regulated megakaryocytic differentiation of K562 cells upon induction by PMA (phorbol 12-myristate 13-acetate) treatment. We also identified that Rac1 was the downstream GTPase which mediated the effect of RhoGDIα on MK differentiation. Various post-translational modifications (PTMs) have been found on RhoGDIα, however, the functional impacts of these PTMs are still largely unknown. We recently identified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) that R111 and R172 of RhoGDIα were di-methylated in K562 cells. My study found that mutation of R111 to K111 significantly reduced the promotive effect of RhoGDIα on PMA-induced MK differentiation. Mutation of R172 also affected differentiation, although to a less extent. Overexpression of these non-methylation RhoGDIα mutant proteins resulted in a dominant negative effect on MK differentiation in K562 cells. My study further showed that R111K mutation greatly decreased the association of RhoGDIα with Rac1 GTPase which likely leads to the activation of Rac1 and a suppression of MK differentiation. In the effort of searching for the potential upstream methyltransferase for RhoGDIα, I performed in vitro methylation assay and showed that protein arginine methyltransferase 6 (PRMT6) could methylate RhoGDIα. Since our previous study has shown that PRMT6 can promote MK differentiation of K562 cells, PRMT6 is a potential regulator of RhoGDIα by methylation on R111, which in turn promote MK differentiation by sequestering more Rac1 GTPase. In the future, we hope to provide a new treatment strategy for diseases related to abnormal blood cell differentiation by understanding the relevant mechanisms of the differentiation of MK.

目錄...........................................i
圖次目錄......................................ii
縮寫表........................................iv
中文摘要......................................vi
英文摘要......................................vii
緒論..........................................1
研究目標......................................9
實驗材料......................................10
實驗方法......................................15
結果..........................................24
討論..........................................31
參考文獻......................................36
圖............................................42

圖次目錄
Figure 1. Schematic representation of the construction of plasmids harboring pcDNA3-HA2-RhoGDIα R172K single mutation....................................................p42
Figure 2. Examination of pcDNA3-HA2-RhoGDIα R172K plasmids....................................................p43
Figure 3. Schematic representation of the construction of plasmids harboring pcDNA3-HA2-RhoGDIα R111K single mutation....................................................p44
Figure 4. Examination of pcDNA3-HA2-RhoGDIα R111K plasmids....................................................p45
Figure 5. Schematic representation of the construction of plasmids harboring pcDNA3-HA2-RhoGDIα R111/172K double mutation....................................................p46
Figure 6. Examination of pcDNA3-HA2-RhoGDIα R111/172K plasmids....................................................p47
Figure 7. The arginine methylation status of RhoGDIα in 50B4 cells.......................................................p48
Figure 8. Analysis of megakaryocytic differentiation upon PMA stimulation in K562 cells...................................p49
Figure 9. Single mutation of R111K or R172K and double mutation of R111/172K reduce megakaryocytic differentiation in K562 cells.......................................................p50
Figure 10. Single mutation of R111K or R172K and double mutation of R111/172K reduce megakaryocytic differentiation in 50B4 cells..................................................p51
Figure 11. Arginine methylation of RhoGDIα was required for the stimulatory effect of PRMT6 on megakaryocytic differentiation.............................................p52
Figure 12. The arginine methylation status of RhoGDIα WT and R111K mutant................................................p53
Figure 13. The Rac1 protein was associated with HA-RhoGDIα in immunoprecipitates from K562 cells..........................p54
Figure 14. PMA stimulation increased binding of RhoGDIα with Rac1........................................................p55
Figure 15. Mutation of RhoGDIα R111K reduced binding with Rac1........................................................p56
Figure 16. PMA treatment did not change the protein levels of PRMT6, RhoGDIα and Rac1................................... p57
Figure 17. Purification of recombinant His-fused RhoGDIα proteins....................................................p58
Figure 18. Immunoprecipitated HA-PRMT6 methylated RhoGDIα in vitro methylation assay.....................................p59

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