精胺酸甲基化對二級結構穩定性、RNA辨認及細胞穿透的影響

Effect of Argnine Methylation on Secondary Structure Stability, RNA Recognition and Cell Penetration

黎哲豪(Jhe-Hao Li)

指導教授 ： 陳平

國立臺灣大學/理學院/化學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.11239

摘要

生物藉著蛋白質後轉譯修飾(post-translational modification)使細胞的蛋白質體的複雜度增加，藉以調控許多精密的生化反應。甲基化是一個很常見且最簡單的後轉譯修飾，而精胺酸(arginine, Arg)甲基化也負責了許多生化過程像是訊息傳遞、mRNA 剪接、基因表現及DNA修復。三種精胺酸甲基化分別為：單一甲基化(monomethylarginine, MMA)、不對稱雙甲基化(asymmetric dimethylargnine, ADMA)以及對稱雙甲基化(symmetric dimethylarginine, SDMA)。而現階段大部分的研究著重在甲基化的蛋白質辨認，對於結構上的影響目前仍不清楚，因此我們利用模式胜肽系統研究精胺酸甲基化對於α螺旋性質以及β摺板穩定性的影響。
在我們的研究中可以發現甲基化精胺酸對於穩定螺旋N端以及C端皆有影響，可能是因為疏水性以及氫鍵作用力的變化，而對於螺旋性質則是 Arg > SDMA > ADMA > MMA、而β摺板穩定性則不受甲基化所影響。
此外我們也研究了精胺酸單一甲基化於HIV-1 Tat(47-57)的六個精胺酸對於 RNA辨認能力以及細胞穿透能力的影響，並藉由膠體電泳分析(Electrophoresis Mobility Shift Assay)量測與TAR RNA的解離常數(Dissociation constant)。當單一甲基化於53及55號位置時，其與TAR RNA 之間的結合能力下降，而其他靠近周圍的精胺酸單一甲基化則使結合能力不變或略微變好。單一甲基化的精胺酸對於細胞穿透能力的影響則是利用流式細胞儀(flow cytometry)作鑑定，而初步結果則顯示在低濃度實驗時，相較於未修飾的Tat 胜肽單一甲基化會有較弱的細胞穿透能力。

關鍵字

精氨酸甲基化 ； 二級結構 ； RNA辨認

並列摘要

Post-translational modification serves to expand the chemical functionalities on proteins, increase the complexity of the proteome, and provide a means to regulate biological processes. Methylation is the simplest post-translational modification, and may be the most abundant chemical modification inside the cell. There are three types of methylated arginines (Arg): monomethylarginine (MMA), symmetric dimethylarginine (SDMA), and asymmetric dimethylarginine (ADMA). These methylated Arg residues play various physiologocal roles, including signal transduction, mRNA splicing, gene expression, and DNA repair. Recent investigations have focused on the recognition and interaction involving protein arginine methylation. However, the structural consequence of the various methylation states remains unknown. As such, we investigated the stability effect of helix capping, helix propensity, and β-sheet stability using model peptide systems. The N-capping and C-capping ability are affected by hydrophobics as well as hydrogen bonding patterns. All three methylation states showed decreased helix propensity, following the trend Arg > SDMA > ADMA > MMA. However, the β-hairpin/sheet stability does not vary with methylation state, suggesting minimal effect on sheet propensity upon methylation. Also, we investigated the effect of monomethylation on HIV-1 Tat(49-57) RNA recognition and cell penetration at all six arginine residues individually. The dissociation constant was measured by gel shift assays, suggesting the importance of hydrogen bonds at specific residues. At positions 53 and 55, the monomethylation resulted in decreased binding affinity compared to wild type, whereas the other flanking residues were around the same. The cellular uptake ability of the monomethylated peptides was measured by flow cytometry. The preliminary results suggested weaker cellular uptake ability compared to the wild type at low concentration (7 μM).

並列關鍵字

Arginine methylation ； Secondary structure ； RNA recognition

參考文獻

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