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Title: | 乙醯化離胺酸對蛋白質二級結構之穩定性及對核糖核酸辨識與細胞穿透之影響 Effect of Lysine Acetylation on Protein Secondary Structure Stability, RNA Recognition, and Cell Penetration |
Authors: | Chin-Yih Chen 陳清怡 |
Advisor: | 陳平(Richard Ping Cheng) |
Keyword: | 乙醯化,離胺酸,Tat衍生胜?,辨識核糖核酸,細胞穿透胜?, Lysine,Acetylation,Tat-derived peptides,RNA recognition,Cellular uptake, |
Publication Year : | 2015 |
Degree: | 碩士 |
Abstract: | 離胺酸乙醯化是在生物體內非常普遍的轉譯後修飾,並參與了許多重要的生 物功能。其反應是將離胺酸側鏈上的胺基所帶的正電荷藉由裝上乙醯基而中和, 此步驟調控許多具有離胺酸的蛋白質內的生化反應。在此研究中,我們設計可偵 測蛋白質二級結構的胜肽模型來測量離胺酸乙醯化對於蛋白質二級結構的影響。 α-螺旋用圓二色光譜儀來測定其螺旋程度。β-摺板則用超導核磁共振光譜儀來觀測 其摺板程度。結果顯示,相對於離胺酸,乙醯化離胺酸可增加α-螺旋的螺旋性質, 也可增進β-摺板的穩定度。乙醯化離胺酸相對於離胺酸,能穩定蛋白質二級結構。
在乙醯化離胺酸生物功能上的研究,我們選用人類免疫缺乏病毒 Tat 蛋白質,它是一個乙醯化離胺酸調節的核糖核酸結合蛋白。我們研究 Tat 蛋白中一段胜 肽序列:Tat49-57,具有與病毒 TAR RNA 結合及穿透細胞膜的能力。Tat49-57 上帶有 許多正電荷的胺基酸,其中的兩個離胺酸(50 號和 51 號位置)都可以在生物體內 被乙醯化酶催化成乙醯化離胺酸。此研究將 Tat49-57上的兩個離胺酸分別以乙醯化 離胺酸取代,來觀察他們對 TAR RNA 結合及穿透細胞膜能力的影響。利用膠體電 泳偏移分析來研究Tat衍生胜肽與TAR RNA的解離常數。同時也以圓二色光譜儀 偵測Tat衍生胜肽的結構。利用流式細胞儀測量Tat衍生胜肽進入Jurkat細胞內部 的效率。結果顯示,乙醯化離胺酸會降低 TAR RNA 結合及穿透細胞膜的能力,且 在 Tat49-57 中,取代 50 號位置的離胺酸成乙醯化離胺酸在 TAR RNA 結合及穿透細 胞膜的能力損壞的程度都比取代 51 號位置的離胺酸成乙醯化離胺酸還要嚴重,顯 示 Tat49-57 中 50 號位置離胺酸是個可能對功能來說相對重要的位置。 Lysine acetylation is a very common and highly regulated post-translational modification (PTM) and is crucial for the protein structures and many biological functions in living organisms. The main effect of lysine acetylation is the neutralization of the lysine positive charge and consequently the regulation of the biochemical properties of lysine-containing proteins. The roles of lysine acetylation in cellular functions have been studied extensively. However, the effect of acetylation on protein secondary structures remains unclear. To study the effect of lysine acetylation on protein secondary structures, α-helical monomeric peptides and β-hairpin peptides were designed as basic models and acetyl-Lysine (AcK) was introduced into each guest position. Hydrogen-bonds have significant impact on protein stability, therefore the effect of replacing a methyl group with an amine group in acetylated Lys residues on secondary structure stability was also studied. The helical content of the α-helical peptides were determined by circular dichorism spectroscopy (CD), and the β-hairpin peptides were analyzed by NMR spectroscopy (TOCSY , ROESY , NOESY , DQF-COSY) to determine the sheet propensity. The result showed that compared to Lys, Lys acetylation increased both α-helix and β-sheet stability. In an α-helix, AcK increased the helix propensity, but lowered the C-Cap parameters. In a β-hairpin, the folding percentage of HPTAcKAla was 1.2 fold higher than that of HPTLysAla. Post-translational modification of RNA binding proteins (RBPs) plays an important role in regulating many cellular functions. The Tat protein, one of the RBPs, is essential for the life cycle of HIV-1 and is known to undergo acetylation on specific lysines to modulate various Tat functions. Tat, like most of the RBPs, contain a region (Tat49-57) rich in positively charged amino acids such as Arg and Lys to mediate the initial recognition of the negatively charged phosphate backbone RNA and the efficiency of cellular uptake. To investigate the effect of Lys side chain charge on RNA recognition and on cellular uptake, each positively charged Lys in Tat49-57 was replaced with one AcK at a time. The dissociation constant for the binding affinity of TAR RNA-Tat derived peptide was studied by gel shift assays, and the cellular uptake efficiency for peptide-treated Jurkat cells was assessed by flow cytometry. The result showed that removing the Lys positive charge by lysine acetylation affected RNA recognition and cell penetration of HIV-1 Tat. Furthermore, the effects showed position dependence. Acetylation of Lys 50 and 51 decreased the binding affinity between Tat49-57 and TAR RNA and could not be recovered by introducing hydrogen bond donors. Acetylation of Lys 50 and 51 on Tat49-57 decreased the cell penetration efficiency. However, adding hydrogen bond donors could compensate this decrease. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55233 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 化學系 |
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