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標題: Structural and Functional Studies of Protein Acetyltransferases from Sulfolobus solfataricus
作者: 卓慶昌
Cho, Ching-Chang
關鍵字: circular dichromism;X射線;X-ray;acetyltransferase;Sulfolobues solfataricus;乙醯轉移酶硫化葉菌
出版社: 分子生物學研究所
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蛋白質在轉譯作用後通常會經由轉譯後修飾作用( Post-translational modification) 成為具功能性的完整蛋白質,這些共價修飾包括:磷酸化反應、甲基化反應和乙醯化反應等。GCN5(General control of amino acid biosynthesis protein 5)相關的氨基端乙醯化轉移酶總科 (GCN5-related N-acetyltransferase superfamily) ,這族群的酵素包含了許多不同種類的乙醯基轉移酶甚至是醯基轉移酶,且廣泛的存在於許多不同種類的生物中,能利用輔因子乙醯輔酶A(acetyl-CoA)對目標蛋白質進行乙醯化作用,其中最著名且研究最多的就是組蛋白乙醯轉移酶 (histone acetyltransferase,HAT) 。在真核生物中,其 DNA 纏繞包裹組蛋白八聚體(histone octamer)形成核小體(nucleosome),並聚合為染色體(chromosome),而在進行複製作用與轉錄作用期間,會利用對組蛋白進行乙醯化作用(acetylation)的方式來調控基因表現。組蛋白乙醯轉移酶能利用乙醯輔酶A對組蛋白的特定離氨酸殘基進行乙醯化作用以降低其對 DNA 的親合性;反之,去乙醯化作用 (deacetylation) 可增加與 DNA 的結合力,以此來調控 DNA 的包裹程度進而影響基因的表現,然而在原核細胞中卻無類似的系統;而古生菌(archaeon)方面, Sulfolobus solfataricus 生長於酸性的高溫環境,是一種古生溫泉菌,其中 ssPat 與 ssArd1 分別為 S. solfataricus 的蛋白質乙醯轉移酶和氨基端乙醯轉移酶,但它們在 S. solfataricus 體內的生理功能都仍未知曉。Alba(Acetylation lower binding affinity)蛋白質為 S. solfataricus 的染色質蛋白,擁有兩種不同的異構體 Alba1 和 Alba2 ,其中Alba1被證明受到乙醯化作用的影響,會降低對 DNA 的親合性。之前研究結果顯示, Sir2(silent information regulator 2)可直接對 Alba1 作用,進行去乙醯化作用,提升 Alba1 的 DNA 結合能力;而 ssPat (S. solfataricus Potein acetyltransferase)為 S. solfataricus 的蛋白質乙醯轉移酶,能對 Alba1 進行乙醯化作用,進而降低 Alba1 對 DNA 的親合性,但ssPat的真實生理功能卻尚未明瞭,因此一方面我們想研究 Alba2 是否亦為 ssPat 之受質蛋白,且探討乙醯化作用對 Alba2 可能造成的影響,再則配合 ssPat 的晶體結構去推測研究 ssPat 的可能催化機轉與生理功能。氨基端乙醯化作用(N-terminal acetylation)是一種普遍存在於真核生物中的修飾作用,在原核物種與古生菌中卻非常罕見,且推測此作用似乎會伴隨著轉譯作用而發生。在 Saccharomyces cerevisiae 中氨基端乙醯轉移酶(N-terminal acetyltransferase,NAT)分為 NatA、NatB 和 NatC三種,且能辨認各自的特定氨基端序列並催化乙醯化作用,ssArd1(S. solfataricus arrest defective 1)為 S. solfataricus 的氨基端乙醯轉移酶活性,其氨基酸序列與 NatA 有同源性,卻同時具有 NatA 和 NatB 的序列專一性,且ssArd1 不需與其他次單元結合就具有催化活性,但氨基端乙醯化作用在生物體內的詳細功能卻不明。為了研究 ssPat 和 ssArd1結構與特性,我們先利用圓二色偏光光譜實驗來測定二級結構組成及討論其生物物理性質,然後利用 X射線結晶學 (X-ray crystallography)實驗提供ssPat 和 ssArd1的蛋白質的晶體結構,結合兩種實驗分析結果進一步探討 ssPat 與 ssArd1 的催化機制及其生理功能。

Covalent modification is another regulatory strategy adopted by the cell to control enzyme activity. Control is achieved by covalent binding of a variety of chemical groups to the enzyme. The addition of the chemical group changes the shape of the enzyme. Posttranslational covalent modifications (acetylation, methylation, and phosphorylation) of the NH2-terminal tail of histones allow regulatable contacts with the underlying DNA and make up the histone code that governs gene transcription.The reversible acetylation of lysine residues has been particularly well studied, and a range of histone acetyltransferases and deacetylases has been identified. the evolution of gene regulatory processes in all three domains of life. Sulfolobues solfataricus grow in terrestrial volcanic hot springs with optimum growth occurring at pH 2-3 and a temperature of 75-80℃. The Alba protein was initially identified as an abundant DNA-binding protein in Sulfolobus extracts. It was proposed to be a major chromatin protein. Alba is a heterodimer, Alba1 and Alba2. The previous work revealed that the Alba1, was acetylated in vivo on Lys-16 and deacetylated by Sir2, and S.solfataricus Pat (ssPat) specifically acetylates Alba1 on lysine 16 and lowers its DNA binding affinity. However, the biological function of ssPat is still unclear. As a result, it aimed us to study whether Alba2 is one of substrates of ssPat and the effect of acetylation on Alba2. In eukarya, N-terminal acetylation is an extremely common modification., affecting 80–90% of cytosolic mammalian proteins. Saccharomyces cerevisiae has three protein N-terminal acetyltransferases (NATs), NatA, NatB and NatC, with catalytic subunits Ard1, Mak3 and Nat3 respectively. The three catalytic subunits arehomologous, and constitute part of the GNAT superfamily of acetyltransferases. In contrast, bacterial N-terminal protein acetylation is highly unusual. ssArd1 is a homolog of the N-terminal protein acetyltransferase in Sulfolobues solfataricus. The biochemical characterization of ssArd1 demonstrates that the protein can acetylate a much wider range of protein N-terminal sequences than any of the eukaryotic NAT proteins. In particular, the strong specific activity against Ser, Ala and Met–Glu termini accounts for the majority of the acetylated protein N-termini detected so far in S. solfataricus, suggesting that ssArd1 is the sole functional NAT in this organism, and that it combines the specificity of the yeast Ard1 and Nat3 proteins. To study the structure and characteristic of ssPat and ssArd1, we analyzed the component of second structure and its property of biophysics by circular dichromism. In addition to X-ray crystallography experiments, which showed the results of crystal structure of ssPat and ssArd1 proteins, we further investigated the mechanism of catalysis and biofunction.
其他識別: U0005-1002200910502700
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