PDCD5蛋白在Sulfolobus solfataricus 古生菌的結構與功能分析;Structural & Functional Activity analysis of Programmed Cell Death 5 from the Hyperthermophile Sulfolobus solfataricus

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Title:	PDCD5蛋白在Sulfolobus solfataricus 古生菌的結構與功能分析;Structural & Functional Activity analysis of Programmed Cell Death 5 from the Hyperthermophile Sulfolobus solfataricus
Authors:	許家源;Hsu,Chia-Yuan
Contributors:	生命科學系
Keywords:	硫化屬葉菌;細胞凋亡;第五型程序凋亡訊息蛋白;X光繞射結晶學;蛋白質結構建構;Sulfolobus solfataricus;Programmed Cell Death;Sso-PDCD5 protein;X-ray crystallization;Structural Building
Date:	2016-07-28
Issue Date:	2016-10-13 13:04:09 (UTC+8)
Publisher:	國立中央大學
Abstract:	本研究是為了了解以古生菌Sulfolobus solfataricus內的DNA結合蛋白PDCD5中的結構進而可了解DNA結合蛋白PDCD5在古生菌中扮演的角色。古生菌Sulfolobus solfataricus屬於Sulfolobaceae科底下分支的一種古生菌，第一次被發現於義大利拿坡里附近的火山島伊斯基亞島是第一批被完成解序的硫化嗜熱古生菌。 PDCD5蛋白質是個最早被發現在人類體內細胞用來上調節的細胞凋亡蛋白。PDCD5蛋白質可從細胞質向細胞核迅速的通過並與Tip60蛋白結合於DNA造成乙醯化，進而傳遞細胞凋亡訊息，其重要性為調節第五型賴氨酸乙酰轉化酶抑制其蛋白酶體相關的降解（參與轉錄、DNA損傷反應和細胞週期控制蛋白）。我們從KEGG數據分析庫確定了SSO0352基因為PDCD5蛋白在古生菌Sulfolobus solfataricus的同源基因，由於目前在古生菌Sulfolobus solfataricus的功能仍然還是未知，為了研究是否有相似的作用及結構。我們表現在質體pET-21a上和找到適合生產PDCD5蛋白質的方法，並表現於大腸桿菌(RIL)菌株，找到其純化條件。利用陽離子交換樹脂和膠體交換法純化出蛋白並使用X-ray繞射與單重原子異常散射（SAD）得知PDCD5蛋白的結構。Sso-PDCD5晶體在X-ray的繞射數據為Space group=C2, a = 100.34 Å, b = 39.71 Å, c = 77.57 Å, α= γ= 90∘, β = 125.15∘的晶胞大小與2.30 Å的解析度。對於解決相位問題，利用點突變蛋白質SsoPDCD5-L45M（L45M）的繞射數據來解決。SeMet-L45M晶體在X-ray的繞射數據為的Space group=C2, a = 109.31 Å, b = 41.03 Å, c = 77.68 Å, α= γ= 90∘, β = 129.51∘的晶胞大小，解析度在2.13 Å。 SsoPDCD5總共具有四個α-螺旋其中三個在N端為低靈活性並可被解析的α-螺旋和另一個是在C端尾部高靈活可動的未解的α-螺旋。 C端的色氨酸(tryptophan)透過結合20bp的雙鏈DNA的熒光光譜實驗表示PDCD5的C端11個胺基酸可能與DNA相互作用。 ;The Programmed Cell Death 5 (PDCD5) is an important signal protein of apoptosis pathway in human. It can interact with Tip60 to induce cell arrest when DNA over damage, and binds with p53 protein from ubiquitination. In previous research, it is known there are triple-helix bundle and two dissociated N-terminal regions in human PDCD5 protein. However, the study of PDCD5 remains unclear in hyperthermophile archaea. Here we identify a PDCD5 homolog, Sso-0352 (SsoPDCD5), in Sulfolobus solfataricus. To explore the differences of PDCD5 homologous protein from S. solfataricus and other species, we express and crystallize the protein S. solfataricus and determine the structure by Single Wavelength Diffraction (SAD) method. The native Sso-PDCD5 crystal appears to the space group C2 with unit cell magnitude of a = 100.34 Å, b = 39.71 Å, c = 77.57 Å, α= γ= 90∘, β = 125.15∘ with 2.30 Å resolution. For the phasing, the structure of single mutant protein SsoPDCD5-L45M (L45M) is also solved as well. The space group of L45M is C2 with unit cell magnitude of a = 109.31 Å, b = 41.03 Å, c = 77.68 Å, α= γ= 90∘, β = 129.51∘with 2.13 Å resolution for SeMet-L45M crystal. SsoPDCD5 has a compact core of low flexibility with four alpha-helices at N-terminal region and a flexible unstructured C-terminal tail. The C-terminal tryptophan can be quenched by 20bp double strand DNA. It indicates PDCD5 may interact with DNA with the C-terminal tail.
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