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標題: | 功能迥異的macrodomain家族蛋白質之結構與功能闡述 Structural and functional elucidations of functionally diverse macrodomain family proteins |
作者: | Chao-Cheng Cho 卓昭成 |
指導教授: | 徐駿森 |
關鍵字: | macrodomain,多聚二磷酸腺?核糖糖水解?,中東呼吸道症候群冠狀病毒,Deinococcus radiodurans,多聚二磷酸腺?核糖化,晶體結構,生物化學, macrodomain,PARG,MERS-CoV,Deinococcus radiodurans,poly ADP-ribosylation,crystal structure,biochemistry, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | Macrodomain是廣泛存在於真核生物、細菌、以及古菌中的蛋白質模組,並且以其和二磷酸腺苷核糖(ADP-ribose),一種蛋白質二磷酸腺苷核糖化的代謝物,之間的交互作用而著名。具有macrodomain的蛋白質展現對於ADP-ribose衍生物如多聚二磷酸腺苷核糖(PAR)、二磷酸腺苷核糖 1’’-磷酸(Appr1p),以及連接在蛋白質上的單體ADP-ribose的酵素活性,並且參與調節各樣的細胞功能,包括DNA修復、基因表現、以及細胞訊息傳遞。在這個研究中,我們利用結構與生物化學的方法來研究兩種典型的macrodomain家族蛋白質,病毒macrodomain和多聚二磷酸腺苷核糖糖水解酶(PARG)。
冠狀病毒以及少數RNA病毒的基因組會轉譯macrodomain,新興中東呼吸道症候群冠狀病毒(MERS-CoV)的非結構性蛋白質中包含一個功能未知且具保守性的macrodomain。我們利用差異掃描螢光法以及等溫滴定量熱法,發現MERS-CoV的macrodomain相較於其他的冠狀病毒的macrodomain來說,是一個更有效率的ADP-ribose結合模組。更進一步地,我們得到了MERS-CoV macrodomain和ADP-ribose複合體的晶體結構,解析度為1.43-埃。與其他人類冠狀病毒的macrodomain進行結構比較,顯示於α1螺旋中存在的結構性差異改變了具有保守性的天門冬胺酸20與ADP-ribose之間的交互作用,並可能解釋了MERS-CoV macrodomain對於二磷酸腺苷核糖有效率結合的原因。 在真核生物中,PARG是催化PAR鏈降解的主要酵素並且負責多聚二磷酸腺苷核糖化(PARylation)的反轉。然而,對於原核生物中的PARylation仍然了解甚少。我們利用免疫墨點法在抗輻射細菌Deinococcus radiodurans中偵測內生性PAR的訊號。免疫墨點法的結果顯示PAR的程度在接受紫外光照射後上升,並且干擾D. radiodurans中PARG同源蛋白質(DrPARG)會造成PAR在細胞內的累積。更進一步地,我們解出屬於不同空間群的DrPARG (apo form)以及其結合ADP-ribose的複合體(bound form)晶體結構。比較apo form與bound form的結果顯示在ADP-ribose結合時蛋白質發生構型改變,可能是由於缺乏來自於具活動性N端的結構支持。細菌型的PARG,不像他們在真核生物的相對應酵素,被認為是絕對的外切糖水解酶。意外地,在bound form結構中我們發現ADP-ribose的2’-羥基是暴露於溶劑的,顯示DrPARG可能具有內切糖水解酶的活性。與這個發現相吻合的,in vitro酵素實驗的產物中可以偵測到PAR的存在。比較DrPARG-PAR模型與絕對外切酶的PARG結構顯示DrPARG的結構活動性與位於267位置的蘇胺酸(T267)會貢獻內切糖水解酶活性。結構與酵素活性的分析顯置換T267會影響內切糖水解酶的活性。最後,我們發現DrPARG會直接的分解D. radiodurans中的內生性PAR。 我們對於MERS-CoV macrodomain以及DrPARG的研究將會對於macrodomain家族的多樣成員之結構與功能提供新的見解以及有助於了解他們在個別物種中所扮演的角色。 Macrodomain is a ubiquitous protein module found in organisms of eukaryotes, bacteria, and archaea and well-known for its interaction with adenosine diphosphate ribose (ADP-ribose), a metabolite of protein ADP-ribosylation. Macrodomain-containing proteins exhibit diverse enzymatic activities to process ADP-ribose derivatives such as poly ADP-ribose (PAR), ADP-ribose 1’’-phosphate (Appr1p), and protein-linked mono ADP-ribose, and often participate in regulating various cellular functions including DNA repair, gene expression, and cellular signaling. In this study, structural and biochemical approaches are taken to study the structure and function of two representatives of macrodomain family, viral macrodomain and bacterial poly ADP-ribose glycohydrolase (PARG). The genomes of coronaviruses and a few other RNA viruses encode macrodomains. The non-structural protein of the newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) harbors the conserved macrodomain with unknown function. Using differential scanning fluorimetry and isothermal titration calorimetry, we characterized the MERS-CoV macrodomain as a more efficient ADP-ribose binding module than macrodomains from other CoVs. Furthermore, the crystal structure of MERS-CoV macrodomain was determined at 1.43-Å resolution in complex with ADP-ribose. Comparison of macrodomains from MERS-CoV and other human CoVs revealed structural differences in the α1 helix alters how the conserved aspartic acid at position 20 interacts with ADP-ribose and may explain the efficient binding of the MERS-CoV macrodomain to ADP-ribose. In eukaryotes, PARG is the major enzyme catalyzing the breakdown of PAR chains and responsible for the turnover of poly ADP-ribosylation (PARylation). However, PARylation of prokaryotic organisms remains poorly understood. We performed immunoblotting using the specific antibody against PAR and detected endogenous PAR signals in the radioresistant bacterium Deinococcus radiodurans. The result of immunoblotting showed that PAR level is upregulated after UV irradiation and disruption of the PARG homologue of D. radiodurans (DrPARG) causes accumulation of PAR in cell. Furthermore, we determined the crystal structures of apo and ADP-ribose bound DrPARG belonging to various space groups. Comparison of apo and bound DrPARG structures revealed conformational changes of the protein during ADP-ribose binding, which may be resulted from lacking structural supports of the flexible N-terminus. The bacterial PARG, unlike its eukaryotic counterpart, was reported to be obligate exo-glycohydrolase. Surprisingly, a solvent accessible 2’-hydroxy group of ADP-ribose was found in the bound form structure, suggesting that DrPARG may possess endo-glycohydrolase activity. In consistent with this finding, in vitro cleavage assay detected PAR in DrPARG processed products. Comparison of DrPARG-PAR model and the obligate exo-PARG structure suggested that the structural flexibility along with the threonine at position 267 (T267) contribute to the endo-glycohydrolase activity. Structural and enzymatic activity analyses were performed and showed that T267 substitution in DrPARG affects endo-glycohydrolase activity. Finally, we showed that DrPARG is capable of processing endogenous PAR of D. radiodurans. Our study of MERS-CoV macrodomain and DrPARG will provide new insights into the structure and function of diverse members of macrodomain family and help to understand their roles in individual organism. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70624 |
DOI: | 10.6342/NTU201802789 |
全文授權: | 有償授權 |
顯示於系所單位: | 基因體與系統生物學學位學程 |
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