Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92201
標題: 探討c-di-AMP合成酶蛋白DacA與DNA受體蛋白LSm14A之生化功能及結構分析。
Biochemical and structural studies of DacA and LSm14A﹐a c-di-AMP synthetase and a DNA sensor﹐respectively.
作者: 康邁文
Mai-Wun Kang
關鍵字: 合成酶蛋白;生化結構;DacA;LSm14A
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摘要: 
【研究主題一】
在近期研究發現,環核苷酸二級傳訊分子中的cyclic diadenosine monophosphate (c-di-AMP),為存在於枯草芽孢桿菌 (Bacillus subtilis,Bsu) 中的一種訊號分子,並且能調控內孢子的形成及細胞壁的代謝。此菌細胞內c-di-AMP濃度的調節主要是由diadenylate cyclase (DAC) 以及phosphodiesterases (PDE) 所負責。其中,DAC具有DGA-domain或是RHR- domain,可將兩分子的ATP合成為一分子的c-di-AMP;而PDE具有DHH-domain或DHHA-domain,皆可將c-di-AMP分解為pApA。
本研究主要探討Bacillus subtilis str. 168中的DAC蛋白BsuDacA(105-268) 的結構及功能分析。目前我們已經得到解析度為2.85A的BsuDacA (105-268) 蛋白DAC domain晶體結構。為了進一步了解DAC domain BsuDacA(105-268) 蛋白與ATP分子之結合能力,我們利用示差掃描熱量分析儀 (DSC) 偵測發現,apo-form與ATP-binding form之BsuDacA(105-268) 蛋白,其Tm值分別為45℃及50℃,顯示BsuDacA(105-268) 蛋白與ATP可能有交互作用。接著再利用pyrophosphate assay進行BsuDacA(105-268) 蛋白酵素動力學的分析,結果顯示了BsuDacA(105-268) 之Vmax約為2.13 umol min-1,Km值約為56.97 uM。因此可知,BsuDacA(105-268 ) 可與ATP結合,並且能驅動DAC活性,進而合成c-di-AMP分子,以利於調控細菌細胞壁的合成。
【研究主題二】
在人體先天免疫系統中,防禦細胞是利用模式辨識受體 (Pattern-Recognition Receptors,PRRs) 來辨識病原性微生物相關分子 (Pathogen-Associated Molecular Patterns,PAMPs),然而不管是位於細胞膜上、內涵體或是細胞質的PRRs,都可當作偵測外源性核酸感應器 (sensor) 的作用。
本研究主要探討哺乳類動物細胞質中hLSm14A蛋白之結構及其功能分析。根據文獻指出hLSm14A蛋白可以偵測到病毒的DNA或RNA,接著透過處理小體 (P-bodies) 與RIG-1/VISA/MITA蛋白結合,進而促進第一型干擾素 (IFNs) 分泌並刺激免疫反應的產生 (如: 引起發炎反應及活化免疫系統)。雖然目前有hLSm14A之N端LSm domain的三級結構已被解出,但其C端 (包括DFDF domain以及FFD/TFG/RGG domain) 之結構尚未被解出,因此無法得知病毒雙股DNA或單股RNA與hLSm14A蛋白之間結合的情形,所以我們利用基因全合成的方式構築hLSm14A (胺基酸261-463) 片段的蛋白表現載體,以進一步探討其與病毒雙股DNA或單股RNA之結合情形。目前雖已篩得hLSm14A(261-463) 蛋白結晶條件,但是由於晶型呈現多晶重疊的現象,因此無法進行X-光晶體繞射。然而由膠體過濾法 (gel-filtration) 的結果顯示,hLSm14A(261-463) 蛋白在水溶液中可能以四聚體或單體的形式存在,進而使蛋白在結晶時有多晶重疊的現象,其造成蛋白晶型不理想。所以現階段建議再研擬構築不同長度的hLSm14A蛋白,期待獲得有品質較好的單晶晶體可供進一步研究。

【Theme one】
Recently, it was found that cyclic diadenosine monophosphate (c-di-AMP), which is one of the cyclic nucleotide second messengers, can exist in Bacillus subtilis (Bsu), in charge of adjusting spore formation and cell wall metabolism. The c-di-AMP concentration in Bacillus subtilis is chiefly adjusted by diadenylate cyclases (DAC) and phosphodiesterases (PDE). It is synthesized from two molecules of ATP by diadenylyl cyclase (DAC) enzymes which have a DGA-domain or RHR- domain, and is degraded to pApA by PDE, which have a DHH-domain or DHHA-domain.
The research aim is to study the structure and the function of DAC protein in Bacillus subtilis str. 168 (BsuDacA). Currently, the crystal structure was determined to a resolution of 2.85A. We further used Differential Scanning Calorimetry (DSC) detect the interaction between the DAC domain BsuDacA(105-268) protein and ATP. From the result, the Tm of BsuDacA(105-268) in its apo-form and ATP-binding form are shown to be 45℃ and 50℃, respectively. The result indicate that ATP bind with BsuDacA to stabilize its conformation. Enzyme Kinetic assay indicates that BsuDacA(105-268) exhibits a Vmax and Km of 2.13 umol min-1 and 56.97 uM, respectively. To sum up, BsuDacA(105-268) can interact with ATP, and synthesize c-di-AMP to potentially assist cell wall formation.


【Theme two】
Pattern recognition receptors (PRRs) are essential for detecting invading Pathogens via the Pathogen-Associated Molecular Patterns (PAMPs). PRRs are located in the membrane or endosome, and can be regarded as the sensor of extracellular nucleic acid.
The research aim is to study the structure and the function of the mammalian cytoplasm protein LSm14A. According to provious results, hLSm14A firstly detects virus DNA or RNA. Then P-bodies will interact with RIG-1/VISA/MITA proteins, initiate the releasing of IFNs and then launch the immune responses, such as immunity, and inflammatory responses. Though the 3D structures of N-terminal LSm domain of hLSm14A has been discovered, the structure of C-terminal (including DFDF domain and FFD/TFG/RGG domain) remained unknown. Thus, little is know about how foreign DNA or RNA of virus interact with hLSm14A protein. To obtain more information about the interaction between hLSm14A and virus DNA or RNA, we have constructed the hLSm14A clone by total gene synthesis and expressed the hLSm14A(261-463) protein fragment. Currently, wa have obtained the hLSm14A(261-463) crystal. But due to the poor crystal quality, we were unable to collect good diffraction data to determine its structure. From the gel-filtration data, hLSm14A(261-463) was found to exist as a mixture of tetramer and monomer in solution, which may lead to the imperfect protein crystals. It may be necessary to study hLSm14A protein in different lengh to get a better crystal quality to determine its structure.
URI: http://hdl.handle.net/11455/92201
Rights: 同意授權瀏覽/列印電子全文服務,2017-01-17起公開。
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