本文選題：腸球菌 + 抑制劑��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：ABC轉(zhuǎn)運(yùn)蛋白(ATP-binding cassette transporter)存在于哺乳動(dòng)物、細(xì)菌、真菌等多種細(xì)胞中,且與多種細(xì)胞的耐藥有關(guān)。耐藥相關(guān)(ARE)蛋白,作為ABC蛋白的亞家族,介導(dǎo)細(xì)菌對(duì)靶向核糖體的一系列抗生素的抗性,在細(xì)菌的耐藥過(guò)程中有著重要的作用。惡唑烷酮類藥物(利奈唑胺和泰地唑胺),被認(rèn)為是治療VRE、MASA引起感染的最有效藥物,而在2015年新發(fā)現(xiàn)的ARE蛋白OptrA可介導(dǎo)腸球菌、MASA對(duì)惡唑烷酮類抗生素的耐藥性,使得惡唑烷酮類抗生素面臨著失效的危險(xiǎn)。以ARE蛋白為靶點(diǎn)的抑制劑的研究越來(lái)越受到人們的重視,因此研究OptrA的耐藥機(jī)制和抑制劑顯得非常重要。本實(shí)驗(yàn)通過(guò)膜蛋白交聯(lián)實(shí)驗(yàn)和金黃色葡萄球菌體外翻譯實(shí)驗(yàn)分別確定OptrA與膜蛋白的相互作用及對(duì)核糖體的保護(hù)情況,并通過(guò)ATP水解酶活性實(shí)驗(yàn)和最低抑菌濃度實(shí)驗(yàn)確定OptrA的ATP水解酶活性中心的關(guān)鍵殘基及其介導(dǎo)細(xì)菌耐藥性的活性中心。使用基于片段的藥物篩選方法篩選了OptrA的抑制劑,并在體外驗(yàn)證了該抑制劑對(duì)OptrA蛋白的ATP水解酶活性的抑制效果。同時(shí),我們?cè)噲D解析OptrA的蛋白質(zhì)晶體結(jié)構(gòu)。雖然并未得到很好的分辨率的晶體結(jié)構(gòu),但是我們使用同源建模和分子對(duì)接初步確定了抑制劑和蛋白質(zhì)相互作用的位點(diǎn)。結(jié)果顯示,在金黃色葡萄球菌RN4220的膜蛋白上并沒有檢測(cè)到OptrA蛋白,說(shuō)明OptrA蛋白不與膜蛋白相互作用。OptrA蛋白可以劑量依賴性的保護(hù)核糖體對(duì)m RNA的翻譯不被抗生素抑制。使用谷氨酰胺替換OptrA全長(zhǎng)氨基酸序列的208和488位置的谷氨酸,不但能使OptrA失去其ATP水解酶活性,而且能使其宿主細(xì)胞RN4220失去對(duì)氟苯尼考的耐藥性�；谄蔚乃幬锖Y選的抑制劑-CP 1可以抑制30%的OptrA的ATP水解酶活性。同源建模和分子對(duì)接結(jié)果顯示CP 1與OptrA蛋白的結(jié)合位點(diǎn)為Ile-496、Ile-292、Ile-289和Lys-271。綜上所述,OptrA通過(guò)保護(hù)核糖體,從而介導(dǎo)細(xì)菌對(duì)抗生素的耐藥性。OptrA全長(zhǎng)氨基酸序列的208和488位置的谷氨酸為OptrA的ATP水解酶活性中心的關(guān)鍵殘基,OptrA的ATP水解酶活性中心為其介導(dǎo)細(xì)菌耐藥的活性中心。CP 1可以作為OptrA的ATP水解酶抑制劑的先導(dǎo)化合物,為進(jìn)一步通過(guò)結(jié)構(gòu)優(yōu)化獲得更高效抑制劑的研究奠定了重要的實(shí)驗(yàn)依據(jù)和理論基礎(chǔ)。
[Abstract]:ABC transporter ATP-binding cassette transporter exists in mammalian, bacteria, fungi and other cells, and is related to drug resistance of many kinds of cells. As a subfamily of ABC proteins, resistance of bacteria to a series of antibiotics targeting ribosomes plays an important role in the process of drug resistance. Oxazolidinones (linazolidomide and tazolidomide) are considered to be the most effective agents in the treatment of VREMASA infection, and the newly discovered ARE protein OptrA in 2015 mediates the resistance of Enterococcus to oxazolidinone antibiotics. This puts oxazolidinone antibiotics at risk of failure. More and more attention has been paid to the study of inhibitors targeting ARE protein, so it is very important to study the mechanism of drug resistance and inhibitors of OptrA. The interaction between OptrA and membrane protein and the protection of ribosome were determined by membrane protein cross-linking test and staphylococcus aureus translation test in vitro, respectively. The key residues of ATP hydrolase active center of OptrA and the active center of bacterial resistance were determined by ATP hydrolase activity test and minimum inhibitory concentration test. A fragment based drug screening method was used to screen the inhibitor of OptrA, and the inhibitory effect of the inhibitor on the ATP hydrolase activity of OptrA protein was verified in vitro. At the same time, we try to analyze the protein crystal structure of OptrA. Although the crystal structure with good resolution has not been obtained, we use homologous modeling and molecular docking to preliminarily identify the sites for the interaction between inhibitors and proteins. The results showed that no OptrA protein was detected on the membrane protein of Staphylococcus aureus RN4220, indicating that the OptrA protein could not interact with the membrane protein. Optra protein could protect the ribosomal translation of m RNA from antibiotic inhibition in a dose-dependent manner. Using glutamine to replace the glutamate at 208 and 488 positions of the OptrA amino acid sequence can not only make OptrA lose its ATP hydrolase activity, but also make its host cell RN4220 lose its resistance to florfenicol. Fragment-based drug screening inhibitor-CP 1 could inhibit the ATP hydrolase activity of 30% of OptrA. The results of homology modeling and molecular docking showed that the binding sites of CP1 to OptrA protein were Ile-496, Ile-292, Ile-289 and Lys-271. To sum up, OptrA protects ribosomes, Thus, the glutamic acid at 208 and 488 positions of the full-length amino acid sequence of OptrA is the key residue of ATP hydrolase active center of OptrA, and the ATP hydrolase activity center of OptrA is involved in the activity of mediated bacterial drug resistance. Heart. CP1 can be used as a leading compound of ATP hydrolase inhibitor of OptrA. It lays an important experimental and theoretical basis for the further study of obtaining more efficient inhibitors through structural optimization.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王華丙;張振義;包銳;陳宇星;;ABC轉(zhuǎn)運(yùn)蛋白的結(jié)構(gòu)與轉(zhuǎn)運(yùn)機(jī)制[J];生命的化學(xué);2007年03期

相關(guān)會(huì)議論文 前1條

1 蔣智鋼;吳聰明;張萬(wàn)江;沈建忠;;蛋白質(zhì)組學(xué)及其在細(xì)菌耐藥性研究中的應(yīng)用[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)獸醫(yī)藥理毒理學(xué)分會(huì)第十次研討會(huì)論文摘要集[C];2009年

相關(guān)碩士學(xué)位論文 前1條

1 袁傳奇;GP5蛋白誘導(dǎo)表達(dá)及其多克隆抗體制備與PRRS和TGE共感染檢測(cè)[D];西北農(nóng)林科技大學(xué);2015年

，

本文編號(hào)：1827710