本文關(guān)鍵詞：金黃色葡萄球菌超抗原樣蛋白Set11的結(jié)構(gòu)與功能研究　出處：《中國(guó)科學(xué)技術(shù)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 金黃色葡萄球菌 超抗原樣蛋白 Set11 生物信息學(xué)分析 結(jié)構(gòu)模型 pull down 補(bǔ)體C5 E3泛素連接酶TRAIP蛋白

【摘要】：金黃色葡萄球菌是一種常見(jiàn)的病原菌,可感染人類并導(dǎo)致一系列的感染和疾病,包括筋膜炎、肺炎、心內(nèi)膜炎、敗血癥、骨髓炎和中毒性休克綜合癥等。為了破壞宿主的天然免疫系統(tǒng),金黃色葡萄球菌分泌大量的毒力因子抑制或阻止中性粒細(xì)胞的招募和激活來(lái)發(fā)揮其功能。金黃色葡萄球菌NCTC 8325菌株能夠分泌 14 種超抗原樣蛋白(staphylococcal superantigen-like(SSLs)proteins),這些 SSLs蛋白最初被命名為金黃色葡萄球菌外毒素樣蛋白(staphylococcal exotoxin-like proteins(Sets)proteins),由于其序列和結(jié)構(gòu)與超抗原蛋白高度相似,之后被重新命名為超抗原樣蛋白。SSLs/Sets蛋白家族在不同的金黃色葡萄球菌菌株中的數(shù)量和命名存在差異。如在NCTC 8325菌株中已經(jīng)鑒定出14種SSLs蛋白,而在Mu50菌株中目前只發(fā)現(xiàn)了 12種超抗原樣蛋白,且以Set命名為主。在己有的研究中,SSLs識(shí)別宿主不同的蛋白來(lái)發(fā)揮多種功能。例如,SSL7結(jié)合補(bǔ)體C5抑制補(bǔ)體系統(tǒng)。SSL10與IgG1相互作用破壞IgG1介導(dǎo)的細(xì)胞吞噬作用。SSL11通過(guò)阻斷PSGL-1與P-選擇素的相互作用來(lái)抑制中性粒細(xì)胞的遷移。SSL3和SSL4抑制Toll-樣受體2在樹(shù)突細(xì)胞和中性粒細(xì)胞表面的表達(dá)。此外,SSL10不僅結(jié)合IgG1,而且與凝血素和FactorXa相互作用來(lái)抑制凝血系統(tǒng)。多數(shù)超抗原樣蛋白識(shí)別的宿主因子仍未知,超抗原樣蛋白在致病菌感染宿主中的作用機(jī)制也知之甚少。Set11是Mu50菌株分泌的超抗原樣蛋白中的一種,其結(jié)構(gòu)和功能尚不明確。在本論文的第一篇中,為鑒定Set11在宿主的血液/血清中潛在的相互作用蛋白并探索其發(fā)揮功能的結(jié)構(gòu)基礎(chǔ)與分子機(jī)制,我們體外重組表達(dá)并純化了 Set11蛋白,首先對(duì)其進(jìn)行了晶體的篩選及大量的晶體優(yōu)化工作。遺憾的是,我們最終沒(méi)有獲得可以用于收集衍射數(shù)據(jù)的晶體,所能得到的晶體衍射分辨率最高為3.6埃。通過(guò)對(duì)Set11進(jìn)行生物信息學(xué)分析,我們發(fā)現(xiàn)Mu50菌株中的Set11與NCTC 8325菌株中的SSL7有90%的序列同源性,暗示Set11是SSL7在Mu50中的同源蛋白。在此前的報(bào)道中,SSL7已經(jīng)能夠與補(bǔ)體C5結(jié)合進(jìn)而抑制補(bǔ)體系統(tǒng)的激活。為了探究Set11是否也能夠與C5結(jié)合,我們分別在已有SSL7(PDB ID:1VIO)和 SSL7-C5(PDBID:3KLS)結(jié)構(gòu)的基礎(chǔ)上構(gòu)建了 Set11 和 Set11-C5 復(fù)合物的同源結(jié)構(gòu)模型。結(jié)構(gòu)分析和序列比對(duì)顯示SSL7中參與C5相互作用的關(guān)鍵氨基酸殘基在Set11中是高度保守的,暗示了 C5是Set11的潛在的靶蛋白。為鑒定Set11與C5的相互作用以及新的Set11結(jié)合蛋白,我們以Set11為誘餌,通過(guò)CNBr-Pull down耦聯(lián)質(zhì)譜分析的實(shí)驗(yàn)方法從新鮮的人血/血清中鑒定了Set11的結(jié)合蛋白。它們分別是纖連蛋白1亞型3前蛋白原、補(bǔ)體C5、補(bǔ)體C3前體、血清白蛋白和α-1-微球蛋白前體。我們的結(jié)果肯定了 Set11與C5的結(jié)合,并鑒定了 Set11潛在的相互作用蛋白,為進(jìn)一步探索Set11的功能提供了線索。E3泛素連接酶TRAIP蛋白是腫瘤壞死因子受體相關(guān)蛋白(tumor necrosis factor(TNF)receptor-associated factors interacting protein)的簡(jiǎn)寫(xiě)。TRAIP 蛋白與腫瘤壞死因子受體相關(guān)因子(TRAF)以及兩個(gè)腫瘤抑制因子CYLD和SYK均存在相互作用。研究發(fā)現(xiàn),TRAIP與TRAF2相互作用抑制TRAF2介導(dǎo)的NF-κB的激活,與SYK和CYLD的相互作用,則負(fù)調(diào)TNF介導(dǎo)的NF-κB的激活,從而抑制細(xì)胞的凋亡。而近幾年研究表明,TRAIP作為一種RING type E3泛素連接酶,通過(guò)RING domain與E2泛素結(jié)合酶UBE2U相互作用,在蛋白質(zhì)泛素化過(guò)程中也發(fā)揮著重要功能,但是泛素化的底物和分子機(jī)制目前仍未知。在本論文的第二篇中,我們通過(guò)體外重組、表達(dá)并純化了小鼠、斑馬魚(yú)、非洲爪蟾和果蠅中E3泛素連接酶TRAIP蛋白R(shí)ING Domain結(jié)構(gòu)域,并嘗試對(duì)E3泛素連接酶TRAIP蛋白R(shí)ING Domain不同截短體蛋白進(jìn)行了晶體的篩選,暫時(shí)沒(méi)有觀察到晶體的生長(zhǎng)。我們希望探究TRAIP蛋白R(shí)ING Domain與E2的相互作用分子機(jī)制,并進(jìn)一步探索泛素化作用的可能底物及機(jī)制。
[Abstract]:Staphylococcus aureus is a common pathogen that can infect humans and cause infection and disease, including a series of fasciitis, pneumonia, septicemia, endocarditis, osteomyelitis and toxic shock syndrome. In order to destroy the host innate immune system, Staphylococcus aureus secretes numerous virulence factors inhibit neutrophil cell recruitment and activation functions. Staphylococcus aureus NCTC 8325 strains to 14 kinds of superantigen like protein secretion (staphylococcal superantigen-like (SSLs) proteins), the SSLs protein was originally named exotoxin like Staphylococcus aureus protein (staphylococcal exotoxin-like proteins (Sets) proteins), because of its sequence and structure and super antigen proteins are highly similar, after being re named superantigen like protein.SSLs/Sets protein family in different strains of Staphylococcus aureus The number of lines and naming differences. As in NCTC 8325 strains have identified 14 SSLs proteins, while Mu50 strains were found at the 12 kinds of superantigen like protein, and named Set. In the existing study, SSLs identify the host specific proteins to play a variety of functions. For example, transfer of.SSL3 and complement C5 SSL4 SSL7 and IgG1.SSL10 inhibition of complement system interaction damage mediated by IgG1 cell phagocytosis.SSL11 to inhibit neutrophils by blocking the interaction between PSGL-1 and P- selectin inhibition of Toll- like receptor 2 in dendritic cells and neutrophil surface expression. In addition, SSL10 not only with IgG1 and prothrombin interacts with FactorXa to inhibit blood coagulation system. Most super antihost factor like protein identification is still unknown, superantigen like protein in the infection mechanism of host pathogen is also known as little.Se T11 is a secreted by Mu50 strain superantigen like protein, its structure and function is not clear. In the first chapter of this paper, for the identification of Set11 in the host blood / interacting protein in serum and explore its potential molecular mechanism and play structural basis function, our body and recombinant expression Set11 protein was purified, first has carried on the crystal crystal screening and lots of optimization work. Unfortunately, we did not get can be used to collect diffraction data of crystal, crystal diffraction resolution can get up to 3.6. The biological information of the Set11 analysis, we found that the sequence homology with Set11 NCTC Mu50 strains in 8325 strains of SSL7 90%, implying that Set11 is SSL7 in Mu50 in the homologous protein. In previous reports, SSL7 has been able to combine and complement C5 activation and inhibition of complement system. To explore whether Set11 can bind to C5, we were in the existing SSL7 (PDB ID:1VIO) and SSL7-C5 (PDBID:3KLS) based on the structure of homology model of Set11 and Set11-C5 complexes. Structure analysis and sequence alignment showed that the key amino acid residues involved in the interaction of C5 in SSL7 is highly conserved in Set 11, suggesting that C5 is a potential target protein of Set11. Interaction for identification of Set11 and C5 and the new Set11 binding protein, we take Set11 as bait, the experimental method by CNBr-Pull down coupled with mass spectrometry from fresh human blood / serum identified Set11 binding protein. They are fibronectin protein 1 isoform 3 precursor protein, complement C5, complement C3 precursor, serum albumin and alpha -1- protein precursor microspheres. Our results confirmed the combination of Set11 and C5, and identified the potential interacting proteins of Set11 in Further investigate the function of Set11 provides a clue of.E3 ubiquitin ligase TRAIP protein is a tumor necrosis factor receptor associated protein (tumor necrosis factor (TNF) receptor-associated factors interacting protein) or.TRAIP protein and tumor necrosis factor receptor associated factor (TRAF) and two tumor suppressor CYLD and SYK interact with each other. The study found that TRAIP the interaction between TRAF2 and activation of TRAF2 mediated inhibition of NF- K B, interaction with SYK and CYLD, activation of TNF mediated negative regulation of NF- K B, then to inhibit cell apoptosis. Studies in recent years show that TRAIP is a RING type E3 ubiquitin ligase, by RING domain and E2 ubiquitin combined with enzyme UBE2U interactions in protein ubiquitination process also plays an important function, but the molecular mechanism and substrate ubiquitination is still unknown. In the second chapter of this paper, we By in vitro recombination, expression and purification of mouse, zebrafish, E3 ubiquitin ligase TRAIP protein RING Domain domain Xenopus and Drosophila, and try to E3 ubiquitin ligase TRAIP protein RING Domain truncated protein crystals were screened, no observed crystal growth. We hope to explore the molecular mechanism of TRAIP protein interaction RING Domain and E2, and further explore the possible mechanism of substrate and ubiquitination.

【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378.11

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