金黄色葡萄球菌超抗原样蛋白Set11的结构与功能研究

发布时间：2018-01-16 17:04

本文关键词：金黄色葡萄球菌超抗原样蛋白Set11的结构与功能研究　出处：《中国科学技术大学》2017年硕士论文　论文类型：学位论文

【摘要】：金黄色葡萄球菌是一种常见的病原菌,可感染人类并导致一系列的感染和疾病,包括筋膜炎、肺炎、心内膜炎、败血症、骨髓炎和中毒性休克综合症等。为了破坏宿主的天然免疫系统,金黄色葡萄球菌分泌大量的毒力因子抑制或阻止中性粒细胞的招募和激活来发挥其功能。金黄色葡萄球菌NCTC 8325菌株能够分泌 14 种超抗原样蛋白(staphylococcal superantigen-like(SSLs)proteins),这些 SSLs蛋白最初被命名为金黄色葡萄球菌外毒素样蛋白(staphylococcal exotoxin-like proteins(Sets)proteins),由于其序列和结构与超抗原蛋白高度相似,之后被重新命名为超抗原样蛋白。SSLs/Sets蛋白家族在不同的金黄色葡萄球菌菌株中的数量和命名存在差异。如在NCTC 8325菌株中已经鉴定出14种SSLs蛋白,而在Mu50菌株中目前只发现了 12种超抗原样蛋白,且以Set命名为主。在己有的研究中,SSLs识别宿主不同的蛋白来发挥多种功能。例如,SSL7结合补体C5抑制补体系统。SSL10与IgG1相互作用破坏IgG1介导的细胞吞噬作用。SSL11通过阻断PSGL-1与P-选择素的相互作用来抑制中性粒细胞的迁移。SSL3和SSL4抑制Toll-样受体2在树突细胞和中性粒细胞表面的表达。此外,SSL10不仅结合IgG1,而且与凝血素和FactorXa相互作用来抑制凝血系统。多数超抗原样蛋白识别的宿主因子仍未知,超抗原样蛋白在致病菌感染宿主中的作用机制也知之甚少。Set11是Mu50菌株分泌的超抗原样蛋白中的一种,其结构和功能尚不明确。在本论文的第一篇中,为鉴定Set11在宿主的血液/血清中潜在的相互作用蛋白并探索其发挥功能的结构基础与分子机制,我们体外重组表达并纯化了 Set11蛋白,首先对其进行了晶体的筛选及大量的晶体优化工作。遗憾的是,我们最终没有获得可以用于收集衍射数据的晶体,所能得到的晶体衍射分辨率最高为3.6埃。通过对Set11进行生物信息学分析,我们发现Mu50菌株中的Set11与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。在此前的报道中,SSL7已经能够与补体C5结合进而抑制补体系统的激活。为了探究Set11是否也能够与C5结合,我们分别在已有SSL7(PDB ID:1VIO)和 SSL7-C5(PDBID:3KLS)结构的基础上构建了 Set11 和 Set11-C5 复合物的同源结构模型。结构分析和序列比对显示SSL7中参与C5相互作用的关键氨基酸残基在Set11中是高度保守的,暗示了 C5是Set11的潜在的靶蛋白。为鉴定Set11与C5的相互作用以及新的Set11结合蛋白,我们以Set11为诱饵,通过CNBr-Pull down耦联质谱分析的实验方法从新鲜的人血/血清中鉴定了Set11的结合蛋白。它们分别是纤连蛋白1亚型3前蛋白原、补体C5、补体C3前体、血清白蛋白和α-1-微球蛋白前体。我们的结果肯定了 Set11与C5的结合,并鉴定了 Set11潜在的相互作用蛋白,为进一步探索Set11的功能提供了线索。E3泛素连接酶TRAIP蛋白是肿瘤坏死因子受体相关蛋白(tumor necrosis factor(TNF)receptor-associated factors interacting protein)的简写。TRAIP 蛋白与肿瘤坏死因子受体相关因子(TRAF)以及两个肿瘤抑制因子CYLD和SYK均存在相互作用。研究发现,TRAIP与TRAF2相互作用抑制TRAF2介导的NF-κB的激活,与SYK和CYLD的相互作用,则负调TNF介导的NF-κB的激活,从而抑制细胞的凋亡。而近几年研究表明,TRAIP作为一种RING type E3泛素连接酶,通过RING domain与E2泛素结合酶UBE2U相互作用,在蛋白质泛素化过程中也发挥着重要功能,但是泛素化的底物和分子机制目前仍未知。在本论文的第二篇中,我们通过体外重组、表达并纯化了小鼠、斑马鱼、非洲爪蟾和果蝇中E3泛素连接酶TRAIP蛋白RING Domain结构域,并尝试对E3泛素连接酶TRAIP蛋白RING Domain不同截短体蛋白进行了晶体的筛选,暂时没有观察到晶体的生长。我们希望探究TRAIP蛋白RING Domain与E2的相互作用分子机制,并进一步探索泛素化作用的可能底物及机制。
[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.

【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378.11

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