兰州大学机构库 >生命科学学院
C-反应蛋白组装机制及折叠相关分子伴侣的鉴定
Alternative TitleAssembly Mechanism and Folding Chaperones Identification of C-reactive protein
姚振宇
Thesis Advisor吉尚戎
2017-09-30
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
KeywordC-反应蛋白 折叠 组装 分子伴侣
Abstract

作为典型的急性期蛋白,天然人C-反应蛋白(C-reactive protein,CRP)主要由肝脏合成和分泌,它由五个相同的亚基通过非共价键形成盘状的五聚体结构。在炎症或组织损伤后的48小时内,其在血浆中的浓度可从基础水平的1μg/ml迅速升高1000多倍,由于CRP血浆浓度与炎症之间的相关性,使得CRP被用作衡量或监视体内的感染、组织损伤以及某些炎症疾病的发展,尤其是作为心血管疾病预测和愈后评价的标志分子受到广泛的关注。然而,更多的体外证据表明,CRP不仅作为标志分子,还通过构像变化形成单体CRP (monomeric CRP,mCRP)直接参与炎症反应,但是五聚体CRP(pentameric CRP,pCRP)和单体CRP在体内的作用仍存在矛盾,这是因为在体内复杂环境下CRP的变构为实验结果增加了复杂性,因此,构建只存在某一种CRP构型的实验动物对于研究CRP的病理生理学作用有重要意义,而这需要对CRP在细胞内的折叠、组装和分泌机制进行深入研究。

在细胞内,野生型CRP只能以五聚体的形式分泌表达,这与体内CRP的表达情况一致,表明CRP亚基只有组装成五聚体才能通过细胞质控体系的监视进入分泌途径。我们通过构建突变体Y40C/V117C,发现其能够形成五聚体并分泌表达,进一步分析发现该突变体能够在保留天然二硫键的情况下,在亚基间形成一个二硫键,这表明CRP亚基的折叠过程发生于五聚体组装之前,只有当亚基折叠成近似天然构像后,才能进一步组装成五聚体,这与pCRP在脲变性溶液中解聚形成mCRP的过程中,CRP五聚体依赖于亚基三级构像的结果相一致。从二硫键突变体、Ca2+位点突变体、离子键突变体的表达和互作研究结果,我们推测CRP亚基的折叠过程大致分为两个主要阶段,首先,CRP从N-端开始折叠,使疏水核心结构形成,相距较远的Cys36和Cys97相互靠近并通过形成二硫键起到稳定疏水核心的作用,二硫键形成后,Ca2+参与亚基的进一步折叠,使其接近天然亚基的构像。在亚基的折叠过程中,DnaK和GroEL是参与肽链折叠的主要分子伴侣,在他们的帮助下,亚基的三级构像基本形成,但此时的亚基仍不能进入组装阶段,可能需要肽脯顺反异构酶的参与,通过对保守的脯氨酸的异构,实现局部区域的微调,使亚基进一步紧密包装,才能进入五聚体的组装过程。

Other Abstract

Natural human C-reactive protein(CRP), composed of five identical subunits which associate each other into a disc-like structure, is a prototypic acute phase protein which is mainly secreted by the liver. CRP concentration in serum increased more than 1000-fold from 1μg/ml baseline level within 48 hours after onset of tissue injury and inflammation. CRP is used to be a marker to evaluate and monitor the development of infection, tissue injury and inflammation related diseases, especially in cardiovascular disease. Emerging evidence in vitro indicate that CRP not only a marker, but also a direct mediator through conformation dissociation to form monomeric CRP. However, contradiction between pCRP and mCRP remained regarding their activities in vivo because of complexity added by CRP conformation changing. To research specific role of CRP in pathophysiology, it is necessary and meaningful to research CRP folding, assembly and secretory for constructing animal models which only express one type of CRP conformation.

Wild type CRP only secreted in pentamer form from cells, which is similar to CRP secretion in vivo. The phenomenon indicate assembly of CRP subunit is necessary to its secretion and monitored by cell quality control system. Mutant Y40C/V117C formed pantamer and be secreted from cells. Further analysis revealed that the mutant formed inter-subunit disulfide bond and mean while the native disulfide bond in subunit was remained. This result indicate subunit folding precedes pentamer assembly and assembly of CRP subunit start only when its structure is similar to native subunit conformation. The conclusion fit the evidence of pCRP dissociation in urea denaturing solution which reflected pentameric CRP depend on the third conformation of subunit. From the result of expression and protein interaction of mutant of disulfide bond, calcium site and ionic bond, we inferred the folding of CRP subunit include two main process. First, the peptide start folding from N-terminal and form hydrophobic core. When Cys36 and Cys97 in contact, the disulfide bond formed and stabilized hydrophobic core. After that, calcium took part in the following folding process and made the subunit more compact and similar to the native conformation. In process of subunit folding, DnaK and GroEL are main folding chaperones which could assisted the third structure formation of subunit. But at this time, the subunit assembly cannot occur and still need the help of peptidyl-prolyl cis-trans isomerase which made the subunit more contact by the isomerization of conservative proline and fine-tuning of local region. Then, assembly of pentamer may occur.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222011
Collection生命科学学院
Recommended Citation
GB/T 7714
姚振宇. C-反应蛋白组装机制及折叠相关分子伴侣的鉴定[D]. 兰州. 兰州大学,2017.
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