【作者】 王文晞；

【导师】 肖庚富；

【作者基本信息】 武汉大学 ， 微生物学， 2009， 博士

【摘要】 朊病毒疾病是一种致死性中枢神经系统退化性疾病,包括了人的库鲁病、吉-斯综合症(GSS)、致死性家族失眠症(FFI)、克雅氏病(CJD)和变异性克雅氏病(vCJD),以及动物的牛海绵状脑病(BSE)和羊瘙痒症(Scrapie)。错误折叠的朊蛋白在大脑特定区域内沉积,会引起朊病毒病。在所有的朊病毒疾病中,致病的关键因素是细胞内正常PrPc蛋白高级构象变化,转变成致病性的PrPSc。Calnexin (CNX)是内质网内Ⅰ型膜蛋白,它可以帮助新生N糖基化蛋白的正确折叠以及内质网质量控制。前人的研究表明,凝集素分子伴侣CNX和糖蛋白底物的结合有两种模式,只依赖与凝集素位点的结合模式以及依赖凝集素位点和多肽结合位点的双重结合模式。在本研究中,我们构建了CNX和PrP的真核和原核表达载体。在随后的试验中,通过非变性凝胶电泳(N-PAGE)和免疫共沉淀(Co-ip)的方法,首次证明了CNX可以和PrP在体内体外相互作用。由于体外大肠杆菌表达出来的PrP是没有糖基化的,因此,实验结果可以推测CNX和PrP的结合是依赖双重结合模式。Williams等人的体外实验结果表明,CNX可以抑制很多蛋白的热聚集。为了证实CNX能否抑制PrP的热聚集,我们采用了浊度法。浊度实验结果表明CNX能够很有效地抑制PrP的热聚集,当CNX和PrP的摩尔比为2比1的时候,PrP的热聚集基本上被完全抑制了。接下来,原子力显微镜(AFM)、分子筛液相色谱(SEC)和静态光散射等实验也进一步验证了这一结果。为了进一步弄清CNX能否降低PrP引起的细胞毒性,我们采用了MTT细胞毒性实验。从实验结果可以看出,单独转染PrP的细胞存活率和空白对照相比有明显的下降,但是PrP和CNX共转染的细胞存活率和空白对照相比却几乎没有变化,说明CNX可以抑制PrP引起的细胞毒性。为了进一步了解CNX抑制细胞毒性是不是由于抑制了PrP诱导的细胞凋亡,运用了流式细胞仪测定转染细胞的凋亡,结果证明CNX可以抑制PrP诱导的细胞凋亡,随着CNX的量的增加,凋亡的抑制效果也越来越明显。对PrP和CNX的相互作用的研究可能可以给朊病毒疾病或其他一些蛋白质构象病诸如老年痴呆症(AD)、帕金森病(Parkinson’s disease)和亨廷顿病(Huntington’s disease)等由蛋白质错误折叠和聚集引起的疾病提供潜在的治疗手段。更多还原

【Abstract】 Prion diseases are fatal, neurodegenerative diseases that include scrapie in sheep; bovine spongiform encephalopathy (BSE) in cattle; Kuru, Gerstmann Straussler Scheinker disease (GSS), fatal familial insomnia (FFI), Creutzfeldt-Jakob disease (CJD), and variant Creutzfeldt-Jakob disease (vCJD)in human. Misfolding and aggregation of prion protein deposit on selected regions of the brain can result in prion diseases. A key event in all prion diseases appears to be the supreme structure changes of normal cellular form of prion protein (PrPc) into the infectious scrapie isoform (PrPSc).Calnexin(CNX) is a type I integral membrane protein in the endoplasmic reticulum (ER) that ensures the proper folding and quality control of newly synthesized N-linked glycoproteins. Previous studies showed us that the lectin chaperone CNX had two mechanisms of association with glycoproteins:lectin-binding only or lectin and polypeptide binding.In this work, we constructed prokaryotic and eukaryotic expression vectors of calnexin (CNX) and prion protein (PrP). Our findings for the first time confirmed that calnexin interacts with PrP. The immunoprecipitation and native polyacrylamide-gel electrophoresis results indicate that calnexin could bind PrP in vivo and in vitro. The E. coli expressed PrP was non-glycosylated, we guess the interaction of PrP with CNX maybe depend on polypeptide binding site.Some researchs demonstrated that CNX could suppresses thermal aggregation of non-glycosylated polypeptides in vitro. To assess whether CNX is capable of supressing aggregation of PrP in vitro, the turbidity assay was employed. Consistent with a molecular chaperone function, CNX effectively suppressed the aggregation of PrP in a concentration-dependent manner with maximal suppression occurring at a molar ratio of one CNX molecule to two PrP molecules. We can see at this molar ratio, the aggregation of PrP was almost suppressed. Then the atom force microscopy (AFM), light scattering and size exclusion chromatograph (SEC) assays proved our conclusion further.To investigate whether CNX could inhibit cytotoxicity induced by PrP, we employed MTT assay. The result showed that the viability of the cell expressed PrP dropped remarkably, while viability of the cells expressed both CNX and PrP kept unchanged compared with that blank control, when the concentration of CNX is the same as PrP, the viability of the cell did not changed significantly. In order to investigate whether CNX could inhibit the cell apoptosis induced by PrP in SK-N-SH cells, we employed flow cytometry analyses. The result showed CNX effectively suppressed the apoptosis induced by PrP in a concentration-dependent manner.Research on PrP interacts with CNX may have clinical benefits in prion-related diseases and other protein conformation diseases such as Alzheimer’s, Parkinson’s and Huntington’s diseases, and other diseases especially resulted from protein misfolding and accumulation.更多还原