【作者】 刘鹏；

【导师】 赵玉芬；

【作者基本信息】 清华大学 ， 化学， 2013， 博士

【摘要】 淀粉样蛋白的错误折叠和异常聚集是2型糖尿病、阿兹海默病、海绵状脑病等淀粉样变性病的重要致病原因之一。淀粉样蛋白的细胞毒性主要来源于淀粉样蛋白的聚集、氧化压力的产生以及与细胞膜的相互作用；细胞毒性的产生过程会受到翻译后修饰、金属离子、共存蛋白以及二维细胞膜表面等多种因素的调控。在本论文中，我们重点研究了磷酸化和泛素化等翻译后修饰、金属铜离子、二维负电表面等因素对淀粉样蛋白细胞毒性产生的调控机理，并同时着重对蛋白定点泛素化合成的新方法进行了探索。本研究对理解淀粉样变性病病理机理具有重要意义，且为相应的治疗手段和药物的开发提供了理论基础。我们首先利用高效原子力显微镜和石英微天平技术，对胰岛淀粉样多肽(hIAPP)和胰岛素(Insulin)在五氧化二钽表面以不同比例共聚集的过程和形貌特征进行了研究，结果表明不同比率的hIAPP/Insulin在负电表面形成不同的聚集纳米结构，表现为成熟纤维-寡聚体纤维-寡聚体-前体原纤维-环状纤维等形式。石英晶体微天平表征结果显示蛋白共混后对表面的吸附作用更强。研究结果对进一步研究2型糖尿病的病理机理有重要的参考价值。我们利用多肽固相合成方法以及BC法、Ellman法和3-CCA法等荧光检测技术对Tau核心片段R3以及Ser324位磷酸化的片段pR3与铜离子作用产生氧化压力情况进行了研究。结果表明R3和pR3都具有还原铜的能力，R3和pR3在铜离子的介导下可以产生羟基自由基，而自身被氧化成由二硫键相连的二聚体，磷酸化会明显加速由R3与铜离子共同介导的活性氧物种产生的速率，这种加速效应会受到pH环境的影响，研究结果有助于我们更深入理解异常磷酸化在Tau病理学中的调控作用。此外，我们通过表达蛋白连接技术和抑制基因引发突变技术，成功获得了泛素蛋白硫酯，并通过生物表达方法将含叠氮基的非天然氨基酸成功引入到泛素蛋白单体的63位之中；我们利用泛素蛋白硫酯的高反应活性在泛素单体C端成功引入了巯基基团和双键基团；利用光催化的巯基－烯基加成反应将含双键的泛素蛋白Ub-AA与含巯基的小分子MESNA进行了偶联；我们利用DNTB试剂作为辅助基团，成功将泛素蛋白单体Ub-SH与淀粉样相关蛋白Prion聚集核心片段prp175-195通过二硫键进行偶联。研究结果为进一步建立具有多位点翻译后修饰蛋白质的合成方法以及生物功能的研究奠定了基础。更多还原

【Abstract】 Misfolding and abnormal aggregation of amyloid protein is an important cause forthe amyloidosis such as type2diabetes, Alzheimer’s disease and spongiformencephalopathy. The amyloid cytotoxicity mainly derived from the aggregation ofamyloid protein,the generation of oxidative stress, as well as the interaction with the cellmembrane. The process of cell toxicity generating will also be related withpost-translationally modified, the metal ions, the coexistence protein interactions as wellas a two-dimensional surface. In this paper, we focus on the effect of thephosphorylation and ubiquitination, metal copper ions, two-dimensional negativelycharged surface on the amyloid cytotoxicity.Moreover,we explored new methodology todesign and get the ubiquitinated protein.this will be helpful to understand the pathogenicmechanisms of the amyloidosis and develop new amyloidosis drugs.We Use high resolution liquid AFM and QCM-D to investigate thenanostructures by co-assembling hIAPP/insulin on Ta2O5surfaces, we have shownthat the structure and morphology of co-assembled aggregates on negativelycharged surfaces depend on the ratio of hIAPP/insulin. By tuning thehIAPP/insulin ratio, the nanostructure morphology changes from fibrils to oligomers, toannular. These findings provide new insights to understand insulin’s affact onthe structure of hIAPP aggregates on biomembranes.Furthermore, we investigated the generation of hydroxyl radicals when R3peptide was co-incubated with Cu(II). We also compared the redox activity ofR3with its phosphorylated form (pR3) at the site of Serine324. Phosphorylationat Ser324was found in paired helical filament in AD brains. Our resultsdemonstrated that both R3and pR3is able to reduce Cu(II). And the speed ofhydroxyl radical generation by R3and Cu(II) is significantly accelerated byphosphorylation. The effect of phosphorylation depends on pH condition.Thiseffect of phosphorylation on ROS generation by tau and copper implies a newperspective of its neurotoxicity.In addition,we use the expressed protein ligation and suppressor mutagenesistechnology to successfully get the ubiquitin thioester,and we also get the ubiquitin monomer containing a non-natural amino acid in the63site. We take advantage of thehigh reactivity of ubiquitin thioester to introduce the mercapto group and double bondgroup to the C terminal of the ubiquitin monomer. the protein Ub-AA coupled with theMESNA through the thiol-ene radical addition reaction under photocatalyticcondition.We use the DTNB reagent as a leaving group to get the Ub-SH monomercoupling with the Prion fragment PrP175-195via a disulfide bond.更多还原