翻译后修饰对人朊蛋白致病结构形成的调控

Transmissible spongiform encephalopathies (TSEs) or prion diseases, including Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep, are infectious and fatal neurodegenerative diseases. The transmission of TSEs between humans and many mammals has posed a serious threat to human health. The cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI) anchored glycoprotein and the conformational conversion of PrPC into its pathogenic isoform PrPSc is believed to be the key event leading to the prion diseases. In the present study, we employ biophysical and cell biological tools to investigate the regulation of the formation of disease-causing structure of human prion protein (PrP) and its pathological mutants by glycosylation, GPI anchoring, and crowded physiological environments, and to compare the molecular biological effect and function of N-glycosylated and GPI-anchored PrP in crowded physiological environments or in dilute solutions with those of unglycosylated PrP. We then observe the location of human PrP aggregates formed either on the cell surface or inside the cell, and detect whether these posttranslational modifications and/or crowded physiological environments have a remarkable enhancing effect on proteinase K resistance activity of human PrP fibrils and whether glycosylation and GPI anchoring mediate prion cytotoxicity and prion infection. We finally employ solid-state NMR, transmission electron microscopy, and atomic force microscopy to determine and compare the structures of aggregates of N-glycosylated and GPI-anchored PrP and unglycosylated PrP, formed either in crowded physiological environments or in dilute solutions, to explain how glycosylation and macromolecular crowding corporately affect the molecular mechanism underlying the formation of disease-causing structure of human PrP, to understand the role of glycosylation and GPI anchoring in the pathogenesis of prion diseases, and to test the "protein-only" hypothesis of prion propagation. Information obtained from this study can enhance our understanding of how posttranslational modifications and/or crowded physiological environments regulate the formation of disease-causing structure of human PrP in vivo, and should be useful in the development of new therapeutic regents for prion diseases.

传染性海绵体脑病(又称prion疾病)是一种能够在人类与许多哺乳动物之间相互感染的致死性神经退行性疾病，其极强的传染性给人类健康带来了重大威胁。本项目以人朊病毒蛋白及其病理突变体为研究对象，研究糖基化修饰、糖基磷脂酰肌醇定位修饰以及生理拥挤环境对人朊蛋白致病结构形成的调控机制，比较生理拥挤环境和稀溶液中翻译后修饰的人朊蛋白与无修饰的人朊蛋白的分子生物学效应和功能差异，观察翻译后修饰的人朊蛋白聚集体在细胞中的定位，检测这些修饰是否显著提高人朊蛋白纤维对蛋白酶K抗性、是否调控朊病毒蛋白的细胞毒性和感染性，用固体核磁共振、透射电子显微和原子力显微等技术在高级结构水平上研究翻译后修饰和生理拥挤环境调控人朊蛋白致病结构的分子基础，阐释糖基化和GPI修饰在人朊蛋白致病结构形成过程中的作用，进而验证朊病毒蛋白protein-only假说，也为prion疾病的发病机制、诊断方法和药物开发提供新的理论依据。

传染性海绵体脑病（又称prion疾病）是一种能够在人类与许多哺乳动物之间相互感染的致死性神经退行性疾病，其极强的传染性给人类健康带来了重大威胁。糖基化修饰是朊蛋白（PrP）最重要的翻译后修饰之一。我们通过confocal实验发现，野生型人朊病毒蛋白定位于细胞膜上，而人PrP糖基化位点附近的病理突变体V180I和F198S则主要定位在细胞质中。我们又构建了糖基化位点单突N181D和N197D、双突N181/197D、三突N181/197D/T199N和糖基磷脂酰肌醇（GPI）突变体，发现N181D、N197D和三突N181/197D/T199N都主要定位于细胞膜上，而N181/197D和GPI突变体则定位在细胞质中。因此GPI定位修饰在PrP的亚细胞定位上发挥着决定性的作用，但N-糖基化修饰对PrP的亚细胞定位也是不可或缺的。我们通过PK梯度酶解、超离和流式细胞实验发现，跟定位于细胞膜上的野生型人朊蛋白相比，定位在细胞质中的N181D/N197D、V180I和F198S更容易发生异常积聚，具有更强的蛋白酶K抗性。因此人朊蛋白N-糖基化程度越低，其对PK的抗性就越强，PrP就越容易积聚，所产生的氧化压力和神经细胞毒性也会越大，越容易诱发prion疾病。我们还发现病理浓度锌离子和野生型人朊病毒蛋白紧密结合，显著改变聚集体构象；然而锌离子只能和八肽重复序列缺失突变体PrPdeltaocta较弱地结合，对聚集体构象也没有明显影响。因此锌离子改变PrP积聚途径和聚集体结构主要通过其与PrP八肽重复序列相结合来实现。我们建立了病理浓度锌离子调控类朊病毒蛋白Tau磷酸化修饰和积聚的细胞模型，锌离子不仅通过与Tau蛋白结合直接促进其积聚，更能对Tau蛋白异常的翻译后修饰起调控作用而进一步促进Tau蛋白的积聚及毒性；纤维形成片段决定了Tau蛋白具有生长纤维、诱导神经细胞凋亡和诱发阿尔茨海默病的能力。上述研究从PrP积聚和细胞毒性两个方面阐释了PrP糖基化修饰与prion疾病之间的关系，揭示了锌离子发挥毒性作用的机理和锌离子修饰朊病毒株型的分子机制，揭示了糖基化修饰调控PrP积聚和细胞毒性的分子机制及其在人朊蛋白致病结构形成过程中的作用，验证了朊病毒蛋白protein-only假说，并有助于对prion疾病的预防、早期诊断和治疗。