Self-Propagating Mechanism of Prion Diseases

描述（由申请人提供）：朊病毒蛋白（PrP）是一系列疾病的基础，没有既定的治疗方法，对人类和经济造成毁灭性的后果。朊病毒疾病与其他神经退行性疾病有几个独特的特征。(i)朊病毒蛋白有一种感染性的同种型PrPSc，它以正常同种型PrPC作为底物，以自催化的方式传播其异常构象。(ii)繁殖过程要求PrPC和PrPSc的氨基酸序列相同（或高度同源）。(iii)在相同的一级结构中，PrP能够在转化为病理性同种型时采用构象上不同的状态，其被称为PrPSc的“菌株”。这些特征表明朊病毒疾病具有独特的分子机制，不同于与蛋白质聚集相关的其他疾病。我们的一般策略是集中在这些功能的PrPSc传播作为理解朊病毒疾病的机制的关键。待检验的假设是这些区别特征是否是朊病毒蛋白的固有特性。特别是，PI将检查非糖基化重组PrP的聚集过程在多大程度上模拟细胞环境外朊病毒疾病的基本特征。使用最先进的生物化学和生物物理学方法，将研究自繁殖过程的三个方面：（1）自繁殖聚集的动力学途径，特别强调转化的早期事件和限速步骤;（2）聚集的自催化机制的生物物理性质;（3）自繁殖聚集体的构象多样性和繁殖的保真度。在拟议的工作中，将使用一系列生物物理工具：蛋白酶K消化结合质谱法，免疫构象测定，分析尺寸排阻色谱与独特的超灵敏检测，光谱技术，动态光散射和电子显微镜。拟议的研究工作将揭示自我传播的机制，将揭示蛋白质折叠的新规则，这些规则控制特定的聚集，并定义使PrP在蛋白质中独一无二的特征。这些知识应该为治疗朊病毒疾病的新分子和药理学方法奠定基础。

DESCRIPTION (provided by applicant): Prion protein (PrP) underlies a spectrum of diseases with no established treatment and devastating human and economic consequences. Several unique features separate prion diseases from other neurodegenerative maladies. (i) There is an infectious isoform of the prion protein, PrPSc, that propagates its abnormal conformation in an autocatalytic manner using the normal isoform, PrPC, as a substrate. (ii) The process of propagation requires the amino acid sequences of PrPC and PrPSc to be identical (or highly homologous). (iii) Within the same primary structure, PrP is capable of adopting conformationally distinct states when it is converted into pathological isoforms, which are known as "strains" of PrPSc. These features indicate that prion diseases have a unique molecular mechanism, distinct from other disorders related to protein aggregation. Our general strategy is to concentrate on these features of PrPSc propagation as a key for understanding the mechanism of prion disease. The hypothesis to be tested is whether these distinguishing features are inherent properties of the prion protein. In particular, the PI will examine the extent to which the process of aggregation of non-glycosylated recombinant PrP mimics basic hallmarks of prion diseases outside of the cellular environment. Using state-of-the-art biochemical and biophysical methods, three aspects of the self-propagating process will be investigated: (1) the kinetic pathway of self-propagating aggregation, with special emphasis on the early events and the rate-limiting step of conversion; (2) the biophysical nature of the autocatalytic mechanism of aggregation; (3) the conformational diversity of self-propagating aggregates and fidelity of propagation. In the proposed work, an array of biophysical tools will be used: proteinase K digestion combined with mass spectrometry, immunoconformational assay, analytical size-exclusion chromatography with unique ultra sensitive detection, spectroscopic techniques, dynamic light scattering, and electron microscopy. The proposed research work will shed light on the mechanism of self-propagation, will reveal novel rules of protein folding that govern specific aggregations, and define the features that make PrP unique among proteins. Such knowledge should lay the foundation for novel molecular and pharmacological approaches for treating prion diseases.