IN VITRO MODEL OF PHENOTYPIC DIVERSITY IN PRION DISEASES

人类普恩病毒疾病是独一无二的，因为它们表现为零星的， 医源性和遗传性神经退行性疾病。他们 表达多种疾病表型，其特征是 存在一种异常的、对蛋白水解酶有抵抗力的蛋白亚型 蛋白质，PrPres。PrPres由与PRNP相同的基因编码 正常的、对蛋白酶敏感的普恩蛋白，PrPc。分子 人类蛋白酪氨酸病PrPc向PrPres转化的基础 在很大程度上是未知的。多重PRNP突变的发现 以及不同类型PrPres亚型的初步发现 疾病表型表明PrP结构的变化可能 这是普恩病毒疾病表型多样性的基础。我们的长期合作 目标是定义关键的确切结构特征 疾病状态下PrPres的形成及其意义 以便合理设计这些致命疾病的有效治疗方法。 自然发生的和致病的PRNP突变有 为我们提供了一个难得的机会来探索这种关系 PrPres结构异常与疾病的关系 它们引起的表型。本研究项目提出 从四个具体目标来审视这个问题。特定目标1交易 关于主要PrPres子类型的特征 对蛋白酶裂解位点和突变特异性的差异 化学修饰。PrPres将从人脑中提纯 家族性Pron病的受试者。初级结构和 各种PrPres亚型的潜在共价修饰将是 通过多种方法进行检测，包括酶消化， 高效液相色谱，N-末端蛋白 测序和质谱分析。在具体目标2中 具有不同突变的PrPres的二级结构将是 已定义。傅里叶变换等光谱方法 红外和圆二色谱将被用来表征和 比较PrPres亚型的构象特性。 具体目标3审查PrPc向PrPre和PrPre的转换 突变影响转换过程的机制。 无细胞转换系统将被用于研究这种形成 通过体外孵育放射性标记的PrPres， 用预先存在的、纯化的PrPres重组PrPc。特定的 目标4重点研究了截断和截断 可能导致淀粉样变性的PrP衍生物，由 突变体PrP的细胞加工。人神经母细胞瘤细胞 表达突变体PrP将用于识别截断形式 SDA/PAGE和免疫印迹分析PrP的表达。被识别的人 PRP衍生物的提纯和N-末端的表征 测序和质谱分析。这些研究将导致 更好地理解表型的分子基础 人类普恩病毒疾病的多样性。

Human prion diseases are unique in that they manifest as sporadic, iatrogenic and inherited neurodegenerative disorders. They express a variety of disease phenotypes characterized by the presence of an abnormal, protease-resistant isoform of the prion protein, PrPres. PrPres is encoded by the same PRNP gene as the normal, protease-sensitive prion protein, PrPc. The molecular basis for the conversion of PrPc to PrPres in human prion disease are largely unknown. The discovery of multiple PRNP mutations and the initial findings of distinct PrPres subtypes in different disease phenotypes suggest that changes in the PrP structure may underlie the phenotypic diversity in prion diseases. Our long-term objective is to define the exact structural features that are critical to the formation of PrPres in the disease state, with implications for rational design of effective treatment for these fatal disorders. The naturally occurring and disease-causing PRNP mutations have provided us with a rara opportunity to probe the relationship between the structural abnormalities of PrPres and the disease phenotypes they cause. The present research project proposes to examine this issue in four specific aims. Specific Aim 1 deals with the characterization of the major PrPres subtypes with respect to the differences in protease cleavage sites and mutation-specific chemical modifications. PrPres will be purified from brains of subjects with familial prion diseases. The primary structure and potential covalent modifications of various PrPres subtypes will be examined by multiple approaches, including enzymatic digestion, high performance liquid chromatography, N-terminal protein sequencing and mass spectrometry. In Specific Aim 2 the secondary structure of PrPres with different mutations will be defined. Spectroscopic methods such as Fourier transform infrared and circular dichroism will be used to characterize and compare the conformational properties of PrPres subtypes. Specific Aim 3 examines the conversion of PrPc to PrP res and the mechanism by which mutations affect the conversion process. A cell-free conversion system will be used to study the formation of nascent PrPres by in vitro incubation of radiolabeled, recombinant PrPc with the pre-existing, purified PrPres. Specific Aim 4 focuses on the characterization of the truncated and potentially amyloidogenic PrP derivatives that result from the cellular processing of the mutant PrP. Human neuroblastoma cells expressing the mutant PrP will be used to identify truncated forms of PrP by SDA/PAGE and immunoblot analysis. The identified PrP derivatives will be purified an characterized by N-terminal sequencing and mass spectrometry. These studies will lead to a better understanding of the molecular basis for phenotypic diversity in human prion diseases.