Structural Determinants of Mammalian Prion Aggregation

项目摘要： 朊病毒病是一种神经退行性疾病，对人类和环境健康构成威胁。这些 疾病有遗传和非遗传原因，可以通过环境获得，并有能力保持 在没有宿主生物体的情况下长时间具有传染性。错误折叠朊病毒蛋白的聚集 （PrPSc）与疾病相关，但聚集背后的机制尚未完全了解，严重 阻碍了预防纤维性颤动和疾病的治疗方法的发展。哺乳动物的β 2 α 2环 朊病毒蛋白先前已被证明是涉及疾病传播的关键区域。一 一个来自9个残基片段的晶体结构，编码了田鼠朊病毒的β 2 α 2环， 全长朊病毒纤维结构特征和稳定性特征。在此基础上， 将追求传递关于稳定性和感染性的信息的PrP聚集体的结构。实现这一 目标，有序聚集的PrP片段，已被描述为在疾病传播中发挥作用， 具有朊病毒疾病易感性范围的各种物种将被生物化学和结构表征。 将重组表达、纯化和纯化并入疾病调节区的构建体， 评估稳定性、蛋白酶K抗性和纤维形态。这些属性将与 在来自患病动物的原纤维中观察到的那些。结合晶体学和单粒子 Cryo-EM将用于确定每个错误折叠PrP的原子排列。这些目标将是 通过应用大分子晶体学的前沿方法，包括电子 微衍射（MicroED）。由此产生的结构将提供一个近三个分子的解释- 这是朊病毒生物学和蛋白质病理学中百年之谜，将有助于区分传染性和非传染性 传染性淀粉样蛋白，揭示了朊病毒传播障碍的结构密码。 !

Project Summary: Prion diseases are neurodegenerative disorders that pose a threat to human and environmental health. These illnesses have genetic and nongenetic causes, can be environmentally acquired, and have the ability to remain infectious in the absence of a host organism for extended periods. Aggregation of misfolded prion protein (PrPSc) correlates with disease, but the mechanism behind aggregation is not fully understood, gravely hindering the development of therapeutics to prevent fibrillation and sickness. The β2α2 loop of mammalian prion protein has previously been demonstrated to be a key region implicated in disease transmission. A crystal structure from a nine-residue segment encoding the β2α2 loop of the bank vole prion has demonstrated structural characteristics and stability characteristic of full-length prion fibers. Building on this structure, structures of PrP aggregates that convey information on stability and infectivity will be pursued. To achieve this goal, ordered aggregates of PrP segments that have been described to play a role in disease transmission in various species with a range of prion disease susceptibility will be biochemically and structurally characterized. Constructs incorporating disease modulating regions will be recombinantly expressed, purified, and fibrillized to assess stability, proteinase K resistance, and fiber morphologies. These properties will be compared against those observed in fibrils derived from diseased animals. A combination of crystallography and single particle Cryo-EM will be used to determine the atomic arrangement of each misfolded PrP. These aims will be achieved through the application of frontier methods in macromolecular crystallography including electron micro-diffraction (MicroED). The resulting structures will provide a molecular explanation for a nearly three- hundred-year-old mystery in prion biology and protein pathology and will help distinguish infectious from non- infectious amyloids, revealing structural code for prion transmission barriers. !