Investigating the Mechanism of Prion Curing by [PSI+] No More Mutations

描述(由申请人提供)：蛋白质的折叠、定位和溶解是基本的过程，对于正常的细胞功能以及生物体的生长和发育是必不可少的；然而，确保这些活动忠实执行的质量控制机制随着年龄的增长而越来越受到损害。毫不奇怪，当这些正常的蛋白质生物发生途径出错时，可能会产生毁灭性的后果。例如，与年龄相关的蛋白质错误折叠疾病，包括阿尔茨海默氏症、帕金森氏病和亨廷顿病，以及一组独特的被称为传染性海绵状脑病的普恩疾病[1，2]，每一种疾病都与严重的神经退化有关，这些疾病是由正常的宿主编码蛋白质的自我永久错误折叠引起的。在健康的个体中，这些蛋白质是可溶的和有功能的，但在患病的个体中，相同的蛋白质错误折叠并形成高度结构化的聚集体，这些聚集体似乎充当正常蛋白质继续错误折叠的模板，从而促进它们自己的组装。萌芽酵母酿酒酵母表达几种蛋白，在类似的自我模板化过程中，这些蛋白也在可溶性和聚集态之间分配。然而，在这些情况下，这些蛋白质的Prion形式与疾病无关，而是作为表观遗传剂来指导新特征的遗传[3]。考虑到酵母在实验中的易操纵性和Pron状态的生存能力，这个系统提供了一个宝贵的平台，可以用来剖析体内蛋白质构象自我复制变化的潜在机制。从大量的体内研究中，我们已经收集到了有效繁殖PrP所需的顺式序列的详细信息。该因子的Prion结构域上的几个突变主要干扰了野生型蛋白质对Prion的有效传播，这表明这些变异专门针对这一过程中的关键步骤。为了深入了解这些事件，我建议通过体内和体外分析相结合的方法来阐明这些变体消除错误折叠形式的机制。总之，这些研究将确定赋予蛋白质自我复制特性的关键性质，进而突出哺乳动物蛋白质错误折叠疾病的潜在治疗干预点。

DESCRIPTION (provided by applicant): The folding, localization, and solubility of proteins are basic processes, essential for proper cellular function as well as organismal growth and development; yet the quality control mechanisms that ensure faithful execution of these events become increasingly compromised with age. Not surprisingly, when these normal pathways of protein biogenesis go awry there can be devastating consequences. For example, the age related protein misfolding diseases, including Alzheimer's, Parkinson's and Huntingdon's diseases, as well as a set of unique prion diseases termed the transmissible spongiform encephalopathy's [1, 2] are each associated with severe neurodegeneration resulting from the self-perpetuated misfolding of a normal, host encoded protein. In healthy individuals, these proteins are soluble and functional, but in afflicted individuals, the same protein misfolds and forms highly structured aggregates which appear to act as templates for the continued misfolding of normal protein, thereby promoting their own assembly. The budding yeast Saccharomyces cerevisiae expresses several prion proteins that also partition between soluble and aggregated forms in an analogous, self-templated process. In these cases, however, the prion form of these proteins is not associated with disease but instead acts as an epigenetic agent to direct the inheritance of a novel trait [3]. Given the experimental tractability of yeast and the viability of the prion state, this system provides an invaluable platform with which to dissect the mechanisms underlying self-replicating changes in protein conformations in vivo. Detailed information on the cis sequence requirements for efficient prion propagation has been gleaned from a number of in vivo studies on the yeast prion protein Sup35. Several mutations in the prion domain of this factor dominantly interfere with efficient prion propagation by the wildtype protein, suggesting that these variants specifically target crucial steps in the process. To gain insight into these events, I propose to elucidate the mechanisms by which these variants eliminate the misfolded form through a combination of in vivo and in vitro analyses. Together, these studies will define the key properties that endow a protein with self-replicating character and by extension highlight potential points of therapeutic intervention for the mammalian protein misfolding diseases.