Investigation of the yeast prion factor (PSI+)

描述（由申请人提供）：某些蛋白质可以以不同的遗传构象存在。在它们的“朊病毒”构象中，它们形成淀粉样聚集体，并将同一蛋白质的非朊病毒分子连接到聚集体上。由于蛋白质构象的类似改变与神经退行性疾病（如阿尔茨海默氏症、亨廷顿氏症、帕金森病和克雅氏病）有关，探索决定朊病毒生物学的潜在机制将有助于更好地了解这些疾病的病因。这种新的朊病毒表观遗传变异机制存在于酵母中。新的酵母朊病毒的研究将阐明遗传蛋白构象的不同作用以及朊病毒之间的关系。候选朊病毒已经并将从遗传和蛋白质组学筛选中检索出来。转录调控和染色质重塑复合体的成员Cyc8和Swi1这两个候选分子的磷酸化将对细胞产生全局影响。一种可能与记忆储存（CPEB）有关的朊病毒候选蛋白和酵母[PSI+]朊病毒蛋白的哺乳动物同源物也将在酵母系统中进行研究。奇怪的是，在哺乳动物和酵母中存在不同的遗传“菌株”或单个朊病毒的变体（具有相同的初级序列）。通过研究体外制备的变异体特异性酵母朊病毒纤维的物理性质和结构，以及鉴定细胞中与朊病毒聚集体相关的蛋白质，将探讨变异体特异性表型差异的原因。一种朊病毒的变体可以“交叉播种”另一种朊病毒的重新形成的效率将在体外使用变体特异性纤维进行测试。将对从细胞中提取的朊病毒聚集体的亚颗粒进行感染性和变异特异性测试，以了解这些亚种是否具有传染性。矛盾的是，某些朊病毒变体可以增强异源[PSI+]朊病毒的新生外观，同时也消除了已经存在的[PSI+]。为了研究这一点，我们将研究在固化过程中[PSI+]“种子”向子细胞的分离。类似的实验将检验异种富qn聚集体破坏[PSI+]的机制。为了研究在没有感染的情况下，朊病毒种子是如何首先产生的，将确定增强或抑制朊病毒新生出现或繁殖的基因，并确定它们在朊病毒生物发生中的作用。这些可能包括参与蛋白质折叠和降解的蛋白质，以及其他。最后，通过体外分析比较了朊病毒的繁殖机制和新生朊病毒种子的形成机制。

DESCRIPTION (provided by applicant): Certain proteins can exist in distinct heritable conformations. In their "prion" conformation, they form amyloid-like aggregates and seed the joining of non-prion molecules of the same protein to the aggregates. Since similar alterations in protein conformations are associated with neurodegenerative diseases such as Alzheimer's, Huntington's, Parkinson's and Creutzfeldt-Jacob disease, exploring the underlying mechanisms that dictate prion biology will provide a better understanding of the etiology of these diseases. This novel prion mechanism of epigenetic variation exists in yeast. Investigations of new yeast prions will clarify the different roles of heritable protein conformations and the relationships between prions. Prion candidates have been, and will be, retrieved from genetic and proteomic screens. Prionization of two candidates, Cyc8 and Swi1, members of transcriptional regulation and chromatin remodeling complexes, would have global effects on the cell. A prion-protein candidate that may be involved with memory storage (CPEB) and mammalian homologs of the yeast [PSI+] prion protein will also be studied in the yeast system. Curiously, different heritable "strains" or variants of individual prions (with identical primary sequences) exist in both mammals and yeast. The causes of variant specific phenotypic differences will be investigated by studying the physical properties and structures of variant-specific yeast prion fibers made in vitro and by identifying the proteins associated with prion aggregates in cells. The efficiencies with which variants of one prion can "cross-seed" the de novo formation of another prion will be tested in vitro using variant specific fibers. Subparticles of prion aggregates extracted from cells will be tested for infectivity and variant specificity to learn if these subspecies are infectious. Paradoxically, certain prion variants that enhance the de novo appearance of the heterologous [PSI+] prion, also eliminate already existing [PSI+]. To investigate this we will examine the segregation of [PSI+] "seeds" into daughter cells during the curing process. Similar experiments will examine the mechanism by which heterologous QN-rich aggregates destabilize [PSI+]. To investigate how prion seeds first arise in the absence of infection, genes that enhance or inhibit de novo appearance or propagation of a prion will be identified and their roles in prion biogenesis will be defined. These are likely to include proteins involved in protein folding and degradation, and others. Finally, the mechanisms of prion propagation and de novo prion seed formation will be compared by in vitro analysis.