Investigation of the yeast prion factor [PSI+]

描述（由申请人提供）：不同细胞蛋白错误折叠成淀粉样聚集体与非感染性神经退行性疾病有关，包括阿尔茨海默氏病、亨廷顿氏病和帕金森病，以及传染性朊病毒疾病，如疯牛病、鹿的慢性消耗症和人类的克雅氏病。对于这些疾病中的每一种，相关的蛋白质聚集体（“种子”）吸引其正常的构象错误折叠并加入聚集体。简单真核生物酵母中的某些蛋白质同样可以错误折叠成传染性淀粉样蛋白聚集体，这些聚集体导致表观遗传变异。在这个提议中，酵母遗传学和分子生物学的力量被用来研究蛋白质是如何错误折叠成淀粉样聚集体的，以及这种错误折叠对细胞的影响。酵母和人类细胞之间的广泛相似性使得酵母模型在理解人类疾病方面做出了重大贡献，这意味着这些研究可能与人类错误折叠的聚集蛋白有关。由于大多数人类蛋白质错误折叠疾病在没有任何外部种子感染的情况下发生，Aim I侧重于围绕自发细胞淀粉样蛋白形成的分子机制。解决的问题是：新出现的朊病毒聚集体首先在细胞中的哪里出现，与它们相关的其他蛋白质是什么，以及先前存在的朊病毒如何增强异种朊病毒的新生外观？Aim I还验证了一个假设，即哺乳动物亨廷顿蛋白相互作用蛋白的酵母同源物Sla2，通过结合种子和待播种蛋白，从而使它们靠近，促进了现有朊病毒交叉播种异种朊病毒蛋白重新聚集的能力。有趣的是，即使蛋白质的氨基酸序列相同，人类和酵母朊蛋白也可以形成结构不同的淀粉样蛋白聚集体的多种变体，并导致不同的表型或疾病病理。Aim II鉴定与几种朊病毒及其变异结合和/或繁殖所需的蛋白质。此外，在合作者的帮助下，将确定同一朊病毒的两个变体的固态核磁共振结构。通过比较异种朊病毒，以及同一朊病毒的不同变体，将确定所有朊病毒的维持和传染性可能共有的因素，因此应该提供有用的药物靶点。虽然淀粉样蛋白的形成与疾病有关，但病理的实际原因尚不清楚。在Aim III中，对两种导致酵母毒性的朊病毒的遗传和分子研究将有助于确定有毒物种。最后，酵母朊病毒的重要性不仅在于它是人类疾病的模型，还在于它提示了在蛋白质构象水平而非核酸水平上运行的一种重要的遗传变异新机制。在Aim IV中，探讨了朊病毒是否有时可以为宿主细胞提供优势的有趣问题，以及这种有利的朊病毒也可能存在于哺乳动物中的可能性。公共卫生相关性：酵母遗传学和分子生物学的力量将用于研究蛋白质错误折叠成淀粉样聚集体，如与几种破坏性神经退行性人类疾病相关的淀粉样聚集体，包括阿尔茨海默氏症、帕金森病、亨廷顿氏病和克雅氏病。酵母和人类细胞之间的广泛相似性使得酵母模型在理解其他人类疾病方面做出了重大贡献，这意味着从这些研究中获得的见解将有助于选择人类疾病的药物靶点。

DESCRIPTION (provided by applicant): The misfolding of different cellular proteins into amyloid-like aggregates is associated with non- infectious neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's, as well as with the infectious prion diseases e.g. Mad Cow, Chronic Wasting in deer and Creutzfeldt-Jacob in humans. For each of these diseases, the associated protein aggregate ("seed') attracts its normal conformers to misfold and join the aggregate. Certain proteins in the simple eukaryote yeast, can likewise misfold into infectious amyloid aggregates, and these aggregates cause epigenetic variation. In this proposal, the power of yeast genetics and molecular biology is used to study how proteins misfold into amyloid-like aggregates and the consequences of this misfolding for the cell. The extensive similarity between yeast and human cells, which has enabled yeast models to make significant contributions in understanding human disease, implies that these studies will likely be relevant to misfolded aggregating proteins in humans. Since most human protein misfolding diseases occur without infection by any external seed, Aim I focuses on the molecular mechanisms surrounding spontaneous cellular amyloid formation. The questions addressed are: where do newly appearing prion aggregates first arise in cells, what other proteins are associated with them, and how do pre-existing prions enhance the de novo appearance of heterologous prions? Aim I also tests the hypothesis that Sla2, the yeast homolog of the mammalian huntingtin interacting protein, facilitates the ability of existing prions to cross-seed the de novo aggregation of heterologous prion proteins, by binding to both the seed and protein to be seeded, thereby placing them in close proximity. Interestingly, human and yeast prion proteins can each form multiple variants of amyloid aggregates that differ in structure and cause distinct phenotypes or disease pathologies, even though the amino acid sequences of the proteins are identical. Aim II identifies proteins bound to, and/or required for, the propagation of several prions and their variants. In addition, solid-state NMR structures of two variants of the same prion will be determined with the help of collaborators. By comparing heterologous prions, as well as different variants of the same prion, factors likely to be common to the maintenance and infectivity of all prions and that should therefore provide useful drug targets, will be identified. While amyloid formation is associated with disease, the actual cause of pathology is unclear. In Aim III, genetic and molecular studies of two prions that cause toxicity in yeast will help define the toxic species. Finally, yeast prions are important not only as a model for human disease, but also because they suggest an important new mechanism of genetic variation operating at the level of protein conformation rather than nucleic acids. In Aim IV the fascinating question of whether prions can sometimes provide the host cell with an advantage is explored, along with the possibility that such advantageous prions may also exist in mammals. PUBLIC HEALTH RELEVANCE: The power of yeast genetics and molecular biology will be used to study the misfolding of proteins into amyloid-like aggregates like those associated with several devastating neurodegenerative human diseases including Alzheimer's, Parkinson's, Huntington's and Creutzfeldt-Jacob's diseases. The extensive similarity between yeast and human cells, which has enabled yeast models to make significant contributions in understanding other human diseases, implies that the insights gained from these studies will be helpful when choosing drug-targets for the human diseases.