The Role of Competitive Forces in Prion Propagation and Appearance

描述(申请人提供)：根据普恩假说，一种正常的、宿主编码的蛋白质的另一种构象，称为普恩，可以作为体内可传递表型状态的表观遗传决定因素。这一过程曾被认为是非典型的，但已与一系列以前令人费解的生物现象有关，例如可传播海绵状脑病(TSE)的病原学和真菌中一组亚稳定性状的非孟德尔遗传，这表明这是一个广泛的过程，有机体可以通过它获得并保持平行的表型状态。虽然这些表型的纯蛋白质基础已经被很好地记录下来，但令人惊讶的是，我们对它们之间如何发生转换的了解有限，尽管它们对正常的细胞生理学具有重要意义。我们的长期目标是揭示在活体中引发蛋白转变的途径。为了获得这一见解，我们将利用酿酒酵母Sup35/[PSI+]Prion的可操纵性。在这个系统中，也可能在哺乳动物中，Pron的传播是通过蛋白质生物发生的多步骤途径发生的，该途径既受Pron的固有属性的影响，也受细胞环境的影响。[PSI+]繁殖通常是有效的，但许多情况下，与哺乳动物相似，诱导表型转换。这项建议的总体目标是将经验研究和计算研究相结合，以确定体内病毒传播途径的哪一步(S)会被这些操作以及这些变化引发开关的细胞途径所改变。到目前为止，大多数分析只从普恩的角度考虑这些影响，但我们建议的研究考虑它们在整个系统的背景下的作用。虽然诱导普鲁恩相关表型转换的条件将不同构象或序列变体引入细胞，但竞争力在普恩转换中的作用从未被探索过。我们假设，Pron传播途径中的关键步骤应该被认为是受制于竞争性力量的酶限制过程，这可以有效地引起表型转变。为了直接验证这一假说，我们将确定：1)显性-负性突变体治愈普恩的途径，2)普恩变异建立显性的途径，以及3)在第二个普恩从头出现时需要一个已存在的普恩的分子基础。通过这个独特的视角，我们将开始揭示Pron相关表型之间潜在的细胞路径，这是Pron生物学中一个关键但知之甚少的方面。

DESCRIPTION (provided by applicant): According to the prion hypothesis, an alternative conformation of a normal, host-encoded protein, known as a prion, can function as an epigenetic determinant of transmissible phenotypic states in vivo. Once considered atypical, this process has been linked to an expanding range of previously enigmatic biological phenomena, such as the etiology of the transmissible spongiform encephalopathies (TSEs) and the non-Mendelian inheritance of a group of metastable traits in fungi, suggesting it is a widespread process through which organisms can access and perpetuate parallel phenotypic states. While the protein-only basis of these phenotypes is well documented, we have surprisingly limited insight into how transitions between them occur, despite their significance for normal cellular physiology. Our long-term goal is to reveal the pathways through which prion transitions are evoked in vivo. To gain this insight, we will exploit the manipulability of the Sup35/[PSI+] prion of S. cerevisiae. Prion propagation in this system, and also likely in mammals, occurs through a multi-step pathway of protein biogenesis that is influenced by both the inherent properties of the prion and by aspects of the cellular environment. [PSI+] propagation is normally efficient, but many conditions, with parallels in mammals, induce phenotypic transitions. The overall objective of this proposal is to combine empirical and computational studies to determine which step(s) of the in vivo prion propagation pathway are altered by these manipulations and the cellular pathways through which these changes evoke the switch. To date, most analyses have considered these effects from the perspective of the prion alone, but our proposed studies consider their action in the context of the entire system. While conditions that induce transitions between prion-associated phenotypes introduce different conformational or sequence variants of the prion into cells, the role of competitive forces in prion transitions has never been explored. We hypothesize that the crucial steps in the prion propagation pathway should be considered as enzyme-limited processes that are subject to competitive forces, which can effectively evoke phenotypic transitions. To directly test this hypothesis, we will determine: 1) the pathways by which dominant-negative mutants cure prions, 2) the pathways by which prion variants establish dominance, and 3) the molecular basis of the requirement for an existing prion in the de novo appearance of a second prion. Through this unique perspective, we will begin to reveal the cellular pathways underlying transitions between prion-associated phenotypes, a crucial yet poorly understood aspect of prion biology.