YEAST PRION DYNAMICS AND CHAPERONE INTERACTIONS

蛋白质构象和组装的变化控制着细胞生物学中的大多数过程。原子或扫描力显微镜(AFM)是一种新的成像手段，它具有强大而独特的能力来直接研究这些过程，因为它具有高空间分辨率(原子在硬表面上)，扩展的作用力范围(高达10-15N)，足以测量几乎任何分子相互作用，纳米级控制，能够被功能化，从而充当特定生物分子的非常特定的传感器，在操作和制备中相对缺乏破坏性，并能够对干燥样品和生理溶液中的样品进行成像。这些能力使原子力显微镜有别于传统的成像模式，并开辟了一种新的结构生物学方法。这项建议汇集了苏珊·林德奎斯特的实验室和阿恩斯多夫实验室的专业知识，前者是研究细胞质遗传因素的领先者之一，后者一直在为生物医学研究开发原子力显微镜。其中最有趣的细胞质遗传基因元件是感染性监狱蛋白，它会导致牛的海绵状脑病等传染性海绵状脑病，以及人类的各种逐渐致命的神经退行性疾病。Prion蛋白通常存在于细胞膜中，最近的研究支持了这样的假设，即构象异常的这种蛋白是导致这些疾病的感染性颗粒。酵母中两个细胞质遗传的遗传元件通过类似的机制繁殖，导致翻译保真度或氮代谢的可遗传变化。[PSI+]是酵母蛋白中最具特性的一种，被认为是翻译终止装置的一个亚基--核编码的Sup35蛋白的一种自持性构象变化。[PSI+]元件的繁殖依赖于特定浓度的热休克蛋白104(HSP104)，热休克蛋白104充当伴侣，但有些自相矛盾的是，过表达HSP104可以“治愈”[PSI+]元件的细胞，并恢复正常的翻译保真度。我们将用原子力显微镜研究：(1)Sup35的表面结构、其组成的亚基“积木”、可能的中间体和聚集体；(2)(1)的时间进程；(3)亚基聚合的自恒定、替代模式及其与应变变化的关系；(4)分子和结构之间的相互作用力，包括(1)中形成纤维的亚基，并评估生理和其他干预对这种相互作用的影响；(5)当Hsp104暴露于Hsp104时，Hsp104颗粒和Sup35的表面形貌；(6)当Hsp104充分过表达时，Sup35的结构随时间发生变化，从而“治愈”[PSI+]元件的细胞；(7)蛋白质的物理特性。

Changes in the conformation and assembly of proteins governs most processes in cell biology. The atomic or scanning force microscope (AFM) is a new imaging modality that has powerful and unique capabilities to study these processes directly because of its high spatial resolution (atomic on hard surfaces), extended force range (up to 10-15N) sufficient to measure virtually any molecular interaction, nanoscale control, capability of being functionalized so it acts as a very specific sensor of specifici biomolecules, relative lack of destructiveness in operation and preparation, and the ability to image specimens dry and in physiologic solutions. These capabilities set AFM apart from traditional imaging modalities and open up a new approach to structural biology. This proposal brings together the expertise of Susan Lindquist's laboratory which is among the leaders in investigating cytoplasmically inherited genetic elements and the Arnsdorf Laboratory which has been developing the AFM for biomedical research. Among the most interesting cytoplasmically inherited genetic elements are the infective prison proteins that cause the transmissible spongiform encephalopathies such as "mad cow disease" in cattle and a variety of progressively lethal neurodegenerative diseases in man. The prion protein is normally found in the cell membrane, and recent investigations support the hypothesis that conformationally abnormal forms of this protein are the infectious particles responsible for these diseases. Two cytoplasmically inherited genetic elements in yeast propagate by a similar mechanism producing heritable changes in translational fidelity or nitrogen metabolism. [PSI+] is the best characterized yeast prion and is believed to be a self-perpetuating conformational alteration in the nuclear-encoded Sup35 protein, a subunit of the translation-termination apparatus. Propagation of the [PSI+] element depends on a specific concentration of heat- shock protein 104 (Hsp 104) which acts as a chaperone, but somewhat paradoxically overexpression of HSP104 can "cure" cells of the [PSI+] element and restore normal translational fidelity. We will investigate with the AFM: (1) the surface structure of Sup35, its component subunit "building blocks", possible intermediates, and aggregates; (2) the time course of (1); (3) the self-perpetuating, alternative modes of subunit polymerization and its relationship to strain variation in [PSI+]; (4) the forces of interactions between molecules and structures including the subunits that form the fibers in (1) and to assess the effects of physiologic and other interventions on such interactions; (5) surface topography of HSP104 particles and of Sup35 when exposed to Hsp104, (6) follow in time structural changes in Sup35 when Hsp104 is sufficiently overexpressed so as to "cure" cells of the [PSI+] element, and (7) physical characteristics of the proteins.