Investigation of the Yeast Prion Factor, [PSI+]

“纯蛋白质”（朊病毒）遗传，在蛋白质构象而不是核酸水平上起作用，已在哺乳动物和真菌等多种生物体中发现。 在这里，酵母被用来研究朊病毒遗传的规则。影响酵母朊病毒外观的因素正在出现。最有趣的是朊病毒样元素[PIN+]，它是[PSI+]朊病毒重新出现所必需的。 编码[PIN+]蛋白质的基因PIN 1将从遗传筛选中鉴定。 破坏和过表达PIN 1将检验2种假设：非朊病毒形式的Pin 1抑制[PSI+]播种，而朊病毒形式的Pin 1催化[PSI+]播种。 将使用纯化的Pin 1在体外检查和模拟Pin 1的体内聚集。 将确定添加的可溶性或聚集的Pin 1对Sup 35纤维形成的动力学的影响。将检查体外Pin 1/Sup 35结合，以及[PSI+]和[PIN+]聚集体的相对细胞定位（使用不同颜色的GFP融合物）。 对于朊病毒从头出现重要的其他因素将被鉴定为当突变或过表达时抑制[PSI+]诱导的基因。此外，将在SUP 35朊病毒结构域中分离突变，其允许在[PIN+]不存在的情况下诱导[PSI+]，并刺激[PIN+]菌株中[PSI+]的自发出现。后者类似于人类PrP朊病毒基因的“家族性”突变，导致朊病毒疾病的遗传易感性。 我们还将研究：1）通过克隆防止不稳定[PSI+]稳定的孟德尔突变来稳定新出现的朊病毒; 2）通过克隆治愈或抑制[PSI+]或与[PSI+]一起合成致死的高拷贝基因来维持和表达现有的朊病毒; 3）不同水平的伴侣蛋白或蛋白酶体活性对[PSI+]和[PIN+]的诱导或维持的影响。朊病毒的跨物种传播威胁着人类健康。 使用由异源Sup 35形成的朊病毒，将检验不同朊病毒株可以不同效率跨越种属屏障的假设。 将选择[PSI+]菌株和SUP 35中改变种属屏障强度的突变。 当跨越物种屏障时，将确定同一细胞中不同物种朊病毒的相对位置。Sup 35的X射线结构将在Sup 35/Sup 45共晶体中确定。 Sup 45应防止通常妨碍晶体形成的Sup 35纤维的形成。 将使用蛋白酶消化和基于变性对免疫反应性的影响的试验研究不同[PSI+]菌株的Sup 35聚集体之间的结构差异。“先锋”酵母朊病毒将被鉴定（通过2D-凝胶分析和质谱）为存在于酵母提取物的沉淀级分中的蛋白质，但可溶于通过GuHCl和HSP 104的缺失治愈朊病毒的等基因菌株的裂解物中。 新朊病毒的鉴定将有助于阐明朊病毒如何工作以及它们为什么存在的原理。

"Protein only" (prion) inheritance, operating at the level of protein conformations rather than nucleic acids, has bee found in such diverse organisms as mammals and fungi. Here, yeast is used to investigate the rules of prion inheritance. Factors influencing the appearance of yeast prions are emerging. The most intriguing is the prion-like element [PIN+], required for the de novo appearance of the [PSI+] prion. The gene encoding the [PIN+] protein, PIN1, will be identified from genetic screens. Disrupting and overexpressing PIN1 will test 2 hypotheses the non-prion form of Pin1 inhibits [PSI+] seeding, vs. the prion form of Pin1 catalyzes [PSI+] seeding. In vivo aggregation of Pin1 will be examined and simulated in vitro using purified Pin1. The effects of added soluble or aggregated Pin1 on the kinetics of Sup35 fiber formation will be determined. Pin1/Sup35 binding in vitro, and the relative cellular localization of [PSI+] and [PIN+] aggregates (using different color GFP fusions) will be examined. Other factors important for the de novo appearance of prions will be identified as genes that inhibit the induction of [PSI+] when mutated or overexpressed. Also, mutations will be isolated in the SUP35 prion domain that permit the induction of [PSI+] in the absence of [PIN+], and that stimulate the spontaneous appearance of [PSI+] in [PIN+] strains. The latter are analogous to "familial" mutations of the human PrP prion gene that cause a heritable predisposition to prion disease. We will also study: 1) stabilization of emerging prions by cloning a Mendelian mutation that prevents stabilization of an unstable [PSI+]; 2) maintenance and expression of existing prions by cloning high copy genes that cure or inhibit [PSI+], or are synthetic-lethal with [PSI+]; 3) effects of varying levels of chaperones or proteasome activity on induction or maintenance of [PSI+] and [PIN+]. The transmission of prions across species lines threatens human health. Using prions formed from heterologous Sup35, the hypothesis that different strains of prions can cross the species barrier with different efficiencies, will be tested. Strains of [PSI+], and mutations in SUP35, that alter the strength of the species barrier will be selected. The relative location of different species of prions in the same cell will be determined when the species barrier is crossed. The X-ray structure of Sup35 will be determined in a Sup35/Sup45 co-crystal. Sup45 should prevent the formation of Sup35 fibers that usually preclude crystal formation. Structural differences between Sup35 aggregates of different [PSI+] strains will be investigated using protease digestion and an assay based on effects of denaturation on immunoreactivity. "Pioneer" yeast prions will be identified (by 2D-gel analysis and mass spectroscopy) as proteins present in pellet fractions of yeast extracts, but soluble in lysates of isogenic strains cured of prions by both GuHCl and deletion of HSP 104. The identification of new prions will help elucidate the principles of how prions work and why they exist.