Investigation of the Yeast Prion Factor, [PSI+]

在哺乳动物和真菌等不同的生物体中，都发现了在蛋白质构象水平而不是在核酸水平上操作的“纯蛋白质”(Prion)遗传。在这里，酵母被用来研究Pron的遗传规律。影响酵母菌蛋白外观的因素正在出现。最耐人寻味的是[PSI]Prion从头出现所需的类Prion元素[PIN]。编码[PIN]蛋白的基因Pin1将从基因筛查中识别出来。干扰和过度表达Pin1将检验两个假说：Pin1的非Pron形式抑制[PSI]种子，而Pin1的Prion形式催化[PSI]种子。用纯化的Pin1在体外检测和模拟Pin1的体内聚集。添加可溶性或聚集态PIN1对Sup35纤维形成动力学的影响将被确定。Pin1/Sup35的体外结合，以及[PSI]和[PIN]聚集体(使用不同颜色的GFP融合)的相对细胞定位。对蛋白从头开始出现的其他重要因素将被确定为当突变或过度表达时抑制[PSI]诱导的基因。此外，将在SUP35蛋白结构域中分离突变，这些突变允许在缺乏[PIN]的情况下诱导[PSI]，并刺激[PIN]菌株自发出现[PSI]。后者类似于人类PrP蛋白基因的“家族性”突变，这种突变导致了PrP蛋白疾病的遗传性。我们还将研究：1)通过克隆孟德尔突变来稳定新出现的Prion，以防止不稳定的[PSI]稳定；2)通过克隆治愈或抑制[PSI]的高拷贝基因，或与[PSI]合成致死的高拷贝基因，维持和表达现有的Prion；3)不同水平的伴侣或蛋白酶体活性对诱导或维持[PSI]和[PIN]的影响。普恩病毒的跨物种传播威胁着人类的健康。使用由异源Sup35形成的普恩病毒，将检验不同菌株的普恩病毒能够以不同的效率跨越物种障碍的假设。将选择能够改变物种屏障强度的[PSI]菌株和SUP35突变。当物种屏障被跨越时，不同物种在同一细胞中的相对位置将被确定。Sup35的X射线结构将在Sup35/Sup45共晶体中确定。Sup45应防止通常排除晶体形成的Sup35纤维的形成。不同[PSI]菌株的Sup35聚集体之间的结构差异将使用蛋白酶消化和基于变性对免疫反应性的影响的分析来研究。“先锋”酵母蛋白将被鉴定为(通过2D-凝胶分析和质谱学)存在于酵母抽提物颗粒组分中的蛋白质，但可溶于同基因菌株的裂解产物，可通过GUHCl和HSP 104的缺失治愈。识别新的普里恩将有助于阐明普恩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.