Investigation of the Yeast Prion Factor, [PSI+]

在哺乳动物和真菌等多种生物中发现了在蛋白质构象而非核酸水平上起作用的“纯蛋白质”（朊病毒）遗传。本文利用酵母研究朊病毒的遗传规律。影响酵母朊病毒外观的因素正在出现。最令人感兴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+]蛋白的基因PIN1将从遗传筛选中鉴定出来。破坏和过表达PIN1将验证两种假设：PIN1的非朊病毒形式抑制[PSI+]播种，而PIN1的朊病毒形式催化[PSI+]播种。将使用纯化的Pin1在体外检测和模拟Pin1的体内聚集。添加可溶性或聚集的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+]聚集体的相对细胞定位（使用不同颜色的GFP融合物）将被检查。朊病毒重新出现的其他重要因素将被确定为当突变或过表达时抑制[PSI+]诱导的基因。此外，突变将在SUP35朊病毒域中分离出来，这些突变允许在缺乏[PIN+]的情况下诱导[PSI+]，并刺激[PIN+]菌株中自发出现[PSI+]。后者类似于人类PrP朊病毒基因的“家族性”突变，可导致朊病毒疾病的遗传易感性。我们还将研究：1)通过克隆孟德尔突变来稳定新出现的朊病毒，以防止不稳定的[PSI+]的稳定；2)通过克隆治疗或抑制[PSI+]或与[PSI+]合成致死的高拷贝基因来维持和表达现有的朊病毒；3)不同水平的伴侣或蛋白酶体活性对诱导或维持[PSI+]和[PIN+]的影响。朊病毒的跨物种传播威胁着人类健康。利用异源Sup35形成的朊病毒，将验证不同菌株的朊病毒以不同的效率跨越物种屏障的假设。会改变物种屏障强度的[PSI+]菌株和SUP35突变将被选择。当物种屏障被跨越时，不同种类的朊病毒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共晶中确定。Sup45应防止通常阻止晶体形成的Sup35纤维的形成。不同[PSI+]菌株的Sup35聚集体之间的结构差异将通过蛋白酶消化和基于变性对免疫反应性影响的测定来研究。“先锋”酵母朊病毒将被（通过2d凝胶分析和质谱）鉴定为存在于酵母提取物颗粒部分的蛋白质，但可溶于经GuHCl和缺失HSP 104固化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的工作原理以及它们存在的原因。

"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.