Investigation of the Yeast Prion Factor, [PSI+]

酵母朊病毒因子的研究，[PSI]

• 批准号: 6525408
• 负责人: SUSAN W LIEBMAN
• 金额: $ 34.15万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525408
• 关键词: Saccharomyces cerevisiae; X ray crystallography; alleles; binding proteins; cell cycle proteins; conformation; fungal genetics; gel electrophoresis; gene expression; gene induction /repression; gene interaction; gene mutation; genetic polymorphism; genetic strain; genetic translation; mass spectrometry; molecular chaperones; molecular cloning; oligonucleotides; phenotype; prions; protein structure function; systematic biology; transposon /insertion element; tumor suppressor proteins; western blottings

"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.

在蛋白质构象水平而不是核酸的水平上运行的“仅蛋白质”（prion）遗传性在哺乳动物和真菌等多种生物中发现了蜜蜂。 在这里，酵母用于研究王室继承规则。影响酵母菌王室出现的因素正在出现。最吸引人的是[PSI+] prion从头出现所需的类似prion的元素[PIN+]。 编码[PIN+]蛋白PIN1的基因将从遗传筛选中鉴定出来。 破坏和表达过表达的PIN1将测试2个假设PIN1的非prion形式抑制[PSI+]播种，与PIN1催化[PSI+]播种的priON形式。 使用纯化的PIN1在体外检查PIN1的体内聚集。 将确定添加可溶性或聚集的PIN1对SUP35纤维形成动力学的影响。 PIN1/SUP35在体外结合，将检查[PSI+]和[PIN+]聚集体的相对细胞定位（使用不同颜色的GFP融合）。 对于从头出现的其他因素，将确定为抑制[PSI+]突变或过表达时诱导[PSI+]的基因。同样，在没有[PIN+]的情况下允许诱导[PSI+]的Sup35 Prion域中的突变，并刺激[PIN+]菌株中[PSI+]的自发外观。后者类似于人类Prp prion基因的“家族性”突变，该突变使遗传性易感性疾病。 我们还将研究：1）通过克隆孟德尔族突变来稳定新出现的王室，从而防止稳定不稳定[PSI+]； 2）通过克隆高拷贝基因来维持和表达现有prions的prions，或与[psi+]一起抑制[psi+]，或者是合成的； 3）不同水平的伴侣或蛋白酶体活动对[PSI+]和[PIN+]的诱导或维持的影响。跨物种线的王室传播威胁人类健康。 使用由异源SUP35形成的prions，将测试不同的王室菌株可​​以以不同的效率越过物种障碍的假设。 [PSI+]的菌株和SUP35中的突变会选择物种屏障的强度。 当物种屏障越过时，将确定不同种类在同一细胞中不同物种的相对位置。 SUP35的X射线结构将在SUP35/SUP45共晶中确定。 SUP45应防止形成通常阻止晶体形成的Sup35纤维。 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研究，将研究不同[PSI+]菌株的SUP35聚集体之间的结构差异。将在酵母提取物中存在的蛋白质（通过2D-GEL分析和质谱法）确定“先驱”酵母prions（通过2D-GEL分析和质谱法），但可溶于通过Guhcl和HSP 104固化Prions固化的Prions的裂解液，对新的Prions的识别将有助于新的Prions的识别。