Curing Prions of Yeast

治愈酵母朊病毒

• 批准号: 7593439
• 负责人: Reed B. WICKNER
• 金额: $ 23.35万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593439
• 关键词: ATP phosphohydrolase; Affect; Alzheimer' s Disease; Amino Acids; Amyloid; Amyloidosis; Amyotrophic Lateral Sclerosis; Binding; Catabolism; Cells; Cellular Stress; DNA; Disease; Genes; Genetic; Guanidines; Guanine Nucleotide Exchange Factors; Heat-Shock Proteins 70; Human; Lead; Molecular Chaperones; Nitrogen Fixation Genes; Non-Insulin-Dependent Diabetes Mellitus; Nucleic Acids; Nucleotides; Parkinson Disease; Phenotype; Play; Polymers; Prion Diseases; Prions; Protein Overexpression; Protein Structure Initiative; Proteins; Rare Diseases; Regulation; Role; Saccharomyces cerevisiae; Science; Screening procedure; Terminator Codon; Translations; Yeast Model System; Yeasts; amyloid formation; analog; base; cell growth; concept; derepression; guanidinium; human disease; inhibitor/antagonist; mutant; structure-specific endonuclease I; termination factor; trait; yeast protein

The word 'prion' means 'infectious protein', a protein which can transmit a disease or trait without the necessity for an accompanying nucleic acid. The prion concept has its origins in studies of the mammalian transmissible spongiform encephalopathies (TSEs), a group of uniformly fatal diseases whose underlying cause appears to be the formation of amyloid composed of the PrP protein. The mammalian prion diseases are not only uncurable, they are so far completely untreatable. Although these are rare diseases, affecting about 1 in a million people per year world wide, they are likely similar in mechanism to the many common amyloid-associated diseases such as Alzheimer's disease, Parkinson's disease, type 2 diabetes mellitus and amyotrophic lateral sclerosis. In 1994, we discovered two prions of the yeast Saccharomyces cerevisiae, called URE3 and PSI, based on a self-propagating inactivation of the Ure2 and Sup35 proteins, respectively(1). Ure2p is a regulator of nitrogen catabolism and URE3 strains show a derepression of the genes normally repressed by this protein. Sup35p is a subunit of the translation termination factor, and PSI+ strains show elevated readthrough of translation termination codons, a phenotype similar to that of a sup35 mutant. Studies by many labs have shown that the yeast prions URE3 and PSI are based on self-propagating amyloid forms of Ure2p and Sup35p, respectively (reviewed in 2). Chaperones play an important role in prion propagation, including the disaggregating chaperone Hsp104 (3), the Hsp70 family of chaperones (4) and the Hsp40s (5). Yeast prions can be cured by low concentrations of the specific Hsp104 inhibitor, guanidine (6), by overexpression of certain fragments of the prion protein (7), by inactivation of Hsp70s (4) or by overexpression of the Hsp40-group chaperone Ydj1p (8). We sought to determine if overexpression of other yeast proteins could cure the URE3 prion, screening for high-copy clones of yeast DNA that eliminate the prion (9). We found that overproduction of the chaperone, Sse1p, can efficiently cure the URE3 prion (9). Sse1p is also a nucleotide exchange factor for the Hsp70 family chaperone Ssa1p, promoting conversion of Ssa1p*GDP to Ssa1p*GTP. Fes1p has a similar activity. We found that deletion of either SSE1 or FES1 completely blocked URE3 propagation, while having little if any effect on cell growth. In addition, deletion of SSE1 also interfered with PSI+ propagation. Thus, an optimal level of Sse1p is necessary for propagation of the URE3 prion (9). Sse1p has both chaperone activity (requiring ATPase) and nucleotide exchange activity with Ssa1p (that requires ATP binding, but not ATPase). We find that a mutant that eliminates ATPase activity but retains ATP binding can still cure URE3, suggesting that it is the nucleotide exchange activity of Sse1p, not its chaperone activity, that is responsible for the curing. Since the Ssa's (cytoplasmic Hsp70s) are known to be required for yeast prion propagation, we believe that Sse1p affects prions by regulation of the Ssa's (9). This information about factors determining prion stability should eventually lead to therapy approaches for human amyloid diseases. We are now characterizing other proteins whose overproduction was found to cure the URE3 prion. References: 1. Wickner, R. B. URE3 as an altered URE2 protein: evidence for a prion analog in S. cerevisiae. Science 264, 566 - 569 (1994). 2. Wickner, R.B., Edskes, H.K., Ross, E.D., Pierce, M.M., Baxa, U., Brachmann, A., and Shewmaker, F. (2004). Prion Genetics: New Rules for a New Kind of Gene. Ann. Rev. Genetics 38, 681-707. 3. Chernoff, Y.O., Lindquist, S.L., Ono, B.-I., Inge-Vechtomov, S.G., and Liebman, S.W. (1995). Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor psi+. Science 268, 880 - 884. 4. Jung, G., Jones, G., Wegrzyn, R.D., and Masison, D.C. (2000). A role for cytosolic Hsp70 in yeast PSI+ prion propagation and PSI+ as a cellular stress. Genetics 156, 559-570. 5. Moriyama, H., Edskes, H.K., and Wickner, R.B. (2000). URE3 prion propagation in Saccharomyces cerevisiae: requirement for chaperone Hsp104 and curing by overexpressed chaperone Ydj1p. Mol. Cell. Biol. 20, 8916 - 8922. 6. Jung, G., Jones, G., and Masison, D.C. (2002). Amino acid residue 184 of yeast Hsp104 chaperone is critical for prion-curing by guanidine, prion propagation, and thermotolerance. Proc. Natl. Acad. Sci. U. S. A. 99, 9936 - 9941. 7. Edskes, H.K., Gray, V.T., and Wickner, R.B. (1999). The URE3 prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments. Proc. Natl. Acad. Sci. U. S. A. 96, 1498 1503. 8. Moriyama, H., Edskes, H.K., and Wickner, R.B. (2000). URE3 prion propagation in Saccharomyces cerevisiae: requirement for chaperone Hsp104 and curing by overexpressed chaperone Ydj1p. Mol. Cell. Biol. 20, 8916 - 8922. 9. Kryndushkin, D., and Wickner, R.B. (2007). Nucleotide exchange factors for Hsp70s are required for URE3 prion propagation in Saccharomyces cerevisiae. Mol. Biol. Cell. 18, 2149 - 2154.

“ prion”一词的意思是“感染蛋白”，一种可以传播疾病或性状的蛋白质，而无需伴有核酸。 Prion概念起源于对哺乳动物可传播的海绵脑病（TSE）的研究，这是一组统一致命的疾病，其根本原因似乎是由PRP蛋白组成的淀粉样蛋白的形成。 哺乳动物的prion疾病不仅是无法育种的，而且到目前为止，它们完全无法治疗。 尽管这些是罕见的疾病，每年影响全世界每年100万人中有100万疾病，但它们的机制可能与许多常见的淀粉样蛋白相关疾病（如阿尔茨海默氏病，帕金森氏病，2型糖尿病）和肌动育型侧面硬化症相似。 1994年，我们基于对URE2和Sup35蛋白的自发失活的酿酒酵母的两个酿酒酵母的两个prions（1）。 URE2P是氮分解代谢的调节剂，URE3菌株表现出通常被该蛋白质抑制的基因消除。 Sup35p是翻译终止因子的亚基，PSI+菌株显示了翻译终止密码子的升高，这是一种类似于Sup35突变体的表型。 许多实验室的研究表明，酵母菌pr ure3和PSI分别基于URE2P和SUP35P的自发淀粉样蛋白形式（审查2）。 伴侣在prion传播中起着重要作用，包括分类的伴侣HSP104（3），HSP70伴侣的HSP70家族（4）和HSP40S（5）。 酵母菌prions可以通过低浓度的特异性HSP104抑制剂鸟苷（6）来治愈，通过过度表达prion蛋白的某些片段（7），通过灭活Hsp70S（4）或通过过表达HSP40-GROUP CAPEROUP CHAPERONE YDJ1P（8）。 我们试图确定其他酵母菌蛋白的过表达是否可以治愈URE3 prion，筛选消除酵母DNA的高拷贝克隆，以消除prion葡萄酒（9）。 我们发现，伴侣SSE1P的过量生产可以有效地治愈URE3 Prion（9）。 SSE1P也是HSP70家族伴侣SSA1P的核苷酸交换因子，可促进SSA1P*GDP转换为SSA1P*GTP。 FES1P具有相似的活动。 我们发现SSE1或FES1的缺失完全阻止了URE3的繁殖，而对细胞生长的影响很小。另外，SSE1的缺失也干扰了PSI+传播。 因此，对于ure3 prion的传播是必要的最佳SSE1P水平（9）。 SSE1P具有伴侣活性（需要ATPase）和SSA1P的核苷酸交换活性（这需要ATP结合，但不需要ATPase）。 我们发现，消除ATPase活性但保留ATP结合的突变体仍然可以治愈URE3，这表明它是SSE1P的核苷酸交换活性，而不是其伴侣活性，而不是其疗法的原因。 由于已知SSA的（细胞质HSP70S）是酵母菌传播所必需的，因此我们认为SSE1P通过调节SSA（9）会影响prions（9）。 有关确定病毒稳定性的因素的此信息最终应导致人类淀粉样蛋白疾病的治疗方法。 现在，我们正在表征其他蛋白质，这些蛋白质的生产过多可治愈URE3 prion。 参考： 1。Wickner，R。B。Ure3作为一种改变的URE2蛋白：酿酒酵母中的Prion类似物的证据。科学264，566-569（1994）。 2。Wickner，R.B.，Edskes，H.K.，Ross，E.D.，Pierce，M.M.，Baxa，U.，Brachmann，A。和Shewmaker，F。（2004）。 prion遗传学：一种新型基因的新规则。安。遗传学修订版38，681-707。 3。Chernoff，Y.O.，Lindquist，S.L.，Ono，B.-I。，Inge-Vechtomov，S.G。和S.W. Liebman （1995）。伴侣蛋白Hsp104在酵母菌样因子PSI+传播中的作用。科学268，880-884。 4。Jung，G.，Jones，G.，Wegrzyn，R.D。和Masison，D.C。（2000）。胞质HSP70在酵母菌+ prion传播中的作用，而PSI+作为细胞应激。遗传学156，559-570。 5。Moriyama，H.，Edskes，H.K。和Wickner，R.B。（2000）。酿酒酵母中的URE3 Prion繁殖：对伴侣HSP104的需求和过表达的伴侣YDJ1P固化。摩尔。细胞。生物。 20，8916-8922。 6。Jung，G.，Jones，G。和Masison，D.C。（2002）。酵母HSP104伴侣的氨基酸残基184对于鸟根，prion传播和耐热的prion蛋白至关重要。 Proc。纳特。学院。科学。 U. S. A. 99，9936-9941。 7。Edskes，H.K.，Gray，V.T。和Wickner，R.B。（1999）。 URE3 Prion是URE2P的汇总形式，可以通过URE2P片段的过表达来治愈。 Proc。纳特。学院。科学。美国A. 96，1498 1503。 8。Moriyama，H.，Edskes，H.K。和Wickner，R.B。（2000）。酿酒酵母中的URE3 Prion繁殖：对伴侣HSP104的需求和过表达的伴侣YDJ1P固化。摩尔。细胞。生物。 20，8916-8922。 9. Kryndushkin，D。和Wickner，R.B。（2007）。 HSP70的核苷酸交换因子是酿酒酵母中URE3 prion繁殖需要的。摩尔。生物。细胞。 18，2149-2154。