Curing Prions of Yeast

治愈酵母朊病毒

• 批准号: 7967189
• 负责人: Reed B. WICKNER
• 金额: $ 21.37万
• 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/7967189
• 关键词: ATP phosphohydrolase; Affect; Alzheimer' s Disease; Amino Acids; Amyloid; Amyloidosis; Amyotrophic Lateral Sclerosis; Binding; Catabolism; Cell Nucleus; Cell division; Cells; Cellular Stress; Chimeric Proteins; DNA; Disease; Employee Strikes; Generations; Genes; Genetic; Guanidines; Guanine Nucleotide Exchange Factors; Heat-Shock Proteins 70; Homologous Gene; Human; Lead; Molecular Chaperones; Nitrogen Fixation Genes; Non-Insulin-Dependent Diabetes Mellitus; Nucleic Acids; Nucleotides; Parkinson Disease; Phenotype; Play; Polymers; Prion Diseases; Prions; Process; Protein Structure Initiative; Proteins; Rare Diseases; Regulation; Reporting; Role; Saccharomyces cerevisiae; Science; Screening procedure; Sorting - Cell Movement; Spielmeyer-Vogt Disease; System; Terminator Codon; Translations; Vacuole; Variant; Yeast Model System; Yeasts; amyloid formation; analog; base; cell growth; derepression; human disease; inhibitor/antagonist; late endosome; mutant; overexpression; prevent; prion seeds; prion-like; structure-specific endonuclease I; sup35; termination factor; trait; yeast prion; 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). Most recently, we found that overproduction of Btn2p, or its homolog Ypr158 (Cur1p), cures URE3 (10). Btn2p is reported to be associated with late endosomes and to affect sorting of several proteins. We find that double deletion of BTN2 and CUR1 stabilizes URE3 against curing by several agents, produces a remarkable increase in the proportion of strong URE3 variants arising de novo and an increase in the number of URE3 prion seeds. Thus normal levels of Btn2p and Cur1p affect prion generation and propagation. Btn2p-GFP fusion proteins appear as a single dot located close to the nucleus and the vacuole. During the curing process, those cells having both Ure2p-GFP aggregates and Btn2p-RFP dots display striking co-localization. Btn2p curing requires cell division, and our results suggest that Btn2p is part of a system, reminiscent of the mammalian aggresome, that collects aggregates preventing their efficient distribution to progeny cells (10). 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., Wegrrzyn, R.D., and Masison, D.C. (2000). Arole 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. 10. Kryndushkin, D. and Wickner, R.B. (2008) Curing of the URE3 prion by Btn2p, a Batten disease-related protein. EMBO J. 27, 2725-35.

“朊病毒”一词的意思是“感染性蛋白质”，一种无需伴随核酸即可传播疾病或性状的蛋白质。朊病毒的概念起源于对哺乳动物传染性海绵状脑病 (TSE) 的研究，这是一组均致命的疾病，其根本原因似乎是由 PrP 蛋白组成的淀粉样蛋白的形成。哺乳动物朊病毒疾病不仅无法治愈，而且迄今为止还完全无法治疗。尽管这些都是罕见疾病，每年影响全世界约百万分之一的人，但它们的机制可能与许多常见的淀粉样蛋白相关疾病相似，如阿尔茨海默病、帕金森病、2 型糖尿病和肌萎缩侧索硬化症。 1994 年，我们分别基于 Ure2 和 Sup35 蛋白的自我繁殖失活，发现了酿酒酵母的两种朊病毒，称为 URE3 和 PSI (1)。 Ure2p 是氮分解代谢的调节剂，URE3 菌株显示出通常受该蛋白抑制的基因的去抑制。 Sup35p 是翻译终止因子的一个亚基，PSI+ 菌株显示翻译终止密码子的通读率升高，其表型与sup35 突变体相似。许多实验室的研究表明，酵母朊病毒 URE3 和 PSI 分别基于 Ure2p 和 Sup35p 的自我繁殖淀粉样蛋白形式（在 2 中进行了综述）。伴侣蛋白在朊病毒传播中发挥着重要作用，包括解聚伴侣蛋白 Hsp104 (3)、伴侣蛋白 Hsp70 家族 (4) 和 Hsp40s (5)。 酵母朊病毒可以通过低浓度的特异性 Hsp104 抑制剂胍 (6)、朊病毒蛋白某些片段的过表达 (7)、Hsp70 的失活 (4) 或 Hsp40 基团伴侣 Ydj1p 的过表达来治愈。 8).我们试图确定其他酵母蛋白的过度表达是否可以治愈 URE3 朊病毒，筛选出消除朊病毒的高拷贝酵母 DNA 克隆 ​​(9)。我们发现伴侣蛋白 Sse1p 的过量产生可以有效治愈 URE3 朊病毒 (9)。 Sse1p 也是 Hsp70 家族伴侣 Ssa1p 的核苷酸交换因子，促进 Ssa1p*GDP 转化为 Ssa1p*GTP。 Fes1p 也有类似的活性。我们发现删除 SSE1 或 FES1 完全阻止了 URE3 的增殖，而对细胞生长的影响很小（如果有的话）。此外，SSE1 的删除也会干扰 PSI+ 的传播。因此，最佳水平的 Sse1p 对于 URE3 朊病毒的传播是必要的 (9)。 Sse1p 具有伴侣活性（需要 ATP 酶）和与 Ssa1p 的核苷酸交换活性（需要 ATP 结合，但不需要 ATP 酶）。我们发现消除 ATP 酶活性但保留 ATP 结合的突变体仍然可以治愈 URE3，这表明是 Sse1p 的核苷酸交换活性，而不是其伴侣活性，负责治愈。由于已知 Ssa（细胞质 Hsp70）是酵母朊病毒繁殖所必需的，因此我们认为 Sse1p 通过调节 Ssa 来影响朊病毒 (9)。 最近，我们发现 Btn2p 或其同源物 Ypr158 (Cur1p) 的过量产生可以治愈 URE3 (10)。 据报道，Btn2p 与晚期内涵体相关并影响多种蛋白质的排序。我们发现，BTN2 和 CUR1 的双重删除可以稳定 URE3，使其免受多种试剂的固化，从而使从头产生的强 URE3 变体的比例显着增加，并增加 URE3 朊病毒种子的数量。因此，Btn2p 和 Cur1p 的正常水平会影响朊病毒的产生和传播。 Btn2p-GFP 融合蛋白显示为靠近细胞核和液泡的单个点。在固化过程中，那些同时具有 Ure2p-GFP 聚集体和 Btn2p-RFP 点的细胞表现出惊人的共定位。 Btn2p 固化需要细胞分裂，我们的结果表明 Btn2p 是一个系统的一部分，让人想起哺乳动物的聚集体，该系统收集聚集物，阻止其有效分布到后代细胞 (10)。 有关决定朊病毒稳定性的因素的信息最终应该会导致人类淀粉样蛋白疾病的治疗方法。我们现在正在鉴定其他蛋白质的特征，这些蛋白质的过量生产被发现可以治愈 URE3 朊病毒。 参考： 1. Wickner, R. B. URE3 作为一种改变的 URE2 蛋白：酿酒酵母中存在朊病毒类似物的证据。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)。朊病毒遗传学：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 Liebman, S.W. （1995）。伴侣蛋白 Hsp104 在酵母类朊病毒因子 psi+ 繁殖中的作用。科学 268, 880 - 884。 4. Jung, G.、Jones, G.、Wegrrzyn, R.D. 和 Maison, D.C. (2000)。 Arole 用于酵母 PSI+ 朊病毒繁殖中的胞质 Hsp70 和 PSI+ 作为细胞应激。遗传学 156, 559-570。 5. Moriyama, H.、Edskes, H.K. 和 Wickner, R.B. (2000)。 URE3 朊病毒在酿酒酵母中的传播：对伴侣 Hsp104 的要求以及过表达伴侣 Ydj1p 的治愈。摩尔。细胞。生物。 20、8916 - 8922。 6.Jung, G.、Jones, G. 和 Maison, D.C. (2002)。酵母 Hsp104 伴侣的氨基酸残基 184 对于胍固化朊病毒、朊病毒繁殖和耐热性至关重要。过程。国家。阿卡德。科学。美国 99, 9936 - 9941。 7. Edskes, H.K.、Gray, V.T. 和 Wickner, R.B. (1999)。 URE3 朊病毒是 Ure2p 的聚集形式，可以通过 Ure2p 片段的过度表达来治愈。过程。国家。阿卡德。科学。美国 96, 1498 1503。 8. Moriyama, H.、Edskes, H.K. 和 Wickner, R.B. (2000)。 URE3 朊病毒在酿酒酵母中的传播：对伴侣 Hsp104 的要求以及过表达伴侣 Ydj1p 的治愈。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 URE3 朊病毒在酿酒酵母中繁殖所必需的。摩尔。生物。细胞。 18, 2149 - 2154. 10. Kryndushkin, D. 和 Wickner, R.B. (2008) 通过 Btn2p（一种巴顿病相关蛋白）治愈 URE3 朊病毒。 EMBO J. 27, 2725-35。