Biological Roles and Structures of Yeast Prions

酵母朊病毒的生物学作用和结构

• 批准号: 9148730
• 负责人: Reed B. WICKNER
• 金额: $ 149.39万
• 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/9148730
• 关键词: Adverse effects; Amino Acid Sequence; Amyloid; Amyloidosis; Automobile Driving; Biochemistry; Biological; Cells; Communicable Diseases; Data; Disease; Filament; Frequencies; Generations; Genes; Genetic; Genets; Goals; Growth; Heat shock proteins; Human; Label; Lead; Mammals; Messenger RNA; Methods; Modeling; National Institute of Diabetes and Digestive and Kidney Diseases; Nature; Organelles; Partner in relationship; Pathogenesis; Pathogenicity; Pathologic; Pathology; Peptide Sequence Determination; Population Genetics; Prion Diseases; Prions; Process; Protein Structure Initiative; Proteins; Publishing; Recording of previous events; Resistance to infection; Role; Saccharomyces cerevisiae; Science; Seeds; Structure; System; Variant; Work; Yeasts; amyloid structure; analog; base; beta pleated sheet; cell growth; design; genetic element; genome sequencing; mutant; neutrophil; novel; prion seeds; prion-like; segregation; solid state nuclear magnetic resonance; suicidal; sup35; transmission process; yeast prion

In 1994 we discovered that yeast can have prions, infectious proteins analogous to the transmissible spongiform encephalopathies of mammals. We showed that the non-Mendelian genetic element, URE3, is a prion of the Ure2 protein, and that PSI+ is a prion of Sup35p (1,2). These prions are amyloids of the respective proteins (3). Our discovery showed that proteins can be genes. Unexpectedly, shuffling the prion domain amino acid sequence of Ure2p or Sup35p did not alter the ability of these domains to support prion formation, suggesting that the amyloid structure is parallel in-register (4). We have shown by solid-state NMR (with Rob Tycko of NIDDK) that the amyloids of Ure2p, Sup35p and Rnq1p are indeed folded in-register parallel beta sheets (5-7). It has not escaped our notice that this folded in-register parallel beta sheet structure can explain how a given protein sequence can encode any of several biologically distinct prion variants based on biochemically distinct self-propagating amyloid structures (8). URE3 prion-forming ability of Ure2p is scattered among yeast species (9). The PSI+ prion can form in some species other than S. cerevisiae, leading some to suggest that PSI+ benefits yeast. We find that the ability of Sup35p to give rise to a PSI+ type prion is sporadically distributed among yeast and fungal species (10). Moreover, the part of Sup35p responsible for prion formation has a normal function in regulating mRNA turnover. This suggests that neither PSI+ - forming nor URE3 - forming ability are conserved, but are rare side effects of domains conserved for normal functions. We find that PSI+ and URE3 are rare in wild strains, though they would be common if they were advantageous (11). We used population genetics to show that cells carrying even the mildest forms of the yeast prions PSI+, URE3 or PIN+ have a >1% growth/survival detriment (12). About 10% of wild strains carry the PIN+ prion (11), but PIN+ arises only very rarely. We find that the presence of PIN+ in wild strains is associated with a history, detected from genome sequences, of outcross mating, indicating that PIN+ is an infectious disease, rather than becoming widespread as a result of conferring an advantage on the host cells (13). But PSI+ and URE3 are not always so mild. We designed a method to find lethal (Suicidal) PSI+s, should they exist. We found that such lethal or near-lethal variants of PSI+ comprise more than half of total isolates (14). We found that common variants of the URE3 prions cause extremely slow growth, although deletion of the URE2 gene in these strains did not slow growth (14). This toxic URE3 must be a due to a pathogenic amyloid, confirming the pathologic nature of the yeast prions PSI+ and URE3. Understanding their mechanisms of pathogenesis may be useful in understanding human amyloidoses. We have sequenced the SUP35 genes of 55 wild S. cerevisiae isolates, finding three groups of common polymorphs (15). PSI+ transmission between polymorphs is largely blocked, suggesting that these changes are selected to protect yeast from the detrimental effects of the prion (15). Indeed, the rate of evolutionary change of the prion domain is much faster than that of the remainder of the molecule suggesting that selection for resistance to infection by prions is driving change in the prion domain. We find that the rare wild PSI+ variants are sensitive to these blocks as well, supporting this interpretation (16). M domain changes in one polymorph are important in blocking prion spread (15). We find that transmission efficiency of PSI+ from a strain with one Sup35p polymorph to one with another polymorph is highly dependent on the variant of PSI+ (15, 16). That is, two PSI+ isolates in the otherwise identical genetic background with the same Sup35p protein sequence can have dramatically different efficiencies of transmission to cells with a different Sup35 protein sequence. Using this transmission frequency as a marker for different prion variants, we demonstrated segregation (separation) of prion variants during growth of the cells under non-selective conditions, and the generation of new variants, presumably due to occasional mis-templating of the amyloid (16). Data suggestive of this "prion cloud" phenomenon in mammalian prions has been published and it is likely to apply as well to the common human amyloid diseases. We found that overproduction of Btn2p or Cur1p could cure the URE3 prion (17), and that in the process of curing URE3, Ure2p aggregates co-localized with Btn2p in a single locus (17). We now find that the large majority of URE3 variants isolated in a btn2 cur1 mutant are cured by restoring just the normal level of Btn2p and Cur1p (18). Moreover, we find that it is specifically URE3 variants of low seed number that are cured by normal levels of these proteins (18). We propose that Btn2p collects prion aggregates, increasing the likelihood that one of the progeny cells will not get any prion seeds and so be cured. We showed that overproduced Hsp42, a small heat shock protein, also cures URE3, and Hsp42 is necessary for curing of URE3 by overproduced Btn2p (18). Btn2p, Cur1p and Hsp42 work together at normal levels to cure URE3 prions that arise. They comprise a anti-prion system. Btn2p has low level homology with mammalian HOOK proteins which are involved in transporting aggregates and organelles around the cell, including formation of the mammalian 'aggresome'. We have used singly- 13C-labeled Sup35NM molecules to confirm our folded parallel in-register beta sheet model of the infectious prion amyloid of Sup35p ( 1. Wickner RB (1994) URE3 as an altered URE2 protein: evidence for a prion analog in S. cerevisiae. Science 264: 566 - 569. 2. Masison DC & Wickner RB (1995) Science 270: 93 - 95. 3. Wickner RB, Edskes HK, Ross ED, Pierce MM, Baxa U, Brachmann A & Shewmaker F (2004) Ann. Rev. Genetics 38: 681-707. 4. Ross ED, Minton AP & Wickner RB (2005) Nature Cell Biol. 7: 1039-1044. 5. Shewmaker F, Wickner RB & Tycko R (2006) Proc. Natl. Acad. Sci. USA 103: 19754 - 19759. 6. Baxa U, Wickner RB, Steven AC, Anderson D, Marekov L, Yau W-M & Tycko R (2007) Biochemistry 46: 13149 - 13162. 7. Wickner RB, Dyda F & Tycko R (2008) Proc Natl Acad Sci U S A 105: 2403 - 2408. 8. Wickner RB, Edskes HK, Shewmaker F, Nakayashiki T 2007 Nat. Rev. Microbiol. 5: 611-618. 9. Edskes HK, Engel A, McCann LM, Brachmann A, Tsai H-F, Wickner RB (2011) Genetics 188:81 90. 10. Edskes HE, Khamar HJ, Winchester C-L, Greenler AJ, Zhou A, McGlinchey RP, Gorkovskiy A, Wickner RB (2014) Genetics, 198: 605-616. 11. Nakayashiki T, Kurtzman CP, Edskes HK, Wickner RB (2005) Proc Natl Acad Sci U S A 102:10575-80. 12. Kelly AC, Shewmaker FP, Kryndushkin D, and Wickner RB (2012) Proc. Natl. Acad. Sci. USA 109: E2683 - E2690. 13. Kelly, A. C., Busby, B. and Wickner, R. B. (2014) Genetics, 197: 1007 - 1024. 14. McGlinchey R, Kryndushkin D, Wickner RB (2011) Proc Natl Acad Sci USA 108:5337 - 41. 15. Bateman, DA and Wickner, RB (2013) Genetics 190:569-579. 16. Bateman, D., and Wickner, R. B. (2013) Plos Genet. 9(1):e1003257. 17. Kryndushkin D, Shewmaker FP, Wickner RB (2008) EMBO J. 27: 2725 - 2735. 18. Wickner RB, Bezsonov E Bateman DA (2014) Proc. Natl. Acad. Sci. USA, 111: E2711-20. 19. Gorgovskiy A, Thurber KR, Tycko R, Wickner RB (2014) Proc. Natl. Acad. Sci. USA, 111:E4615-22.

1994 年，我们发现酵母可能含有朊病毒，这是一种类似于哺乳动物传染性海绵状脑病的传染性蛋白质。我们证明非孟德尔遗传元件 URE3 是 Ure2 蛋白的朊病毒，而 PSI+ 是 Sup35p 的朊病毒 (1,2)。这些朊病毒是各自蛋白质的淀粉样蛋白 (3)。我们的发现表明蛋白质可以是基因。出乎意料的是，改组 Ure2p 或 Sup35p 的朊病毒结构域氨基酸序列并没有改变这些结构域支持朊病毒形成的能力，表明淀粉样蛋白结构是平行对齐的 (4)。我们通过固态 NMR（与 NIDDK 的 Rob Tycko 合作）证明，Ure2p、Sup35p 和 Rnq1p 的淀粉样蛋白确实是按套准平行 β 片层折叠的 (5-7)。我们注意到，这种折叠的对齐平行 β 片层结构可以解释给定的蛋白质序列如何编码基于生化上不同的自我繁殖淀粉样蛋白结构的几种生物学上不同的朊病毒变体中的任何一种 (8)。 Ure2p 的 URE3 朊病毒形成能力分散于酵母菌种中 (9)。 PSI+ 朊病毒可以在酿酒酵母以外的某些物种中形成，因此一些人认为 PSI+ 对酵母有益。我们发现 Sup35p 产生 PSI+ 型朊病毒的能力零星分布在酵母和真菌物种中 (10)。此外，Sup35p中负责朊病毒形成的部分在调节mRNA更新方面具有正常功能。这表明 PSI+ 形成能力和 URE3 形成能力均不保守，而是正常功能保守结构域的罕见副作用。 我们发现 PSI+ 和 URE3 在野生菌株中很少见，尽管如果它们具有优势的话它们会很常见 (11)。我们利用群体遗传学证明，即使携带最温和形式的酵母朊病毒 PSI+、URE3 或 PIN+ 的细胞也会产生 >1% 的生长/生存损害 (12)。大约 10% 的野生菌株携带 PIN+ 朊病毒 (11)，但 PIN+ 出现的情况非常罕见。我们发现野生菌株中 PIN+ 的存在与从基因组序列中检测到的异交交配历史有关，表明 PIN+ 是一种传染病，而不是 由于赋予宿主细胞优势而变得普遍 (13)。 但 PSI+ 和 URE3 并不总是那么温和。我们设计了一种方法来查找致命（自杀性）PSI+（如果存在）。我们发现 PSI+ 的这种致死或近致死变体占总分离株的一半以上 (14)。我们发现 URE3 朊病毒的常见变体会导致生长极其缓慢，尽管删除这些菌株中的 URE2 基因并不会减慢生长 (14)。这种有毒的 URE3 一定是由致病性淀粉样蛋白引起的，证实了酵母朊病毒 PSI+ 和 URE3 的病理性质。了解其发病机制可能有助于了解人类淀粉样变性。 我们对 55 个野生酿酒酵母分离株的 SUP35 基因进行了测序，发现了三组常见的多态性 (15)。多晶型物之间的 PSI+ 传播很大程度上被阻断，这表明选择这些变化是为了保护酵母免受朊病毒的有害影响 (15)。 事实上，朊病毒结构域的进化变化速度比分子其余部分的进化变化速度快得多，这表明对朊病毒感染的抵抗力的选择正在推动朊病毒结构域的变化。我们发现罕见的野生 PSI+ 变体也对这些块敏感，支持了这种解释 (16)。一种多晶型物中 M 结构域的变化对于阻止朊病毒传播非常重要 (15)。 我们发现 PSI+ 从具有一种 Sup35p 多晶型物的菌株到具有另一种多晶型物的菌株的传播效率高度依赖于 PSI+ 的变体 (15, 16)。也就是说，在其他方面相同的遗传背景中具有相同 Sup35p 蛋白序列的两个 PSI+ 分离株在向具有不同 Sup35 蛋白序列的细胞传播时可能具有显着不同的效率。使用这种传输频率作为不同朊病毒变体的标记，我们证明了在非选择性条件下细胞生长过程中朊病毒变体的分离（分离），以及新变体的产生，这可能是由于淀粉样蛋白偶尔的错误模板造成的。 16）。表明哺乳动物朊病毒中这种“朊病毒云”现象的数据已经发表，并且它可能也适用于常见的人类淀粉样蛋白疾病。 我们发现，Btn2p 或 Cur1p 的过量产生可以治愈 URE3 朊病毒 (17)，并且在治愈 URE3 的过程中，Ure2p 聚集体与 Btn2p 共定位在单个基因座中 (17)。我们现在发现，在 btn2 cur1 突变体中分离的绝大多数 URE3 变体可以通过恢复 Btn2p 和 Cur1p 的正常水平来治愈 (18)。此外，我们发现，这些蛋白质的正常水平可以治愈低种子数的 URE3 变体 (18)。我们建议 Btn2p 收集朊病毒聚集体，从而增加其中一个子代细胞不会获得任何朊病毒种子并因此被治愈的可能性。我们发现，过量产生的 Hsp42（一种小型热休克蛋白）也能治愈 URE3，并且 Hsp42 是过量产生的 Btn2p 治愈 URE3 所必需的 (18)。 Btn2p、Cur1p 和 Hsp42 在正常水平下协同作用，治愈出现的 URE3 朊病毒。它们组成了一个抗朊病毒系统。 Btn2p 与哺乳动物 HOOK 蛋白具有低水平同源性，后者参与细胞周围聚集体和细胞器的运输，包括哺乳动物“聚集体”的形成。 我们使用单 13C 标记的 Sup35NM 分子来确认我们的 Sup35p 感染性朊病毒淀粉样蛋白的折叠平行登记 β 片层模型（ 1. Wickner RB (1994) 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。 2.Maison DC 和 Wickner RB (1995)《科学》270：93 - 95。 3. Wickner RB、Edskes HK、Ross ED、Pierce MM、Baxa U、Brachmann A 和 Shewmaker F (2004) Ann。遗传学修订版 38：681-707。 4. Ross ED、Minton AP 和 Wickner RB (2005)《自然细胞生物学》。 7：1039-1044。 5. Shewmaker F、Wickner RB 和 Tycko R (2006) Proc。国家。阿卡德。科学。美国 103：19754 - 19759。 6. Baxa U、Wickner RB、Steven AC、Anderson D、Marekov L、Yau W-M 和 Tycko R (2007) 生物化学 46：13149 - 13162。 7. Wickner RB、Dyda F 和 Tycko R (2008) Proc Natl Acad Sci U S A 105：2403 - 2408。 8. Wickner RB、Edskes HK、Shewmaker F、Nakayashiki T 2007 年全国微生物学牧师。 5：611-618。 9. Edskes HK、Engel A、McCann LM、Brachmann A、Tsai H-F、Wickner RB (2011) 遗传学 188:81 90。 10. Edskes HE、Khamar HJ、Winchester C-L、Greenler AJ、Zhou A、McGlinchey RP、Gorkovskiy A、Wickner RB (2014) 遗传学，198：605-616。 11. Nakayashiki T、Kurtzman CP、Edskes HK、Wickner RB (2005) Proc Natl Acad Sci U S A 102：10575-80。 12. Kelly AC、Shewmaker FP、Kryndushkin D 和 Wickner RB (2012) Proc。国家。阿卡德。科学。美国 109：E2683 - E2690。 13. Kelly, A. C.、Busby, B. 和 Wickner, R. B. (2014) 遗传学，197：1007 - 1024。 14. McGlinchey R、Kryndushkin D、Wickner RB (2011) 美国国家科学院院报 108:5337 - 41。 15. DA 贝特曼和 RB 威克纳 (2013) 遗传学 190:569-579。 16. Bateman, D. 和 Wickner, R. B. (2013) Plos Genet。 9（1）：e1003257。 17. Kryndushkin D、Shewmaker FP、Wickner RB (2008) EMBO J. 27：2725 - 2735。 18. Wickner RB、Bezsonov E Bateman DA (2014) Proc。国家。阿卡德。科学。美国，111：E2711-20。 19. Gorgovskiy A、Thurber KR、Tycko R、Wickner RB (2014) Proc。国家。阿卡德。科学。美国，111：E4615-22。