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.

在1994年，我们发现酵母菌可以具有类似于哺乳动物的可传染性海绵状脑病的传染蛋白。我们表明，非孟德尔遗传元素URE3是URE2蛋白的prion，PSI+是Sup35p的prion（1,2）。这些王室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，将URE2P或SUP35P的Prion结构域氨基酸序列改组并没有改变这些结构域支持prion形成的能力，这表明淀粉样蛋白结构是平行于内置的（4）。我们已经通过固态NMR（NIDDK的Rob Tycko）表明，URE2P，SUP35P和RNQ1P的淀粉样蛋白确实被折叠为注册的并行β（5-7）。它并没有逃脱我们的注意到，这种折叠内的平行β片结构可以解释给定蛋白序列如何基于生物化学上不同的自发性淀粉样蛋白结构来编码几种生物学上不同的prion变体中的任何一种（8）。 URE2P的URE3 Prion形成能力分散在酵母菌中（9）。 PSI+ prion可以在酿酒酵母以外的某些物种中形成，导致一些人表明PSI+有益于酵母。我们发现，Sup35p产生PSI+类型prion的能力在酵母和真菌物种之间散发地分布（10）。此外，负责prion形成的Sup35p的部分在调节mRNA更新方面具有正常的功能。这表明PSI+ - 形成和URE3的形成能力都不是保守的，而是针对正常功能保守的域的罕见副作用。 我们发现PSI+和URE3在野生菌株中很少见，尽管如果它们有利，它们会很常见（11）。我们使用种群遗传学表明，即使携带最轻度的酵母菌psi+，Ure3或Pin+的细胞也具有> 1％的生长/生存损害（12）。大约10％的野生菌株带有销钉+ prion（11），但很少出现PIN+。我们发现，野生菌株中PIN+的存在与从基因组序列，杂交交配中检测到的历史有关，表明PIN+是一种感染性疾病，而不是 由于赋予宿主细胞的优势而变得广泛（13）。 但是PSI+和URE3并不总是那么温和。我们设计了一种方法，可以找到致命的（自杀）psi+s。我们发现，PSI+的这种致命或近致死变体占总分离株的一半以上（14）。我们发现，URE3 Prions的常见变异会导致极高的生长，尽管这些菌株中URE2基因的缺失并没有减慢生长的速度（14）。这种有毒的URE3必须是由于致病性淀粉样蛋白的原因，证实了酵母菌psi+和Ure3的病理性质。了解其发病机理的机制可能有助于理解人类淀粉样蛋白。 我们已经测序了55个野生葡萄球菌分离株的Sup35基因，发现了三组常见的多晶型物（15）。多晶型物之间的PSI+传播在很大程度上被阻断，这表明选择了这些变化是为了保护酵母免受病毒的不利影响（15）。 实际上，prion域的进化变化速度比分子其余部分的进化速度要快得多，这表明选择因prions毒而抗性感染的抗性正在推动prion域的变化。我们发现罕见的野生PSI+变体对这些块也很敏感，支持这种解释（16）。一个多晶型物中的M域变化对于阻断prion河的扩散很重要（15）。 我们发现，PSI+从一个Sup35p多晶型物的菌株的传输效率高度取决于PSI+的变体（15，16）。也就是说，具有相同SUP35P蛋白序列的原本相同遗传背景中的两个PSI+分离株可以具有不同的SUP35蛋白序列向细胞传播的效率急剧不同。使用此传输频率作为不同的prion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prion变体的隔离（分离），以及新变体的产生，可能是由于淀粉样蛋白的偶尔误解了（16）。哺乳动物prions中这种“ prion云”现象的数据已发表，并且可能也适用于人类常见的人淀粉样蛋白疾病。 我们发现BTN2P或CUR1P的过量生产可以治愈URE3 Prion（17），并且在固化URE3的过程中，URE2P聚集物与BTN2P共定位在单个位点（17）。现在，我们发现在BTN2 CUR1突变体中分离出的绝大多数URE3变体是通过仅恢复BTN2P和CUR1P的正常水平（18）来固化的。此外，我们发现它特别是低种子数的URE3变体，这些变体被这些蛋白质的正常水平固化（18）。我们建议BTN2P收集prion骨聚集体，增加了一个后代细胞不会获得任何prion种子并因此可以治愈的可能性。我们表明，生产过量的Hsp42，一种小热激蛋白，也可以治愈URE3，而HSP42对于过量生产的BTN2P固化URE3是必需的（18）。 BTN2P，CUR1P和HSP42在正常水平上一起使用，以治愈产生的URE3 prions。它们包括一个反派系系统。 BTN2P与哺乳动物钩蛋白具有低水平的同源性，该蛋白与细胞周围的聚集体和细胞器有关，包括形成哺乳动物的“杂种”。 我们已经使用单一标记的SUP35NM分子来确认我们的折叠平行型内注册β模型Sup35p的传染性prion淀粉样蛋白（ 1。WicknerRB（1994）URE3作为URE2蛋白的改变：酿酒酵母中的Prion类似物的证据。科学264：566-569。 2。MasisonDC＆Wickner RB（1995）科学270：93-95。 3。WicknerRB，Edskes HK，Ross ED，Pierce MM，Baxa U，Brachmann A＆Shewmaker F（2004）Ann。 Rev. Genetics 38：681-707。 4。RossEd，Minton AP＆Wickner RB（2005）自然细胞生物。 7：1039-1044。 5。ShewmakerF，Wickner RB和Tycko R（2006）Proc。纳特。学院。科学。美国103：19754-19759。 6。BaxaU，Wickner RB，Steven AC，Anderson D，Marekov L，Yau W -M＆Tycko R（2007）生物化学46：13149-13162。 7。WicknerRB，Dyda F＆Tycko R（2008）Proc Natl Acad Sci U S A 105：2403-2408。 8。WicknerRB，Edskes HK，Shewmaker F，Nakayashiki T 2007 Nat。 Microbiol牧师。 5：611-618。 9。EdskesHK，Engel A，McCann LM，Brachmann A，Tsai H-F，Wickner RB（2011）遗传学188：81 90。 10。EdskesHe，Khamar HJ，Winchester C-L，Greenler AJ，Zhou A，McGlinchey RP，Gorkovskiy A，Wickner RB（2014）Genetics，198：605-616。 11。NakayashikiT，Kurtzman CP，Edskes HK，Wickner RB（2005）Proc Natl Acad Sci U S 102：10575-80。 12。KellyAC，Shewmaker FP，Kryndushkin D和Wickner RB（2012）Proc。纳特。学院。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。McGlincheyR，Kryndushkin D，Wickner RB（2011）Proc Natl Acad Sci USA 108：5337-41。 15。Bateman，DA和Wickner，RB（2013）遗传学190：569-579。 16。Bateman，D。和Wickner，R。B.（2013）PLOS Genet。 9（1）：E1003257。 17。KryndushkinD，Shewmaker FP，Wickner RB（2008）Embo J. 27：2725-2735。 18。WicknerRB，Bezsonov E Bateman DA（2014）Proc。纳特。学院。科学。美国，111：E2711-20。 19。GorgovskiyA，Thurber KR，Tycko R，Wickner RB（2014）Proc。纳特。学院。科学。美国，111：E4615-22。