Prions of Yeast and Anti-Prion Systems

酵母朊病毒和抗朊病毒系统

基本信息

批准号：
10919386
负责人：
Reed B. WICKNER
金额：
$ 135.78万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10919386
关键词：
Alzheimer&apos s Disease Amino Acid Sequence Amyloid Amyloidosis Architecture Bacterial Infections Binding Proteins Carbon Cells Complex DNA Defect Diphosphates Disadvantaged Disease F-Box Proteins Filament Frequencies Generations Genes Genetic Glutamate-Ammonia Ligase Glycerol Growth Human In Vitro Infection Innate Immune System Inositol Learning Mammals Mediating Minority Mitochondria Modeling Molecular Chaperones Molecular Conformation Mutagenesis Mutation National Institute of Diabetes and Digestive and Kidney Diseases Nature Non-Insulin-Dependent Diabetes Mellitus Normal Cell Parkinson Disease Pathogenicity Pathology Pathway interactions Polyphosphates Prevention Prion Diseases Prions Process Production Protein Conformation Protein Structure Initiative Proteins Ribosomes Science Son Source Stress Structure System Testing Time Variant Virus Diseases Work Yeasts amyloid formation amyloid structure beta pleated sheet expectation human disease in vivo mRNA Decay multicatalytic endopeptidase complex mutant next generation sequencing non-prion overexpression prevent prion seeds prion-like protein protein interaction pyrophosphatase restoration solid state nuclear magnetic resonance transcription factor ubiquitin-protein ligase yeast prion

项目摘要

In 1994 we discovered prions (infectious proteins) infecting yeast (1) analogous to the transmissible spongiform encephalopathies of mammals. URE3 is a prion of Ure2p, and PSI+ is a prion of Sup35p (1,2), each an amyloid of the respective protein (reviewed in ref. 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 showed by solid-state NMR (with Rob Tycko of NIDDK) that the infectious amyloids of Ure2p, Sup35p and Rnq1p are indeed folded in-register parallel beta sheets (5,6). From this architecture we explained how a given protein sequence can template its conformation, and thus how a protein can act as a gene (7). This is the first and only explanation that has been offered for the templating of protein conformation that is central to the prion phenomenon and amyloid diseases. Prion-forming ability of Ure2p and Sup35p are not conserved among yeast and fungal species (8, 9), and the prion (amyloid)-forming parts of Ure2p and Sup35p have normal non-prion functions. PSI+ and URE3 are rare in wild strains (10,11) and most variants are toxic or even lethal, showing that these are diseases of yeast (12). Overproducing Btn2p or Cur1p, acting with Hsp42, cures all variants of the URE3 prion, but normal levels of each cure nearly all URE3 prions except those with the highest seed number (13, 14). Btn2p cures by collecting Ure2p aggregates at a single locus (sequestration), increasing the likelihood that one of the progeny cells will not get any prion seeds and so be cured (13). These are antiprion systems. We find that overactive proteasomes prevent prion curing by Btn2 or Cur1 (15) and partially inactive proteasomes result in URE3 curing as a result of the dramatically elevated levels of Btn2 and Cur1 (16). We propose that proteasomes overwhelmed by denatured proteins in stress conditions automatically turn on the Btn2 and Cur1 systems to help in the clean-up. The disaggregase Hsp104 is necessary for the propagation of all amyloid-based yeast prions, but cures PSI+ if overexpressed. We find that mutant Hsp104 specifically lacking the prion-curing activity generatates PSI+ 15x more often, and that most PSI+ variants isolated in the Hsp104 mutant are cured by restoration of normal levels of w.t. Hsp104, showing that this activity is an antiprion system (17). We found Siw14p, a pyrophosphatase specific for 5-diphosphoinositol pentakisphosphate (5PP-IP5) is anti-prion for PSI+ (18). We showed that most PSI+ prions require 5PP-IP5 or related inositol polyphosphates for their propagation, and 1PP-IP5 has a prion-inhibiting action in the absence of the inositol-5 pyrophosphates (18). We found that nonsense-mediated mRNA decay pathway components Upf1, Upf2 and Upf3, at normal expression levels, cure most PSI+ prions arising in their absence (19). The Upf proteins normally complex with Sup35p, and thereby block most PSI+ prion formation and cure most of those PSI+ prions arising in their absence (19). Upf1p blocks amyloid formation by Sup35p in vitro, and co-localizes with Sup35p aggregates in vivo in PSI+ cells. We infer that normal protein-protein interactions prevent the abnormal protein-protein interactions that produce prions. We also find that ribosome-associated chaperones Ssb, Ssz1 and Zuo1, that insure proper folding of nascent proteins, cure most of the PSI+ prions arising in their absence (20). Mutation of any results in 15-fold elevated prion generation frequency. Triple mutant ssz1 upf1 hsp104T160M cells produce PSI+ up to 5000-fold more often than wild type, but most prions arising are cured by replacing any one of the defective genes (21). Thus these antiprion systems act independently, and the real frequency of prions arising in normal cells is much higher than had been appreciated, but most variants arising are cured by these systems before they can be detected in the usual type of test. We used transposon mutagenesis and next-generation sequencing to find proteins that prevent growth defects that would otherwise be produced by the URE3 prion (22). We found that Lug1p/Ylr352wp prevents a growth defect on non-fermentable carbon sources (e.g. glycerol) that is produced by the URE3 prion in the absence of Lug1p (22). This effect is suppressed by overproduction of Hap4p, a transcription factor promoting expression of mitochondrial-bound proteins. A defect in Gln1p (glutamine synthase) also suppress the growth defect of lug1 URE3 strains. This also identifies a new function for Ure2p. Lug1p is an F-box protein, a substrate-directing subunit of an E3 ubiquitin ligase. We also found that mutation of any of a wide array of chaperones results in a selective disadvantage for URE3 - carrying cells. Thus, cells act to limit the pathology produced on prion infection. We recently identified 19 human proteins whose expression in yeast cures URE3 or PSI+ or both. We find that at least one such protein acts by impeding interaction of Hsp40s with Hsp70s in the process of Hsp104-catalyzed cleavage of prion amyloid filaments, a process essential for prion propagation. Multiple systems prevent prion formation, cure most of the prions that do manage to arise, and limit the pathology from the few prions that evade the other systems. Just as we utilize humoral, cellular and innate immune systems to treat or cure or limit the damage from viral and bacterial infections, we suggest that these anti-prion systems will prove to be useful in treatment or prevention of prion/amyloid diseases of humans. 1. Wickner RB (1994) 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) PNAS 103: 19754 - 19759. 6. Gorkovskiy A, Thurber KR, Tycko R, Wickner RB (2014) PNAS 111:E4615-22. 7. Wickner RB, Edskes HK, Shewmaker F, Nakayashiki T 2007 Nat. Rev. Microbiol. 5: 611-618. 8. Edskes HK, Engel A, McCann LM, Brachmann A, Tsai H-F, Wickner RB (2011) Genetics 188:81 90. 9. Edskes HE, Khamar HJ, Winchester C-L, Greenler AJ, Zhou A, McGlinchey RP, Gorkovskiy A, Wickner RB (2014) Genetics, 198: 605-616. 10. Nakayashiki T, Kurtzman CP, Edskes HK, Wickner RB (2005) PNAS 102:10575-80. 11. Kelly AC, Shewmaker FP, Kryndushkin D, Wickner RB (2012) PNAS 109: E2683 - E2690. 12. McGlinchey R, Kryndushkin D, Wickner RB (2011) PNAS 108:5337 - 41. 13. Kryndushkin D, Shewmaker FP, Wickner RB (2008) EMBO J. 27: 2725 - 2735. 14. Wickner RB, Bezsonov E Bateman DA (2014) PNAS 111: E2711-20. 15. Bezsonov EE, Edskes HK, Wickner RB (2021) Genetics 217: doi: 10.1093/genetics/iyab013. 16. Edskes HK, Stroobant EE, DeWilde M, Bezsonov EE, Wickner RB (2021) Genetics 218: doi:10.1093/genetics/iyab037 17. Gorkovskiy A, Reidy M, Masison DC, Wickner RB (2017) PNAS 114: E4193-E4202. 18. Wickner RB, Kelly AC, Bezsonov EE, Edskes HE (2017) 114: E8402-E8410. 19. Son M, Wickner RB (2018) PNAS 115: E1184-E1193. 20. Son M, Wickner RB (2020) PNAS 117: 26298-26306. 21. Son M, Wickner RB (2022) PNAS 119: e2205500119. 22. Edskes HK, Mukhamedova M, Edskes BK, Wickner RB (2018) Genetics 209:789-800.

1994 年，我们发现了感染酵母的朊病毒（传染性蛋白质）(1)，类似于哺乳动物的传染性海绵状脑病。 URE3 是 Ure2p 的朊病毒，PSI+ 是 Sup35p (1,2) 的朊病毒，每种蛋白都是各自蛋白质的淀粉样蛋白（参考文献 3 中进行了综述）。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 的感染性淀粉样蛋白确实是按套准平行 β 片层折叠的 (5,6)。从这个架构中，我们解释了给定的蛋白质序列如何模板化其构象，以及蛋白质如何充当基因 (7)。这是对蛋白质构象模板的第一个也是唯一的解释，而蛋白质构象是朊病毒现象和淀粉样蛋白疾病的核心。 Ure2p 和 Sup35p 的朊病毒形成能力在酵母和真菌物种中并不保守 (8, 9)，并且 Ure2p 和 Sup35p 的朊病毒（淀粉样蛋白）形成部分具有正常的非朊病毒功能。 PSI+ 和 URE3 在野生菌株中很少见 (10,11)，大多数变体有毒甚至致命，表明这些是酵母疾病 (12)。过量产生 Btn2p 或 Cur1p，与 Hsp42 一起作用，可以治愈 URE3 朊病毒的所有变体，但每种正常水平的 Btn2p 或 Cur1p 几乎可以治愈所有 URE3 朊病毒，除了种子数最高的那些 (13, 14) 之外。 Btn2p 通过在单个位点收集 Ure2p 聚集体（隔离）来治愈，增加了子代细胞之一不会获得任何朊病毒种子并因此被治愈的可能性 (13)。这些是抗朊病毒系统。我们发现，过度活跃的蛋白酶体可阻止 Btn2 或 Cur1 固化朊病毒 (15)，而部分失活的蛋白酶体则由于 Btn2 和 Cur1 水平显着升高而导致 URE3 固化 (16)。我们提出，在应激条件下被变性蛋白质淹没的蛋白酶体会自动打开 Btn2 和 Cur1 系统来帮助清理。解聚酶 Hsp104 对于所有基于淀粉样蛋白的酵母朊病毒的繁殖都是必需的，但如果过度表达，则可以治愈 PSI+。我们发现，特别缺乏朊病毒治愈活性的突变体 Hsp104 产生 PSI+ 的频率提高了 15 倍，并且在 Hsp104 突变体中分离出的大多数 PSI+ 变异体可以通过恢复正常水平的重量来治愈。 Hsp104，表明该活性是一种抗朊病毒系统 (17)。我们发现 Siw14p（一种对 5-二磷酸肌醇五磷酸 (5PP-IP5) 具有特异性的焦磷酸酶）可抗 PSI+ 朊病毒 (18)。我们表明，大多数 PSI+ 朊病毒的繁殖需要 5PP-IP5 或相关的肌醇多磷酸盐，并且 1PP-IP5 在缺乏 5 肌醇焦磷酸盐的情况下具有朊病毒抑制作用 (18)。我们发现无义介导的 mRNA 衰变途径组分 Upf1、Upf2 和 Upf3 在正常表达水平下可以治愈大多数在其缺失时出现的 PSI+ 朊病毒 (19)。 Upf 蛋白通常与 Sup35p 复合，从而阻止大多数 PSI+ 朊病毒的形成，并治愈大多数在它们不存在时产生的 PSI+ 朊病毒 (19)。 Upf1p 在体外阻断 Sup35p 形成淀粉样蛋白，并在体内与 PSI+ 细胞中的 Sup35p 聚集体共定位。我们推断正常的蛋白质-蛋白质相互作用可以防止产生朊病毒的异常蛋白质-蛋白质相互作用。我们还发现核糖体相关伴侣 Ssb、Ssz1 和 Zuo1 可确保新生蛋白质的正确折叠，从而治愈大多数在其缺失时产生的 PSI+ 朊病毒 (20)。任何突变都会导致朊病毒产生频率提高 15 倍。三重突变体 ssz1 upf1 hsp104T160M 细胞产生 PSI+ 的频率比野生型细胞高 5000 倍，但大多数产生的朊病毒可以通过替换任何一个缺陷基因来治愈 (21)。因此，这些反朊病毒系统独立作用，正常细胞中产生的朊病毒的实际频率比人们想象的要高得多，但大多数产生的变异在通常类型的测试中被检测到之前就被这些系统治愈了。我们使用转座子诱变和新一代测序来寻找能够防止 URE3 朊病毒产生的生长缺陷的蛋白质 (22)。我们发现 Lug1p/Ylr352wp 可以防止在没有 Lug1p 的情况下 URE3 朊病毒产生的不可发酵碳源（例如甘油）的生长缺陷 (22)。 Hap4p 的过量产生会抑制这种效应，Hap4p 是一种促进线粒体结合蛋白表达的转录因子。 Gln1p（谷氨酰胺合酶）的缺陷也会抑制 lug1 URE3 菌株的生长缺陷。这也确定了 Ure2p 的一个新功能。 Lug1p 是一种 F-box 蛋白，是 E3 泛素连接酶的底物导向亚基。我们还发现，任何多种伴侣蛋白的突变都会导致携带 URE3 的细胞处于选择性劣势。因此，细胞起到限制朊病毒感染产生的病理学的作用。我们最近鉴定了 19 种人类蛋白质，它们在酵母中的表达可以治愈 URE3 或 PSI+ 或两者。我们发现，在 Hsp104 催化的朊病毒淀粉样蛋白丝裂解过程中，至少有一种这样的蛋白质通过阻碍 Hsp40 与 Hsp70 的相互作用来发挥作用，这是朊病毒繁殖所必需的过程。多个系统可以防止朊病毒形成，治愈大多数设法产生的朊病毒，并限制少数逃避其他系统的朊病毒的病理学。正如我们利用体液、细胞和先天免疫系统来治疗或治愈或限制病毒和细菌感染的损害一样，我们建议这些抗朊病毒系统将被证明可用于治疗或预防人类朊病毒/淀粉样蛋白疾病。 1. Wickner RB (1994)《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) PNAS 103：19754 - 19759。 6. Gorkovskiy A、Thurber KR、Tycko R、Wickner RB (2014) PNAS 111：E4615-22。 7. Wickner RB, Edskes HK, Shewmaker F, Nakayashiki T 2007 全国微生物学牧师。 5：611-618。 8. Edskes HK、Engel A、McCann LM、Brachmann A、Tsai H-F、Wickner RB (2011) 遗传学 188:81 90。 9. Edskes HE、Khamar HJ、Winchester C-L、Greenler AJ、Zhou A、McGlinchey RP、Gorkovskiy A、Wickner RB (2014) 遗传学，198：605-616。 10. Nakayashiki T、Kurtzman CP、Edskes HK、Wickner RB (2005) PNAS 102：10575-80。 11.Kelly AC、Shewmaker FP、Kryndushkin D、Wickner RB (2012) PNAS 109：E2683 - E2690。 12. McGlinchey R、Kryndushkin D、Wickner RB (2011) PNAS 108：5337 - 41。 13. Kryndushkin D、Shewmaker FP、Wickner RB (2008) EMBO J. 27：2725 - 2735。 14. Wickner RB、Bezsonov E Bateman DA (2014) PNAS 111：E2711-20。 15. Bezsonov EE、Edskes HK、Wickner RB (2021) 遗传学 217：doi：10.1093/Genetics/iyab013。 16. Edskes HK、Stroobant EE、DeWilde M、Bezsonov EE、Wickner RB (2021) 遗传学 218：doi:10.1093/Genetics/iyab037 17. Gorkovskiy A、Reidy M、Maison DC、Wickner RB (2017) PNAS 114：E4193-E4202。 18. Wickner RB、Kelly AC、Bezsonov EE、Edskes HE (2017) 114：E8402-E8410。 19. Son M，Wickner RB (2018) PNAS 115：E1184-E1193。 20. Son M，Wickner RB (2020) PNAS 117：26298-26306。 21.Son M，Wickner RB (2022) PNAS 119：e2205500119。 22. Edskes HK、Mukhamedova M、Edskes BK、Wickner RB (2018) 遗传学 209:789-800。

项目成果

期刊论文数量（53）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The yeast Sup35NM domain propagates as a prion in mammalian cells.

酵母 Sup35NM 结构域作为朊病毒在哺乳动物细胞中繁殖。

DOI：
发表时间：
2009-01-13
期刊：
影响因子：
11.1
作者：
Krammer, Carmen;Kryndushkin, Dmitry;Suhre, Michael H;Kremmer, Elisabeth;Hofmann, Andreas;Pfeifer, Alexander;Scheibel, Thomas;Wickner, Reed B;Schatzl, Hermann M;Vorberg, Ina
通讯作者：
Vorberg, Ina

Sex, prions, and plasmids in yeast.

酵母中的性、朊病毒和质粒。

DOI：
发表时间：
2012-10-02
期刊：
影响因子：
11.1
作者：
Kelly, Amy C;Shewmaker, Frank P;Kryndushkin, Dmitry;Wickner, Reed B
通讯作者：
Wickner, Reed B

Effect of domestication on the spread of the [PIN+] prion in Saccharomyces cerevisiae.

驯化对酿酒酵母中[PIN]朊病毒传播的影响。

DOI：
发表时间：
2014-07
期刊：
影响因子：
3.3
作者：
Kelly, Amy C;Busby, Ben;Wickner, Reed B
通讯作者：
Wickner, Reed B

Parallel in-register intermolecular β-sheet architectures for prion-seeded prion protein (PrP) amyloids.

朊病毒种子朊病毒蛋白 (PrP) 淀粉样蛋白的平行注册分子间 β-片结构。

DOI：
发表时间：
2014-08-29
期刊：
影响因子：
0
作者：
Groveman, Bradley R;Dolan, Michael A;Taubner, Lara M;Kraus, Allison;Wickner, Reed B;Caughey, Byron
通讯作者：
Caughey, Byron

Yeast prions: proteins templating conformation and an anti-prion system.

酵母朊病毒：蛋白质模板构象和抗朊病毒系统。

DOI：
发表时间：
2015-02
期刊：
影响因子：
6.7
作者：
Wickner, Reed B;Edskes, Herman K;Bateman, David A;Gorkovskiy, Anton;Dayani, Yaron;Bezsonov, Evgeny E;Mukhamedova, Maryam
通讯作者：
Mukhamedova, Maryam