Prions Of Yeast

酵母朊病毒

• 批准号: 6983645
• 负责人: Reed B. WICKNER
• 金额: --
• 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/6983645
• 关键词: Candida; DNA binding protein; Saccharomyces cerevisiae; amyloid proteins; amyloidosis; fungal genetics; fungal proteins; gene expression; molecular pathology; prions; protein structure function; spongiform encephalopathy; virus infection mechanism; virus protein; yeasts

We initiated a new field of yeast genetics with our discovery that two non-chromosomal genetic elements, [URE3] and [PSI], were prions (infectious proteins), analogous to the agent causing the transmissible spongiform encephalopathies of mammals. The first, [URE3], is an altered form of Ure2p, the protein product of the chromosomal URE2 gene important in regulation of nitrogen catabolism. The second, [PSI], is an altered form of Sup35p, a subunit of the translation release factor and product of the chromosomal SUP35 gene. We found that Ure2p is more resistant to protease digestion in [URE3] strains than in wild-type strains, and is aggregated specifically in cells carrying the prion, supporting the prion model and suggesting amyloid formation as its molecular basis. The N-terminal 65 aminoacid residues of Ure2p is sufficient to propagate [URE3], or to induce the de novo appearance of [URE3]. We showed that the Ure2p prion domain forms amyloid filaments in vitro. Moreover, just as the prion domain induces prion formation in vivo, it induces the otherwise stably soluble native Ure2p to form amyloid in vitro. The properties of Ure2p amyloid formation in vitro reflect and explain the prion properties of [URE3] in vivo. We thus proposed that the [URE3] prion is an infectious amyloidosis. We showed that fragments of Ure2p or fusions with other proteins cure the prion efficiently. This phenomenon may be due to interruption of the growth of the amyloid 'crystals' due to the fragments or fusion proteins, and suggests a new approach to the treatment of amyloid diseases. We find that the Mks1 protein is essential for the de novo formation of the [URE3] prion. Mks1 activity is negatively regulated by the Ras - cAMP pathway, and we find that activation of Ras2p prevents de novo [URE3] prion formation by inactivating Mks1. We showed that the Hsp104 chaperone is necessary for [URE3] prion propagation, and that overexpression of the Hsp40-family chaperone Ydj1p can cure the [URE3] prion. We recently showed that the Hsp70-family chaperone Ssa2p is also necessary for the propagation of [URE3]. In collaboration with Drs. Vladislav Speransky and Alasdair Steven (NIAMS), we further showed that cells with the [URE3] prion contain networks of filaments consisting of the Ure2 protein. Further, most of the Ure2p in extracts of [URE3] strains is in a form insoluble even after boiling in 3M urea and 2% SDS, confirming that it is in an amyloid state. Our collaborators, Drs. Tim Umland and David Davies (LMB, NIDDK), have determined the structure of the nitrogen regulation domain of Ure2p and find that it is closely similar to glutathione-S-transferases (GST). Ure2p is inactivated by prion (amyloid) formation in vivo. We find that Ure2p is not inactivated by a conformational change in the functional part of the molecule, but by a steric effect or diffusion limitation on the interaction of Ure2p with Gln3p. The Ure2-GFP fusion protein forms amyloid filaments with a helical form. The length of the helical repeat is constant within each filament, but this length varies by more than 2 fold from one filament to another. This may be the basis of prion strains, that have different infectious properties and different effects on the host. We have isolated homologs of the URE2 gene from other strains of S. cerevisiae, from various pathogenic Candida species and from a filamentous fungus. While the C-terminal domain is highly conserved and the homologs can substitute for the cerevisiae Ure2p, the N-terminal domain (up to residue 99) is highly variable. Nonetheless, there is a conserved part of the prion domain from residues 10 to 39. This region apparently interacts with the Ure2p C-terminus as judged by inactivation of Ure2p when the fragment is overexpressed. This region also is responsible for the curing of the [URE3] prion by fusions with GFP mentioned above. We find that the prion domain forms the centralll core of the amyloid filaments, with residues 1 to 65 comprising the highly protease resistant part. Ure2p residues 71-95 serve as a linker between the amyloid core and the peripherally arrayed functional domains (residues 95-354). Monomers of Ure2p are bound to eachother by interactions between the prion domains. We have recently described an entirely new class of prions, based not on amyloid formation, but on the requirement for autoactivation in trans of the vacuolar protease B (PrB) of yeast. Cells that lack active PrB remain in that state, except for the rare (10^-5) spontaneous activation of the enzyme. Cells with active PrB give rise to progeny nearly all of whom have active enzyme. These cells can also infect cells without active enzyme by the transfer of active PrB. Thus PrB, in its active form is an infectious protein (a prion). There are many proteins that are necessary for their own activation, which could thus potentially act as prions. The N-terminal prion domain of Ure2p (residues 1-90) is rich in N and Q residues and we showed that these are important for prion formation and propagation. However, we find that there are no essential amino acid sequence elements in the prion domain: five random shuffles of these amino acids leave a protein that can form prions in vivo and amyloid in vitro. This surprising result implies that amino acid composition, rather than sequence, is the critical driving force for prion formation in the case of Ure2p. We are now testing other prion-forming proteins to extend this result. Amyloid of Ure2p has an amyloid core that is high in beta sheet structure with beta strands perpendicular to the filament axis as judged by X-ray diffraction and electron diffraction (with U. Baxa, D. R. Davies, A. C. Steven). This result confirms the amyloid nature of the filaments formed by Ure2p. Our studies of the mechanism of the Mks1p requirement for [URE3] prion generation show that this requirement is independent of its role in regulating glutamate biosynthesis, and that altered expression of the cAMP-dependent protein kinases alters [URE3] generation as predicted from our earlier studies. Using expression chip analysis, we find that the [URE3] prion does not induce any transcriptional changes other than those attributable to the role of Ure2p in regulation of nitrogen catabolism. Using the yeast two hybrid method, we have detected a set of proteins that interact with the Ure2p prion domain in vivo. Possible effects of these proteins on prion generation or propagation are now being examined. In another study, we have used the yeast deletion bank to look for genes involved in prion generation. A number of candidates are now being further studied to determine the mechanisms of their involvement.

我们发现两种非染色体遗传元件 [URE3] 和 [PSI] 是朊病毒（传染性蛋白质），类似于引起哺乳动物传染性海绵状脑病的病原体，从而开创了酵母遗传学的新领域。第一个，[URE3]，是 Ure2p 的一种改变形式，Ure2p 是染色体 URE2 基因的蛋白质产物，对氮分解代谢的调节非常重要。第二种，[PSI]，是 Sup35p 的一种改变形式，Sup35p 是翻译释放因子的一个亚基，也是染色体 SUP35 基因的产物。我们发现 Ure2p 在 [URE3] 菌株中比野生型菌株更能抵抗蛋白酶消化，并且在携带朊病毒的细胞中特异性聚集，支持朊病毒模型并表明淀粉样蛋白形成为其分子基础。 Ure2p N 端 65 个氨基酸残基足以繁殖 [URE3]，或诱导 [URE3] 从头出现。我们发现 Ure2p 朊病毒结构域在体外形成淀粉样蛋白丝。此外，正如朊病毒结构域在体内诱导朊病毒形成一样，它也会在体外诱导稳定可溶的天然 Ure2p 形成淀粉样蛋白。体外 Ure2p 淀粉样蛋白形成的特性反映并解释了体内 [URE3] 的朊病毒特性。因此我们提出[URE3]朊病毒是一种传染性淀粉样变性。我们证明 Ure2p 片段或与其他蛋白质的融合可以有效治愈朊病毒。这种现象可能是由于片段或融合蛋白中断了淀粉样蛋白“晶体”的生长，并提出了治疗淀粉样蛋白疾病的新方法。我们发现 Mks1 蛋白对于 [URE3] 朊病毒的从头形成至关重要。 Mks1 活性受到 Ras - cAMP 通路的负向调节，我们发现 Ras2p 的激活通过灭活 Mks1 来阻止 [URE3] 朊病毒的从头形成。我们表明，Hsp104 分子伴侣对于 [URE3] 朊病毒的繁殖是必需的，并且 Hsp40 家族分子伴侣 Ydj1p 的过度表达可以治愈 [URE3] 朊病毒。我们最近表明，Hsp70 家族伴侣 Ssa2p 对于 [URE3] 的繁殖也是必需的。与博士合作。 Vladislav Speransky 和 ​​Alasdair Steven (NIAMS) 博士进一步证明，带有 [URE3] 朊病毒的细胞含有由 Ure2 蛋白组成的细丝网络。此外，[URE3]菌株提取物中的大部分Ure2p即使在3M尿素和2%SDS中煮沸后仍处于不溶形式，证实其处于淀粉样蛋白状态。我们的合作者，博士。 Tim Umland 和 David Davies（LMB，NIDDK）确定了 Ure2p 氮调节结构域的结构，并发现它与谷胱甘肽-S-转移酶 (GST) 非常相似。 Ure2p 因体内朊病毒（淀粉样蛋白）的形成而失活。我们发现 Ure2p 并不是由于分子功能部分的构象变化而失活，而是由于 Ure2p 与 Gln3p 相互作用的空间效应或扩散限制而失活。 Ure2-GFP 融合蛋白形成螺旋状淀粉样蛋白丝。每根细丝内螺旋重复的长度是恒定的，但该长度从一根细丝到另一根细丝的变化超过 2 倍。这可能是朊病毒株的基础，它们具有不同的感染特性并对宿主产生不同的影响。 我们从其他酿酒酵母菌株、各种致病性念珠菌属物种和丝状真菌中分离出了 URE2 基因的同源物。虽然 C 端结构域高度保守且同源物可以替代酿酒酵母 Ure2p，但 N 端结构域（最多残基 99）却高度可变。尽管如此，从残基 10 到 39 的朊病毒结构域有一个保守部分。根据当该片段过表达时 Ure2p 失活来判断，该区域显然与 Ure2p C 末端相互作用。该区域还负责通过与上述 GFP 融合来固化 [URE3] 朊病毒。 我们发现朊病毒结构域形成淀粉样蛋白丝的中央核心，残基1至65构成高度蛋白酶抗性部分。 Ure2p 残基 71-95 充当淀粉样蛋白核心和外围排列的功能域（残基 95-354）之间的接头。 Ure2p 的单体通过朊病毒结构域之间的相互作用相互结合。 我们最近描述了一类全新的朊病毒，它不是基于淀粉样蛋白的形成，而是基于酵母液泡蛋白酶 B (PrB) 反式自动激活的需要。缺乏活性 PrB 的细胞会保持这种状态，除了罕见的 (10^-5) 自发激活酶之外。具有活性 PrB 的细胞产生的后代几乎全部都具有活性酶。这些细胞还可以通过活性 PrB 的转移来感染没有活性酶的细胞。因此，活性形式的 PrB 是一种感染性蛋白（朊病毒）。有许多蛋白质是自身激活所必需的，因此它们可能充当朊病毒。 Ure2p 的 N 端朊病毒结构域（残基 1-90）富含 N 和 Q 残基，我们表明这些残基对于朊病毒的形成和传播很重要。然而，我们发现朊病毒结构域中没有必需的氨基酸序列元件：这些氨基酸的五次随机改组留下了可以在体内形成朊病毒和在体外形成淀粉样蛋白的蛋白质。这一令人惊讶的结果表明，就 Ure2p 而言，氨基酸组成（而不是序列）是朊病毒形成的关键驱动力。我们现在正在测试其他朊病毒形成蛋白以扩展这一结果。 根据 X 射线衍射和电子衍射（与 U. Baxa、D. R. Davies、A. C. Steven 合作）判断，Ure2p 的淀粉样蛋白具有高度 β 片层结构的淀粉样蛋白核心，其 β 链垂直于丝轴。该结果证实了 Ure2p 形成的细丝的淀粉样蛋白性质。 我们对 [URE3] 朊病毒生成的 Mks1p 需求机制的研究表明，这种需求与其在调节谷氨酸生物合成中的作用无关，并且 cAMP 依赖性蛋白激酶的表达改变会改变 [URE3] 生成，正如我们之前预测的那样研究。 使用表达芯片分析，我们发现除了 Ure2p 在氮分解代谢调节中的作用之外，[URE3] 朊病毒不会诱导任何转录变化。 使用酵母两种杂交方法，我们检测到了一组在体内与 Ure2p 朊病毒结构域相互作用的蛋白质。目前正在研究这些蛋白质对朊病毒产生或传播的可能影响。在另一项研究中，我们使用酵母缺失库来寻找与朊病毒生成有关的基因。目前正在对一些候选人进行进一步研究，以确定他们的参与机制。