Prions Of Yeast

朊病毒是一种感染性蛋白质，这一概念来源于对哺乳动物海绵状脑病的研究。我们发现酿酒酵母可以被两种朊病毒感染，这两种朊病毒是两种非染色体遗传元件，它们的特性是预测的感染性蛋白质，而不是核酸复制子或病毒。首先，[URE3]是Ure2p的改变形式，Ure2p是染色体URE2基因的蛋白质产物，在氮代谢的调节中很重要。第二个，[PSI]，是Sup35p的改变形式，Sup35p是翻译释放因子的亚基和染色体SUP35基因的产物。我们发现Ure2p在[URE3]菌株中比在野生型菌株中对蛋白酶消化更有抗性，支持[URE3]的朊病毒模型。这种蛋白酶抗性不是氮代谢去抑制的恒定伴随物。在完全不存在C-末端氮调节结构域的情况下，Ure2p的N-末端65个氨基酸残基足以繁殖[URE3]。URE2蛋白的过表达，而不是URE2 RNA，是诱导[URE3]从头形成的原因。C-末端氮调节结构域仅在共价连接到N-末端结构域时失活，表明[URE3]通过N-末端朊病毒结构域之间的相互作用传播。Ure2p的C-末端氮调节结构域内的某些缺失导致[URE3]朊病毒出现的频率增加100倍。事实上，Ure2p的两个非重叠部分中的任何一个在过表达时都可以诱导[URE3]的从头出现。我们发现Ure2p在体内聚集在携带朊病毒的细胞中[URE3]。这种聚集需要朊病毒结构域，并在[URE3]治愈的细胞中消失。我们发现Ure2p朊病毒结构域（残基1 - 65）在体外形成淀粉样蛋白丝。其他不包括朊病毒结构域的类似大小的Ure2p片段不形成淀粉样蛋白。朊病毒结构域通过纯化的天然可溶性Ure2p促进淀粉样蛋白丝的形成。该反应是特异性的，因为Ure2p朊病毒结构域不促进其他蛋白质的淀粉样蛋白形成，淀粉样蛋白形成肽Abeta也不促进Ure2p的淀粉样蛋白形成。体外Ure2p淀粉样蛋白形成的性质反映并解释了体内[URE3]的朊病毒性质。我们认为[URE3]朊病毒是一种传染性淀粉样变性。我们发现Ure2p片段的过表达，或某些Ure2p-GFP融合蛋白在正常水平的表达导致[URE3]朊病毒的有效治愈。这种现象可能是由于淀粉样蛋白“晶体”的生长被碎片或融合蛋白所中断，并提出了一种治疗淀粉样蛋白疾病的新方法。我们发现，Mks1蛋白是必不可少的从头形成的[URE3]朊病毒。Mks1活性受Ras-cAMP途径负调控，我们发现Ras2p的激活通过灭活Mks1阻止了从头[URE3]朊病毒的形成。 我们现在发现Hsp104分子伴侣是[URE3]朊病毒传播所必需的。我们还发现Hsp40家族伴侣Ydj1p的过表达可以治愈[URE3]朊病毒。 在与Vladislav Speransky和Alasdair Steven（NIAMS）博士的合作中，我们进一步证明了带有[URE3]朊病毒的细胞含有由Ure2蛋白组成的细丝网络。这种丝状网络在没有朊病毒的细胞中并不存在。此外，[URE3]菌株的提取物中的大多数Ure2p即使在3M尿素和2%SDS中煮沸后也是不溶的形式，证实其处于淀粉样状态。 我们的合作者Tim Umland和大卫戴维斯（LMB，NIDDK）博士已经确定了Ure2p的氮调节结构域的结构，并发现它与谷胱甘肽-S-转移酶非常相似。

A prion is an infectious protein, a concept derived from studies of mammalian spongiform encephalopathies. We discovered that the yeast Saccharomyces cerevisiae can be infected with two prions, two non-chromosomal genetic elements whose properties are those predicted for an infectious protein, not a nucleic acid replicon or a virus. The first, [URE3] is an altered form of Ure2p, the protein product of the chromosomal URE2 gene important in regulation of nitrogen metabolism. 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, supporting the prion model for [URE3]. This protease resistance is not a constant concomitant of derepression of nitrogen metabolism. The N-terminal 65 aminoacid residues of Ure2p is sufficient to propagate [URE3], in the complete absence of the C-terminal nitrogen regulation domain. The overexpression of the URE2 protein, and not the URE2 RNA, is what induces the de novo formation of [URE3]. The C-terminal nitrogen regulation domain is only inactivated when covalently attached to the N-terminal domain, showing that [URE3] is propagated by interactions between the N-terminal prion domains. Certain deletions within the C-terminal nitrogen regulation domain of Ure2p result in a 100-fold increase the frequency with which the [URE3] prion arises. In fact, either of two non-overlapping parts of Ure2p can, when overexpressed, induce the de novo appearance of [URE3]. We showed that Ure2p is aggregated in vivo in cells carrying the prion [URE3]. This aggregation requires the prion domain, and disappears in cells cured of [URE3]. We showed that the Ure2p prion domain (residues 1-65) forms amyloid filaments in vitro. Other similar size fragments of Ure2p that do not include the prion domain do not form amyloid. The prion domain promotes the formation of amyloid filaments by the purified native soluble Ure2p. This reaction is specific in that the Ure2p prion domain does not promote amyloid formation by other proteins, nor does the amyloid-forming peptide Abeta promote amyloid formation by Ure2p. The properties of Ure2p amyloid formation in vitro reflect and explain the prion properties of [URE3] in vivo. We propose that the [URE3] prion is an infectious amyloidosis. We find that overexpression of fragments of Ure2p, or expression of certain Ure2p-GFP fusion proteins at normal levels results in efficient curing of the [URE3] prion. 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 now find that the Hsp104 chaperone is necessary for [URE3] prion propagation. We also find that overexpression of the Hsp40-family chaperone Ydj1p can cure the [URE3] prion. 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. Such filamentous networks were not present in cells without the prion. 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.