Prions Of Yeast

Prion是一种具有感染性的蛋白质，这一概念源于对哺乳动物海绵状脑病的研究。我们发现，酿酒酵母可以感染两个普里恩，两个非染色体的遗传元件，它们的性质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的一种改变形式，Ure2p是染色体上URE2基因的蛋白质产物，在氮代谢调节中起重要作用。第二个，[PSI]，是Sup35p的改变形式，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，与野生型菌株相比，[URE3]菌株中的Ure2p对蛋白酶消化的抵抗力更强，支持[URE3]的普恩模型。这种对蛋白水解酶的抵抗力并不是氮代谢去抑制的持续伴随。在完全缺乏C-末端氮调节域的情况下，Ure2p的N-端65个氨基酸残基足以繁殖[URE3]。诱导[URE3]从头形成的是URE2蛋白的过度表达，而不是URE2RNA的过度表达。只有当共价连接到N-末端结构域上时，C-末端氮调节结构域才被失活，这表明[URE3]是通过N-末端蛋白结构域之间的相互作用而传播的。在Ure2p的C-末端氮调节域内的某些缺失导致[URE3]蛋白出现的频率增加100倍。事实上，Ure2p的两个非重叠部分中的任何一个在过度表达时都可以诱导[URE3]从头开始出现。我们发现Ure2p在体内聚集在携带Prion[URE3]的细胞中。这种聚集需要Prion结构域，并在[URE3]固化的细胞中消失。我们发现Ure2p蛋白结构域(残基1-65)在体外形成淀粉样丝。其他类似大小的Ure2p片段，如果不包括Pron结构域，则不会形成淀粉样蛋白。Pron结构域通过纯化的天然可溶性Ure2p促进淀粉样细丝的形成。这一反应的特异性在于，Ure2p蛋白结构域不会通过其他蛋白质促进淀粉样蛋白的形成，淀粉样蛋白形成肽Abeta也不会通过Ure2p蛋白促进淀粉样蛋白的形成。Ure2p淀粉样蛋白的体外形成特性反映和解释了体内[URE3]的Prion特性。我们认为[URE3]Prion是一种传染性淀粉样变性。我们发现，Ure2p片段的过度表达，或某些Ure2p-GFP融合蛋白在正常水平的表达，会导致[URE3]Prion的有效治愈。这种现象可能是由于碎片或融合蛋白导致淀粉样蛋白“晶体”的生长中断，并提示了一种治疗淀粉样蛋白疾病的新方法。我们发现Mks1蛋白对于[URE3]蛋白的从头形成是必不可少的。Mks1的活性受Ras-cAMP途径的负调控，我们发现Ras2p的激活通过使Mks1失活来阻止从头形成[URE3]Pron。我们现在发现Hsp104是[URE3]Pron繁殖所必需的。我们还发现，Hsp40家族伴侣Ydj1p的过表达可以治愈[URE3]Prion。在与Vladislav Speransky博士和Alasdair Steven博士(NIAMS)的合作中，我们进一步表明，带有[URE3]Pron的细胞包含由Ure2蛋白组成的细丝网络。这样的丝状网络不存在于没有普鲁恩的细胞中。此外，[URE3]菌株提取物中的大部分Ure2p在3M尿素和2%SDS中煮沸后仍处于不溶状态，证实其处于淀粉样蛋白状态。我们的合作者Tim Umland和David Davies博士(LMB，NIDDK)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.