Mechanism of inactivation on prion conversion of the Saccharomyces cerevisiae Ure2 protein
Mechanism of inactivation on prion conversion of the Saccharomyces cerevisiae Ure2 protein
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DOI：
10.1073/pnas.082097899
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发表时间：
2002-04-16
影响因子：
11.1
通讯作者：
Wickner, RB
中科院分区：
文献类型：
--
作者：
Baxa, U;Speransky, V;Wickner, RB
酿酒酵母的[URE 3]感染性蛋白（朊病毒）是Ure 2 p的自繁殖淀粉样蛋白形式。Ure 2 p的C-末端结构域通过与转录因子Gln 3 p复合来控制氮催化剂，而富含天冬酰胺的N-末端“朊病毒”结构域负责淀粉样蛋白丝的形成（朊病毒转化）。在细丝形成时，Ure 2 p失活，反映了C-末端结构域的结构变化或其与Gln 3 p相互作用的空间阻断。我们融合了朊病毒结构域与四种蛋白质，其活动不应受到空间阻碍的聚集，因为他们的底物是非常小的：芽孢杆菌RNA酶，碳酸酐酶，谷胱甘肽S-转移酶，和绿色荧光蛋白。所有形成淀粉样纤维在体外，其直径增加的附加酶的质量。螺旋重复长度在单个细丝内是一致的，但随着构建体和来自单个构建体的细丝之间而变化。CID数据表明，在可溶性融合蛋白中，朊病毒结构域没有规则的二级结构，而早期的数据表明，在细丝中，它几乎都是β-折叠。在细丝中，附加蛋白质的活性最多温和地降低，当底物扩散效应被考虑在内时，表明它们保留了它们的天然结构。这些观察结果表明，这些细丝的淀粉样蛋白含量仅限于其含有朊病毒结构域的骨架，并暗示Ure 2 p在[URE 3]细胞中通过空间阻断机制失活。
The [URE3] infectious protein (prion) of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The C-terminal domain of Ure2p controls nitrogen catabolism by complexing with the transcription factor, Gln3p, whereas the asparagine-rich N-terminal ''prion'' domain is responsible for amyloid filament formation (prion conversion). On filament formation, Ure2p is inactivated, reflecting either a structural change in the C-terminal domain or steric blocking of its interaction with Gln3p. We fused the prion domain with four proteins whose activities should not be sterically impeded by aggregation because their substrates are very small: barnase, carbonic anhydrase, glutathione S-transferase, and green fluorescent protein. All formed amyloid filaments in vitro, whose diameters increased with the mass of the appended enzyme. The helical repeat lengths were consistent within a single filament but varied with the construct and between filaments from a single construct. CID data suggest that, in the soluble fusion proteins, the prion domain has no regular secondary structure, whereas earlier data showed that in filaments, it is virtually all beta-sheet. In filaments, the activity of the appended proteins was at most mildly reduced, when substrate diffusion effects were taken into account, indicating that they retained their native structures. These observations suggest that the amyloid content of these filaments is confined to their prion domain-containing backbones and imply that Ure2p is inactivated in [URE3] cells by a steric blocking mechanism.