Synthesis and structural characterization of a mimetic membrane-anchored prion protein
Synthesis and structural characterization of a mimetic membrane-anchored prion protein
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DOI：
10.1111/j.1742-4658.2006.05152.x
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发表时间：
2006-03-01
期刊：
影响因子：
5.4
通讯作者：
Pinheiro, TJT
中科院分区：
文献类型：
--
作者：
Hicks, MR;Gill, AC;Pinheiro, TJT
在传染性海绵状脑病（TSE）的发病过程中，宿主编码的朊病毒蛋白（PrP（C））的异常形式（PrP（Sc））积累在不溶性纤维和斑块中。PrP的两种形式似乎具有相同的共价结构，但二级和三级结构不同。PrP（C）和PrP（Sc）都具有糖基磷脂酰肌醇（GPI）锚，蛋白质通过该锚被拴系到细胞膜。膜附着被认为在PrP（C）向PrP（Sc）的转化中起作用，但是大多数PrP的功能、结构、折叠和稳定性的体外研究使用缺乏GPI锚的重组蛋白。为了研究膜对PrP结构的影响，我们合成了GPI锚模拟物（GPIm），我们将其共价偶联到重组PrP C-末端的基因工程半胱氨酸残基上。脂质锚将蛋白质放置在与天然存在的GPI锚相同的距离处。我们表明，PrP耦合到GPIm（PrP-GPIm）插入模型脂质膜，结构信息可以从这个膜锚定的PrP。我们发现，在磷脂酰胆碱和筏膜重构的PrP-GPIm的结构类似于PrP，没有GPI锚，在溶液中。结果提供了实验证据，支持以前的建议，NMR结构的可溶性，锚无形式的PrP代表细胞，膜锚定的PrP的结构。PrP的脂质锚定结构的可用性为研究不同脂质环境对PrP结构和转化机制的影响提供了独特的模型。
During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrP(Sc)) of the host encoded prion protein (PrP(C)) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrP(C) and PrP(Sc) have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrP(C) to PrP(Sc), but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.