Copper(II) inhibits in vitro conversion of prion protein into amyloid fibrils
Copper(II) inhibits in vitro conversion of prion protein into amyloid fibrils
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DOI：
10.1021/bi050251q
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发表时间：
2005-05-10
期刊：
影响因子：
2.9
通讯作者：
Baskakov, IV
中科院分区：
文献类型：
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作者：
Bocharova, OV;Breydo, L;Baskakov, IV
在最近的研究中，从含有89-230残基的重组PrP蛋白(rPrP 89-230)体外产生的淀粉样纤维被证明在转基因小鼠中产生可传播形式的Prion疾病(Legname等人，(2004)Science 305,673-676)。然而，在接种淀粉样蛋白纤维时观察到较长的孵育时间，这表明在体外产生的纤维蛋白具有较低的感染性滴度。这些结果强调了在体外确定Pron转化的最佳条件的必要性，在这种条件下，在无细胞系统中可以产生高水平的感染性。由于铜(II)参与了PrP蛋白的正常和病理功能，因此我们研究了Cu2+对重组PrP无细胞转化的影响。我们的结果表明，在pH 7.2和微摩尔浓度下，Cu2+抑制全长重组PrP(rPrP23-230)向淀粉样纤维的转化。这一影响对Cu2+的影响最为明显，对Zn2+的影响较小，而对Mn2+的影响不明显。在pH为6.0时，依赖Cu2+的抑制淀粉样蛋白形成的效果较差，此时rPrP23-230表现出较低的Cu2+结合能力。利用rPrP 89-230，我们发现，即使在没有八重复区的情况下，也会发生Cu2+依赖的抑制作用，但效果较差。我们的进一步研究表明，Cu2+通过稳定非淀粉样蛋白原PK抗性形式的α-rPrP来抑制转化。值得注意的是，Cu2+对预制的淀粉样纤维也有深远的影响。当Cu2+加入到纤维中时，诱导了单个纤维的长程卷曲，并增强了它们对PK的抗性。然而，它只对它们的二级结构产生了微小的变化。此外，Cu2+诱导淀粉样蛋白纤维进一步聚集成大团块，可能是通过八重复序列的铜离子纤维间配位作用。综上所述，我们的研究表明，Cu2+在Pron疾病发病机制中的作用是复杂的。由于Cu2+可以抑制病毒复制，同时稳定疾病特异性异构体，使其不被蛋白质降解清除，因此铜诱导的对病毒疾病进展的影响可能并不是一帆风顺的。
In recent studies, the amyloid fibrils produced in vitro from recombinant prion protein encompassing residues 89-230 (rPrP 89-230) were shown to produce transmissible form of prion disease in transgenic mice (Legname et al., (2004) Science 305, 673-676). Long incubation time observed upon inoculation of the amyloid fibrils, however, suggests that the fibrils generated in vitro have low infectivity titers. These results emphasize the need to define optimal conditions for prion conversion in vitro, under which high levels of infectivity can be generated in a cell-free system. Because copper(II) has been implicated in normal and pathological functions of the prion protein, here we investigated the effect of Cu2+ on cell-free conversion of recombinant PrP. Our results show that at pH 7.2 and at micromolar concentrations, Cu2+ inhibited conversion of full-length recombinant PrP (rPrP 23-230) into amyloid fibrils. This effect was most pronounced for CU2+, and less so for Zn2+, while Mn2+ had no effect on the conversion. CU2+ -dependent inhibition of the amyloid formation was less effective at pH 6.0, at which rPrP 23-230 displays lower Cu2+-binding capacity. Using rPrP 89-230, we found that Cu2+-dependent inhibition occurred even in the absence of octarepeat region; however, it was less effective. Our further studies indicated that Cu2+ inhibited conversion by stabilizing a nonamyloidogenic PK-resistant form of alpha-rPrP. Remarkably, Cu2+ also had a profound effect on preformed amyloid fibrils. When added to the fibrils, Cu2+ induced long-range coilin g of individual fibrils and enhanced their PK-resistance. It, however, produced only minor changes in their secondary structures. In addition, Cu2+ induced further aggregation of the amyloid fibrils into large clumps, presumably, through interfibrillar coordination of copper ions by octarepeats. Taken together, our studies suggest that the role of Cu2+ in the pathogenesis of prion diseases is complex. Because Cu2+ may inhibit prion replication, while at the same time stabilize disease-specific isoform against proteolytic clearance, the final outcome of copper-induced effect on progression of prion disease may not be straightforward.