结构域置换如何影响兔/人朊病毒蛋白的原纤维形成？
How does domain replacement affect fibril formation of the rabbit/human prion proteins.
作者信息
Yan Xu, Huang Jun-Jie, Zhou Zheng, Chen Jie, Liang Yi
机构信息
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
出版信息
PLoS One. 2014 Nov 17;9(11):e113238. doi: 10.1371/journal.pone.0113238. eCollection 2014.
BACKGROUND
It is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and macromolecular crowding has different effects on fibril formation of the rabbit/human PrPs, we do not know which domains of PrPs cause such differences. In this study, we have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs.
METHODOLOGY/PRINCIPAL FINDINGS: As revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes fibril formation of both chimeras. As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP. However, amyloid fibrils formed by rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP. The results from transmission electron microscopy show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.
CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time that the domains beyond PrP-H2H3 (β-strand 1, α-helix 1, and β-strand 2) have a remarkable effect on fibrillization of the rabbit PrP but almost no effect on the human PrP. Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different effects on fibrillization of PrPs from different species.
背景
已知人体内的朊病毒蛋白（PrP）有形成纤维状沉积物的倾向，并与传染性致命朊病毒疾病相关，而兔PrP不易形成纤维，不太可能引发朊病毒疾病。尽管我们之前已经证明，兔PrP和人PrP形成的淀粉样纤维具有不同的二级结构，且大分子拥挤对兔/人PrP的纤维形成有不同影响，但我们尚不清楚PrP的哪些结构域导致了这些差异。在本研究中，我们构建了两种PrP嵌合体，兔嵌合体和人嵌合体，并研究了结构域替换如何影响兔/人PrP的纤维形成。
方法/主要发现：硫黄素T结合试验和 Sarkosyl 可溶性 SDS-PAGE 结果表明，强拥挤剂的存在显著促进了两种嵌合体的纤维形成。圆二色性、傅里叶变换红外光谱和蛋白酶K消化试验证明，人嵌合体形成的淀粉样纤维具有与人PrP形成的纤维相似的二级结构和蛋白酶K抗性特征。然而，兔嵌合体形成的淀粉样纤维在拥挤的生理环境中具有与兔PrP形成的纤维不同的蛋白酶K抗性特征和二级结构，在稀溶液中的二级结构与兔PrP相似。透射电子显微镜结果显示，大分子拥挤导致人嵌合体而非兔嵌合体形成短纤维和非纤维颗粒。
结论/意义：我们首次证明，PrP-H2H3（β链1、α螺旋1和β链2）之外的结构域对兔PrP的纤维化有显著影响，但对人PrP几乎没有影响。我们的研究结果有助于解释为什么兔PrP和人PrP形成的淀粉样纤维具有不同的二级结构，以及为什么大分子拥挤对不同物种PrP的纤维化有不同影响。
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