Villa Valentina, Corsaro Alessandro, Thellung Stefano, Paludi Domenico, Chiovitti Katia, Venezia Valentina, Nizzari Mario, Russo Claudio, Schettini Gennaro, Aceto Antonio, Florio Tullio
Department of Oncology, Biology and Genetics, University of Genova, Viale Benedetto XV, 16132 Genova, Italy.
Ann N Y Acad Sci. 2006 Dec;1090:276-91. doi: 10.1196/annals.1378.030.
Prion diseases comprise a group of fatal neurodegenerative disorders that affect both animals and humans. The transition of the prion protein (PrP) from a mainly alpha-structured isoform (PrPC) to a prevalent beta-sheet-containing protein (PrPSc) is believed to represent a major pathogenetic mechanism in prion diseases. To investigate the linkage between PrP neurotoxicity and its conformation, we used a recombinant prion protein fragment corresponding to the amino acidic sequence 90-231 of human prion protein (hPrP90-231). Using thermal denaturation, we set up an experimental model to induce the process of conversion from PrPC to PrPSc. We report that partial thermal denaturation converts hPrP90-231 into a beta-sheet-rich isoform, displaying a temperature- and time-dependent conversion into oligomeric structures that share some physico-chemical characteristics with brain PrPSc. SH-SY5Y cells were chosen to characterize the potential neurotoxic effect of hPrP90-231 in its different structural conformations. We demonstrated that hPrP90-231 in beta-conformation, but not when alpha-structured, powerfully affected the survival of these cells. hPrP90-231 beta-structured caused DNA fragmentation and a significant increase in caspase-3 proteolytic activity (maximal effects+170%), suggesting the occurrence of apoptotic cell death. Finally, we investigated the involvement of MAP kinases in the regulation of beta-hPrP90-231-dependent apoptosis. We observed that the p38 MAP kinase blocker SB203580 prevented the apoptotic cell death evoked by hPrP90-231, and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. In conclusion, we demonstrate that the hPrP90-231 elicits proapoptotic activity when in beta-sheet-rich conformation and that this effect is mediated by p38 and caspase-3 activation.
朊病毒疾病是一组影响动物和人类的致命性神经退行性疾病。朊病毒蛋白（PrP）从主要为α结构的异构体（PrPC）转变为普遍含β折叠的蛋白（PrPSc），被认为是朊病毒疾病的主要致病机制。为了研究PrP神经毒性与其构象之间的联系，我们使用了与人类朊病毒蛋白（hPrP）氨基酸序列90 - 231相对应的重组朊病毒蛋白片段（hPrP90 - 231）。通过热变性，我们建立了一个实验模型来诱导从PrPC到PrPSc的转化过程。我们报告称，部分热变性将hPrP90 - 231转化为富含β折叠的异构体，呈现出温度和时间依赖性地转化为寡聚结构，这些寡聚结构与脑PrPSc具有一些物理化学特征。选择SH - SY5Y细胞来表征hPrP90 - 231在其不同结构构象下的潜在神经毒性作用。我们证明，处于β构象的hPrP90 - 231，而非α结构时，会强烈影响这些细胞的存活。β结构的hPrP90 - 231导致DNA片段化以及caspase - 3蛋白水解活性显著增加（最大效应增加170%），表明发生了凋亡性细胞死亡。最后，我们研究了丝裂原活化蛋白激酶（MAP激酶）在β - hPrP90 - 231依赖性凋亡调节中的作用。我们观察到p38 MAP激酶抑制剂SB203580可阻止hPrP90 - 231诱发的凋亡性细胞死亡，蛋白质印迹分析显示细胞暴露于该肽会诱导p38磷酸化。总之，我们证明hPrP90 - 231在富含β折叠的构象时会引发促凋亡活性，且这种效应是由p38和caspase - 3激活介导的。
