Cecchi Cristina, Pensalfini Anna, Baglioni Serena, Fiorillo Claudia, Caporale Roberto, Formigli Lucia, Liguri Gianfranco, Stefani Massimo
Department of Biochemical Sciences, University of Florence, Italy.
FEBS J. 2006 May;273(10):2206-22. doi: 10.1111/j.1742-4658.2006.05234.x.
Considerable attention has been paid to the high cytotoxic potential of small, prefibrillar aggregates of proteins/peptides, either associated or not associated with amyloid diseases. Recently, we reported that different cell types are variously affected by early aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N), a protein not involved in any disease. In this study, we provide detailed information on a chain of events triggered in Hend murine endothelial cells and IMR90 fibroblasts, which have previously been shown to be highly vulnerable or very resistant, respectively, to HypF-N aggregates. Initially, both cell lines displayed impaired viability upon exposure to HypF-N toxic aggregates; however, at longer exposure times, IMR90 cells recovered completely, whereas Hend cells did not. In particular, significant initial mitochondrial permeability transition (MPT) pore opening was found in IMR90 cells followed by a sudden repair of membrane integrity with rapid and efficient inhibition of cytochrome c and AIF release, and upregulation of Bcl-2. The greater resistance of IMR90 fibroblasts may also be due to a higher cholesterol content in the plasma membrane, which disfavours interaction with the aggregates. In contrast, Hend cells, which have less membrane cholesterol, showed delayed MPT opening with prolonged translocation of cytochrome c into the cytosol. Finally, the caspase 9 active fragment was increased significantly in both Hend and IMR90 cells; however, only Hend cells showed caspase 8 and caspase 3 activation with DNA fragmentation. From our data, the different responses of the two cell types to the same aggregates appear to be associated with two key events: (a) aggregate interaction with the plasma membrane, disfavoured by a high level of membrane cholesterol; and (b) alterations in mitochondrial functionality, leading to the release of pro-apoptotic stimuli, which are counteracted by upregulation of Bcl-2.
蛋白质/肽的小的、原纤维前聚集体的高细胞毒性潜力已受到相当多的关注，这些聚集体与淀粉样疾病有关或无关。最近，我们报道了不同细胞类型受到原核氢化酶成熟因子HypF的N端结构域（HypF-N）早期聚集体的不同影响，HypF是一种与任何疾病都无关的蛋白质。在本研究中，我们提供了关于在亨德氏小鼠内皮细胞和IMR90成纤维细胞中引发的一系列事件的详细信息，这两种细胞先前已分别被证明对HypF-N聚集体高度敏感或非常耐受。最初，两种细胞系在暴露于HypF-N毒性聚集体时均表现出活力受损；然而，在较长的暴露时间下，IMR90细胞完全恢复，而亨德氏细胞则没有。特别是，在IMR90细胞中发现了显著的初始线粒体通透性转换（MPT）孔开放，随后膜完整性突然修复，细胞色素c和凋亡诱导因子（AIF）的释放迅速且有效地受到抑制，同时Bcl-2上调。IMR90成纤维细胞更强的耐受性也可能归因于质膜中较高的胆固醇含量，这不利于与聚集体相互作用。相比之下，膜胆固醇含量较低的亨德氏细胞显示MPT开放延迟，细胞色素c向细胞质的转位延长。最后，亨德氏细胞和IMR90细胞中的半胱天冬酶9活性片段均显著增加；然而，只有亨德氏细胞显示出半胱天冬酶8和半胱天冬酶3的激活以及DNA片段化。根据我们的数据，两种细胞类型对相同聚集体的不同反应似乎与两个关键事件有关：（a）聚集体与质膜的相互作用，高水平的膜胆固醇不利于这种相互作用；（b）线粒体功能的改变，导致促凋亡刺激的释放，而Bcl-2的上调可抵消这种刺激。
