Polajnar Mira, Zavašnik-Bergant Tina, Kopitar-Jerala Nataša, Tušek-Žnidarič Magda, Zerovnik Eva
Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia.
Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
Biochim Biophys Acta. 2014 Sep;1843(9):2089-99. doi: 10.1016/j.bbamcr.2014.05.018. Epub 2014 Jun 5.
EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death. We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.
EPM1是一种罕见的进行性肌阵挛癫痫，患者小脑会出现细胞凋亡。stefin B（胱抑素B）基因的突变是EPM1潜在主要缺陷的病因。以stefin B聚集体为模型，我们探究了是蛋白质聚集还是氧化应激先出现，以及这两个过程如何与细胞死亡相关联。我们之前研究了stefin B野生型、G4R突变体和R68X片段在细胞中的聚集情况（Ceru等人，2010年，《生物细胞》）。本研究对人类stefin B的另外两个错义突变体G50E和Q71P进行了研究，它们在过表达时同样显示出大量聚集体。研究表明，在带有聚集体的细胞中，突变体和寡聚体依赖性的氧化应激及细胞死亡增加。另一方面，聚集体的大小和数量与细胞死亡之间没有相关性。我们认为聚集体毒性的差异取决于它们是处于寡聚体/原纤维形式还是纤维状形式。这反过来可能取决于突变体的三维结构，其中未折叠的蛋白质毒性较低。透射电子显微镜成像显示，细胞中的聚集体有不同类型：较大的核周聚集体，被膜包围，有时呈现囊泡状内陷，或者较小、点状且分散在整个细胞质中。所研究的所有EPM1突变体作为半胱氨酸蛋白酶抑制剂均无活性，以此方式导致stefin B功能丧失。本文还讨论了通过毒性功能增强与EPM1疾病的相关性。
