Department of Biochemistry, Molecular and Structural Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
Biol Cell. 2010 Mar 17;102(6):319-34. doi: 10.1042/BC20090163.
Protein aggregation is a major contributor to the pathogenic mechanisms of human neurodegenerative diseases. Mutations in the CSTB (cystatin B) gene [StB (stefin B)] cause EPM1 (progressive myoclonus epilepsy of type 1), an epilepsy syndrome with features of neurodegeneration and increased oxidative stress. Oligomerization and aggregation of StB in mammalian cells have recently been reported. It has also been observed that StB is overexpressed after seizures and in certain neurodegenerative conditions, which could potentially lead to its aggregation. Human StB proved to be a good model system to study amyloid fibril formation in vitro and, as we show here, to study protein aggregation in cells.
Endogenous human StB formed smaller, occasional cytoplasmic aggregates and chemical inhibition of the UPS (ubiquitin-proteasome system) led to an increase in the amount of the endogenous protein and also increased its aggregation. Further, we characterized both the untagged and T-Sapphire-tagged StB on overexpression in mammalian cells. Compared with wild-type StB, the EPM1 missense mutant (G4R), the aggregate-prone EPM1 mutant (R68X) and the Y31 StB variant (both tagged and untagged) formed larger cytosolic and often perinuclear aggregates accompanied by cytoskeletal reorganization. Non-homogeneous morphology of these large aggregates was revealed using TEM (transmission electron microscopy) with StB detected by immunogold labelling. StB-positive cytoplasmic aggregates were partially co-localized with ubiquitin, proteasome subunits S20 and S26 and components of microfilament and microtubular cytoskeleton using confocal microscopy. StB aggregates also co-localized with LC3 and the protein adaptor p62, markers of autophagy. Flow cytometry showed that protein aggregation was associated with reduced cell viability.
We have shown that endogenous StB aggregates within cells, and that aggregation is increased upon protein overexpression or proteasome inhibition. From confocal and TEM analyses, we conclude that aggregates of StB show some of the molecular characteristics of aggresomes and may be eliminated from the cell by autophagy. Intracellular StB aggregation shows a negative correlation with cell survival.
蛋白质聚集是人类神经退行性疾病发病机制的主要原因之一。CSTB（半胱氨酸蛋白酶抑制剂 B）基因[StB（Stefin B）]的突变导致 EPM1（1 型进行性肌阵挛性癫痫），这是一种具有神经退行性和氧化应激增加特征的癫痫综合征。最近有报道称，哺乳动物细胞中的 StB 发生寡聚化和聚集。也观察到癫痫发作后和某些神经退行性疾病中 StB 表达过度，这可能导致其聚集。人类 StB 被证明是研究体外淀粉样纤维形成的良好模型系统，正如我们在这里展示的，也是研究细胞内蛋白质聚集的良好模型系统。
内源性人 StB 形成较小的、偶尔的细胞质聚集体，化学抑制 UPS（泛素-蛋白酶体系统）导致内源性蛋白含量增加，同时也增加了其聚集。此外，我们对哺乳动物细胞中过表达的未标记和 T-Sapphire 标记的 StB 进行了特征描述。与野生型 StB 相比，EPM1 错义突变体（G4R）、易聚集的 EPM1 突变体（R68X）和 Y31 StB 变体（标记和未标记）形成较大的细胞质和常位于核周的聚集体，同时伴有细胞骨架重排。使用 TEM（透射电子显微镜）和免疫金标记检测 StB，揭示了这些大聚集体不均匀的形态。使用共聚焦显微镜，StB 阳性的细胞质聚集体部分与泛素、蛋白酶体亚基 S20 和 S26 以及微丝和微管细胞骨架的成分共定位。StB 聚集体也与 LC3 和蛋白接头 p62 共定位，后者是自噬的标志物。流式细胞术显示，蛋白质聚集与细胞活力降低有关。
我们已经表明，内源性 StB 在细胞内聚集，并且在蛋白过表达或蛋白酶体抑制时聚集增加。通过共聚焦和 TEM 分析，我们得出结论，StB 的聚集体显示出一些聚集物的分子特征，并且可能通过自噬从细胞中被清除。细胞内 StB 聚集与细胞存活呈负相关。
