Bader Reto, Seeliger Markus A, Kelly Sadie E, Ilag Leopold L, Meersman Filip, Limones Alejandra, Luisi Ben F, Dobson Christopher M, Itzhaki Laura S
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
J Biol Chem. 2006 Jul 7;281(27):18816-24. doi: 10.1074/jbc.M603628200. Epub 2006 May 4.
The Saccharomyces cerevisiae Cks protein Cks1 has a COOH-terminal glutamine-rich sequence not present in other homologues. Cks proteins domain swap to form dimers but unique to Cks1 is the anti-parallel arrangement of protomers within the dimer. Despite the differences in Cks1 compared with other Cks proteins, we find the domain swapping properties are very similar. However, aggregation of Cks1 occurs by a route distinct from the other Cks proteins studied to date. Cks1 formed fibrillar aggregates at room temperature and neutral pH. During this process, Cks1 underwent proteolytic cleavage at a trypsin-like site into two fragments, the globular Cks domain and the glutamine-rich COOH terminus. At high protein concentrations, the rate of fibril formation was the same as the rate of proteolysis. The dominant species present within the fibrils was the glutamine-rich sequence. Consistent with this result, fibril formation was enhanced by addition of trypsin. Moreover, a truncated variant lacking the glutamine-rich sequence did not form fibrils under the same conditions. A lag phase at low protein concentrations indicates that fibril formation occurs through a nucleation and growth mechanism. The aggregates appear to resemble amyloid fibrils, in that they show the typical cross-beta x-ray diffraction pattern. Moreover, infrared spectroscopy data indicate that the glutamine side chains are hydrogen-bonded along the axis of the fibril. Our results indicate that the proteolytic reaction is the crucial step initiating aggregation and demonstrate that Cks1 is a simple, tunable model system for exploring aggregation mechanisms associated with polyglutamine deposition diseases.
酿酒酵母Cks蛋白Cks1具有一个COOH末端富含谷氨酰胺的序列，该序列在其他同源物中不存在。Cks蛋白通过结构域交换形成二聚体，但Cks1独有的是二聚体内原体的反平行排列。尽管Cks1与其他Cks蛋白存在差异，但我们发现其结构域交换特性非常相似。然而，Cks1的聚集通过一条不同于迄今研究的其他Cks蛋白的途径发生。Cks1在室温及中性pH条件下形成纤维状聚集体。在此过程中，Cks1在一个类胰蛋白酶位点发生蛋白水解切割，形成两个片段，球状的Cks结构域和富含谷氨酰胺的COOH末端。在高蛋白浓度下，纤维形成速率与蛋白水解速率相同。纤维内存在的主要物种是富含谷氨酰胺的序列。与该结果一致，添加胰蛋白酶可增强纤维形成。此外，缺乏富含谷氨酰胺序列 的截短变体在相同条件下不形成纤维。低蛋白浓度下的延迟期表明纤维形成通过成核和生长机制发生。这些聚集体似乎类似于淀粉样纤维，因为它们显示出典型的交叉β x射线衍射图案。此外，红外光谱数据表明谷氨酰胺侧链沿纤维轴形成氢键。我们的结果表明蛋白水解反应是引发聚集的关键步骤，并证明Cks1是一个用于探索与多聚谷氨酰胺沉积疾病相关的聚集机制的简单且可调控的模型系统。
