Folding and fibril formation of the cell cycle protein Cks1
Folding and fibril formation of the cell cycle protein Cks1
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DOI：
10.1074/jbc.m603628200
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发表时间：
2006-07-07
影响因子：
4.8
通讯作者：
Itzhaki, Laura S.
中科院分区：
文献类型：
--
作者：
Bader, Reto;Seeliger, Markus A.;Itzhaki, Laura S.
酿酒酵母Cks蛋白Cks1具有在其他同源物中不存在的COOH末端富含谷氨酰胺的序列。Cks蛋白结构域交换形成二聚体，但Cks1的独特之处在于二聚体内的原聚体的反平行排列。尽管Cks1与其他Cks蛋白相比存在差异，但我们发现结构域交换性质非常相似。然而，聚集的Cks1发生的路径不同的其他Cks蛋白质研究的日期。Cks1在室温和中性pH下形成纤维状聚集体。在此过程中，Cks1在胰蛋白酶样位点进行蛋白水解裂解成两个片段，球状Cks结构域和富含谷氨酰胺的COOH末端。在高蛋白质浓度下，原纤维形成的速率与蛋白质水解的速率相同。原纤维内的优势物种是富含谷氨酰胺的序列。与该结果一致，通过添加胰蛋白酶增强原纤维形成。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原纤维。在低蛋白质浓度下的滞后期表明原纤维形成通过成核和生长机制发生。聚集体看起来类似于淀粉样蛋白原纤维，因为它们显示出典型的交叉β射线衍射图案。此外，红外光谱数据表明，谷氨酰胺侧链是氢键沿着轴的原纤维。我们的研究结果表明，蛋白水解反应是启动聚集的关键步骤，并表明Cks1是一个简单的，可调的模型系统，用于探索与多聚谷氨酰胺沉积疾病相关的聚集机制。
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-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.