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首页> 外文会议>American Institute of Chemical Engineers annual meeting >(66c) Design and Construction of a Protein Degradation Switch
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(66c) Design and Construction of a Protein Degradation Switch

机译：（66C）蛋白质降解开关的设计和构建

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Targeted protein degradation in eukaryotic cells requires recruitment of the target protein to proteasome which is usually triggered by ubiquitination of the target protein. It has recently been shown that ubiquitination is not necessary and localization to the proteasome is sufficient for degradation [1]. One intriguing way to bypass the ubiquitination step is artificial induction of target protein and proteasome dimerization. In this method, both the target protein and the proteasome are fused to particular protein domains and upon addition of a small molecule that can simultaneously bind to both domains, dimerization and consequently localization of the target protein to the proteasome occur. To implement this idea, we took advantage of a naturally occurring dimerizing molecule, rapamycin. We fused the Rpn10 subunit of Saccharomyces cerevisiae's proteasome with Fprl, and on the other hand we fused the target protein, such as Ura3, with the Fprl-rapamycin binding domain of Tori. Upon adding rapamycin to the cell culture medium, Fprl and Tori domains come together and hence the target protein is localized to the proteasome and degrades. We have shown previously that the above approach is applicable for certain target proteins [2]. However, its efficiency varies substantially across different proteins and the method remains ineffective for proteins that are inherently difficult to degrade. To overcome this limitation, we propose to utilize multiple binding modules to speed up the degradation process. After confirmation from simulation studies that increasing the number of binding modules would lead to faster degradation, we constructed a series of differently tagged proteins to test our hypothesis. To study systematically the effect of the number of binding modules, we made use of a mutated version of Tori that cannot bind to rapamycin. Triple tags with different combinations of wild-type and mutated Tori domains (eight combinations in total) were constructed and fused to Ura3. The temporal profile of Ura3 in response to rapamycin has been measured in various constructs and the half-life of the Ura3 protein has been estimated accordingly.
机译：真核细胞中的靶向蛋白质降解需要募集靶蛋白至蛋白酶体，该蛋白酶通常通过靶蛋白的普遍突出而引发。最近已经表明，泛素化不是必需的，并且对蛋白酶体的定位足以降解[1]。一种绕过泛素化步骤的一种有趣方式是人工诱导靶蛋白和蛋白酶体二聚化。在该方法中，靶蛋白和蛋白酶均融合到特定蛋白质结构域，并且在加入可以同时结合域，二聚化并因此将靶蛋白的定位同时结合到蛋白酶体中的小分子。为了实现这个想法，我们利用了天然存在的二聚，雷帕霉素。我们用FPRL融合了酿酒酵母酿酒酵母的蛋白酶蛋白酶体的RPN10亚基，另一方面，我们将靶蛋白如URA3与FPRL-雷帕霉素结合结构域融合。在向细胞培养基中加入雷帕霉素时，FPRL和TORI结​​构域均匀化，因此靶蛋白质局部化为蛋白酶体并降解。我们以前表明，上述方法适用于某些靶蛋白[2]。然而，其效率基本上变化在不同的蛋白质上，并且该方法对固有难以降解的蛋白质保持无效。为了克服这种限制，我们建议利用多个绑定模块来加速劣化过程。在确认仿真研究之后，增加绑定模块的数量会导致更快的降解，我们构建了一系列不同标记的蛋白质来测试我们的假设。为了系统地学习结合模块的数量的效果，我们使用了不能与雷帕霉素结合的突变的扭曲形式。构建和融合具有不同野生型和突变的TORI结​​构域（总共八种组合）的三重标签（总共八种组合）并融合到URA3。在各种构建体中测量了响应于雷帕霉素的URA3的时间轮廓，并且估计了URA3蛋白的半衰期。

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