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A cell based screening approach for identifying protein degradation regulators

机译：一种鉴定蛋白质降解调节剂的基于细胞的筛选方法

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Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover.
机译：细胞转变通过调节降解途径的齐节作用来实现。在细胞周期的情况下，泛素介导的降解确保从一个相到另一个相的单向转变。例如，细胞周期调节器细胞周期蛋白B1的换档在中期后发生以诱导有丝分裂出口。为了更好地理解控制细胞周期蛋白B1周转的途径，将细胞周期蛋白B1的N-末端结构域融合给荧光素酶，以产生N-Cyclin B1-Luciferase蛋白，可用作蛋白质转换的报告。现有研究证明，使用本报告的基于细胞的筛网鉴定了抑制泛素连接酶控制细胞周期蛋白B1转交的小分子。我们的组适用于G2-M调节剂WEE1的这种方法，其中用于鉴定抑制控制WEE1转换的上游激酶的选择性小分子。在本研究中，我们提出了一种筛选方法，其中细胞周期调节剂与荧光素酶融合并用CDNA过表达以识别蛋白质周转的特定调节剂。我们用N-Cyclin B1-Luciferase融合蛋白过表达约14,000个CDNA，并相对于其他荧光素酶融合蛋白确定其稳态水平。我们将已知的APC / C调节剂CDH1和F箱蛋白FBX115鉴定为N-Cyclin B1-Luciferase稳态水平和周转的特定调节剂。统称，我们的研究表明，并行分析岩藻酶融合蛋白的稳态水平有助于鉴定蛋白质周转的特定调节剂。

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