Short half-life proteins regulate many essential processes, including cell cycle, transcription, and apoptosis. However, few well-characterized protein-turnover pathways have been identified because traditional methods to measure protein half-life are time and labor intensive. To overcome this barrier, we developed a protein stability probe and high-content screening pipeline for novel regulators of short half-life proteins using automated image analysis. Our pilot probe consists of the short half-life protein c-MYC (MYC) fused to Venus fluorescent protein (MYC-Venus). This probe enables protein half-life to be scored as a function of fluorescence intensity and distribution. Rapid turnover prevents maximal fluorescence of the probe due to the relatively longer maturation time of the fluorescent protein. Cells expressing the MYC-Venus probe were analyzed using a pipeline in which automated confocal microscopy and image analyses were used to score MYC-Venus stability by two strategies: assaying the percentage of cells with Venus fluorescence above background, and phenotypic comparative analysis. To evaluate this high-content screening pipeline and our probe, a kinase inhibitor library was screened by confocal microscopy to identify known and novel kinases that regulate MYC stability. Compounds identified were shown to increase the half-life of both MYC-Venus and endogenous MYC, validating the probe and pipeline. Fusion of another short half-life protein, myeloid cell leukemia 1 (MCL1), with Venus also demonstrated an increase in percent Venus-positive cells after treatment with inhibitors known to stabilize MCL1. Together, the results validate the use of our automated microscopy and image analysis pipeline of stability probe-expressing cells to rapidly and quantitatively identify regulators of short half-life proteins. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

中文翻译：

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基于图像的蛋白质稳定性分析。

短半衰期蛋白质调节许多重要过程，包括细胞周期、转录和细胞凋亡。然而，由于测量蛋白质半衰期的传统方法费时费力，因此很少有明确的蛋白质周转途径被确定。为了克服这一障碍，我们使用自动图像分析开发了一种蛋白质稳定性探针和高内涵筛选流程，用于短半衰期蛋白质的新型调节剂。我们的先导探针由与金星荧光蛋白 (MYC-Venus) 融合的短半衰期蛋白 c-MYC (MYC) 组成。该探针能够根据荧光强度和分布对蛋白质半衰期进行评分。由于荧光蛋白的成熟时间相对较长，快速周转会阻碍探针的最大荧光。使用管道分析表达 MYC-Venus 探针的细胞，其中使用自动共焦显微镜和图像分析通过两种策略对 MYC-Venus 稳定性进行评分：测定高于背景的 Venus 荧光的细胞百分比，以及表型比较分析。为了评估这种高内涵筛选流程和我们的探针，通过共聚焦显微镜筛选了激酶抑制剂库，以识别调节 MYC 稳定性的已知和新型激酶。鉴定出的化合物可延长 MYC-Venus 和内源 MYC 的半衰期，从而验证了探针和管道。另一种短半衰期蛋白髓样细胞白血病 1 (MCL1) 与 Venus 的融合也表明，在使用已知可稳定 MCL1 的抑制剂治疗后，Venus 阳性细胞的百分比有所增加。总之，这些结果验证了我们使用稳定性探针表达细胞的自动化显微镜和图像分析管道来快速定量地识别短半衰期蛋白质的调节因子。© 2019 作者。细胞计数法 A 部分由 Wiley periodicals, Inc. 代表国际细胞计数法促进会出版。