Enhanced sensitivity of an Ah-receptor system in yeast through condition modification and use of mammalian modifiers.
Enhanced sensitivity of an Ah-receptor system in yeast through condition modification and use of mammalian modifiers.
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DOI：
10.1016/j.toxrep.2022.03.012
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发表时间：
2022
影响因子：
--
通讯作者：
Bradfield CA
中科院分区：
文献类型：
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作者：
Vazquez-Rivera E;Rojas BL;Carney PR;Marrero-Valentin JL;Bradfield CA
Ah 受体 (AHR) 等蛋白质具有作为传感器检测环境和生物样品中配体的潜力，也可以作为调节生物合成和工业过程的工具。 AHR 也是 PAS 超家族的原型系统，可以感知和调节对光、电压、氧气和一系列小分子等多种信号的适应。酿酒酵母已被证明是研究 AHR 等传感器信号转导的重要模型，因为它易于使用、有多种可用的遗传操作策略以及异源表达的能力。为了更好地了解传感器蛋白作为酵母检测系统组件的效用，我们表征了嵌合 AHR-LexA 系统，该系统驱动 Lex 算子 (LexO) 驱动的 β-半乳糖苷酶 (β-Gal) 报告基因的表达。在本报告中，我们证明了通过仔细优化酵母生长阶段和配体暴露持续时间可以提高检测灵敏度和药理学。我们还报告说，来自哺乳动物细胞的异型修饰剂（例如 ARA9 和 ARA3 蛋白）的共表达可以提高酵母分析性能。我们建议，用其他人描述的先前报告的酵母突变来补充这些测定改进将扩大 AHR 在生物技术应用中的效用。基于酵母的 AHR 配体嵌合测定的优化。描述测定条件，以加强许多实验室之间的合作并协调相关数据。通过减少配体暴露时间和 AHR 相关蛋白的共表达，可以提高测定的灵敏度。
Proteins, such as the Ah receptor (AHR), hold potential as sensors to detect ligands in environmental and biological samples, and may also serve as tools to regulate biosynthetic and industrial processes. The AHR is also a prototype system for the PAS superfamily that can sense and mediate adaptation to signals as diverse as light, voltage, oxygen and an array of small molecules. The yeast, S. cerevisiae, has proven to be an important model to study the signal transduction of sensors like the AHR because of its ease of use, numerous available strategies for genetic manipulation, and capacity for heterologous expression. To better understand the utility of sensor proteins as components of yeast detection systems, we characterized a chimeric AHR-LexA system that drives expression from a Lex operator (LexO) driven, beta-galactosidase (β-Gal) reporter. In this report, we demonstrate that improvements in assays sensitivity and pharmacology can arise from the careful optimization of yeast growth phase and the duration of ligand exposure. We also report that the coexpression of heterotypic modifiers from mammalian cells (e.g., the ARA9 and ARA3 proteins), can improve yeast assay performance. We propose that complementing these assay improvements with previously reported yeast mutations described by others will expand the utility of the AHR for biotechnology applications. Optimization of a yeast based chimeric assay for AHR ligands. Description of assay conditions to enhance collaboration and harmonize the related data between many laboratories. Sensitivity increases in the assay by reduction of ligand exposure times, andcoexpression of of AHR associated proteins.