Development of a live cell assay for real-time monitoring the interactions between the Hippo pathway components 14-3-3 and TAZ

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery Pub Date : 2024-11-05 DOI:10.1016/j.slasd.2024.100191

Blaž Andlovic , Alexander Wolf , Malgorzata Hiltmann , Bert M. Klebl , Jan Eickhoff , Christian Ottmann

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引用次数: 0

Abstract

The Hippo pathway plays an important role in organ size control and tissue homeostasis. Dysregulation is involved in many pathologies, including cancer, which has attracted interest in targeting the Hippo pathway. Since the upstream components are bona fide tumor suppressors, it is feasible to target oncogenic downstream targets such as TAZ, a key downstream effector in the Hippo pathway. Its activity is regulated by phosphorylation on multiple sites, with Ser89 playing a critical role in regulation of TAZ activity. Phosphorylation of TAZ at Ser89 promotes binding to 14–3–3 scaffolding proteins, preventing nuclear translocation and abolishing target gene transcription. Here we describe the development of a cell-based assay suitable for high-throughput screening, based on a split NanoLuc luciferase, for monitoring interactions between 14 3–3 and TAZ in living cells. We have validated the assay by screening of a kinase-biased library. The assay can be quickly adapted for higher throughput and thus offers a valuable tool to study new signal inputs involved in regulation of TAZ activity as well as for identification of molecules that modulate the Hippo pathway.

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开发实时监测 Hippo 通路成分 14-3-3 和 TAZ 之间相互作用的活细胞检测方法。

希波通路在器官大小控制和组织稳态中发挥着重要作用。失调涉及包括癌症在内的多种病症，这引起了人们对靶向 Hippo 通路的兴趣。由于上游成分是真正的肿瘤抑制因子，因此以致癌的下游靶点为目标是可行的，例如希波通路的关键下游效应物 TAZ。它的活性受多个位点的磷酸化调控，其中 Ser89 在 TAZ 活性调控中起着关键作用。TAZ在Ser89上的磷酸化会促进与14-3-3支架蛋白的结合，阻止核转位并取消靶基因转录。在此，我们介绍了一种基于细胞的检测方法的开发情况，该方法适用于高通量筛选，它基于分裂的 NanoLuc 荧光素酶，用于监测活细胞中 14-3-3 和 TAZ 之间的相互作用。我们通过筛选激酶偏倚库验证了该检测方法。该检测方法可以快速适应更高的通量，因此为研究参与 TAZ 活性调控的新信号输入以及鉴定调节 Hippo 通路的分子提供了一种有价值的工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

SLAS Discovery Chemistry-Analytical Chemistry

CiteScore

7.00

自引率

3.20%

发文量

审稿时长

39 days

期刊介绍： Advancing Life Sciences R&D: SLAS Discovery reports how scientists develop and utilize novel technologies and/or approaches to provide and characterize chemical and biological tools to understand and treat human disease. SLAS Discovery is a peer-reviewed journal that publishes scientific reports that enable and improve target validation, evaluate current drug discovery technologies, provide novel research tools, and incorporate research approaches that enhance depth of knowledge and drug discovery success. SLAS Discovery emphasizes scientific and technical advances in target identification/validation (including chemical probes, RNA silencing, gene editing technologies); biomarker discovery; assay development; virtual, medium- or high-throughput screening (biochemical and biological, biophysical, phenotypic, toxicological, ADME); lead generation/optimization; chemical biology; and informatics (data analysis, image analysis, statistics, bio- and chemo-informatics). Review articles on target biology, new paradigms in drug discovery and advances in drug discovery technologies. SLAS Discovery is of particular interest to those involved in analytical chemistry, applied microbiology, automation, biochemistry, bioengineering, biomedical optics, biotechnology, bioinformatics, cell biology, DNA science and technology, genetics, information technology, medicinal chemistry, molecular biology, natural products chemistry, organic chemistry, pharmacology, spectroscopy, and toxicology. SLAS Discovery is a member of the Committee on Publication Ethics (COPE) and was published previously (1996-2016) as the Journal of Biomolecular Screening (JBS).