Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells.

Jeahee Ryu,Chungwon Kang,Dae Yeon Lee,Minhyeong Lee,Soyoun Kim,Seungil Park,Youngeun Kwon,Euiyeon Lee

doi:10.3390/ijms22094747

Jeahee Ryu, Chungwon Kang + Show 6 more

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https://doi.org/10.3390/ijms22094747

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Abstract

Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.

Highlights

Physical or emotional stress results in a surge of chemicals in the human body, including the stress hormone cortisol
To screen the glucocorticoid receptor (GR) effectors, we designed a sensing system based on the well-studied pair of Nostoc punctiforme (Npu) DnaE split inteins, which mediate fast and efficient protein trans-splicing (PTS) reactions [30,31,32]
To obstruct the spontaneous PTS of Npu DnaE intein, the GR-containing protein 2 was localized in the cytoplasm, whereas the mCherry-containing protein 1 was positioned in the nucleus by using the C-terminal nuclear localization signal (NLS) peptide

Summary

Introduction

Physical or emotional stress results in a surge of chemicals in the human body, including the stress hormone cortisol. Cortisol is a glucocorticoid (GC), an adrenal steroid hormone, which helps the body to respond to stress appropriately. Cortisol plays a key role in maintaining homeostasis and metabolism in the human body and coordinating the immune system’s negative feedback mechanism to lower immune activity [1,2,3,4,5,6]. Abnormal cortisol levels can trigger various adverse symptoms including bone and muscle breakdown, fatigue, depression, pain, and memory impairments and lead to endocrine disorders such as Addison’s and Cushing’s diseases [7,8,9]. The discovery of cortisol analogues has attracted considerable interest in drug development research [10,11]. A cortisol-receptor agonist, dexamethasone (Dex), has been marketed as an anti-inflammatory agent for over 50 years; the cortisol-receptor antagonist, mifepristone (RU-486), is prescribed in the USA against hyperglycemia secondary to hypercortisolism in patients with Cushing disease

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