期刊
BIOCHEMISTRY
卷 58, 期 6, 页码 799-808出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.8b01073
关键词
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资金
- MIUR, Ministero dell'Istruzione, dell'Universita e della Ricerca (FIRB grant) [RBFR12FI27 005]
- Fondo di Ateneo per la Ricerca (FAR2015)
- University of Modena
- Reggio Emilia in the Departments of Excellence Programme - MIUR
Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant (K-d) values of 5.6 +/- 3.2 and 13.7 +/- 0.8 mu M were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS- rhodamine conjugate by sildenafil; the K-i values were 3.6 +/- 0.3 nM (IC50 = 2.3 nM) in HEK293 cells and 10 +/- 1.0 nM (IC50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.
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