当前位置:
X-MOL 学术
›
Biochemistry
›
论文详情
Our official English website, www.x-mol.net, welcomes your
feedback! (Note: you will need to create a separate account there.)
Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach
Biochemistry ( IF 2.9 ) Pub Date : 2018-12-11 00:00:00 , DOI: 10.1021/acs.biochem.8b01073
Livio Casarini 1, 2 , Laura Riccetti 1 , Silvia Limoncella 1 , Clara Lazzaretti 1 , Federica Barbagallo 3 , Salvatore Pacifico 4 , Remo Guerrini 4 , Simonetta Tagliavini 5 , Tommaso Trenti 5 , Manuela Simoni 1, 2, 6 , Marco Sola 7 , Giulia Di Rocco 7
Biochemistry ( IF 2.9 ) Pub Date : 2018-12-11 00:00:00 , DOI: 10.1021/acs.biochem.8b01073
Livio Casarini 1, 2 , Laura Riccetti 1 , Silvia Limoncella 1 , Clara Lazzaretti 1 , Federica Barbagallo 3 , Salvatore Pacifico 4 , Remo Guerrini 4 , Simonetta Tagliavini 5 , Tommaso Trenti 5 , Manuela Simoni 1, 2, 6 , Marco Sola 7 , Giulia Di Rocco 7
Affiliation
![]() |
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 (Kd) values of 5.6 ± 3.2 and 13.7 ± 0.8 μM 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 Ki 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.
中文翻译:
用细胞内FRET / BRET联合方法探讨西地那非对孕酮和睾丸激素产生的影响
最近,基于Forster共振能量转移(FRET)的生物传感器已应用于生物学途径的研究。在这项研究中,首次在活HEK293和类固醇生成的MLTC-1细胞系中验证了一种新的生物传感器,以研究PDE5抑制剂对hCG / LH诱导的类固醇生成途径的影响。该传感器改善了供体(D),可以共价结合与PDE5催化结构域融合的四半胱氨酸基序的荧光素样二砷探针和受体(A)之间的FRET,受体(A)与缀合在伪底物cGMPS上的罗丹明探针之间。用HEK293和MLTC-1细胞获得的亲和常数(K d)值分别为5.6±3.2和13.7±0.8μM。该检测基于西地那非对cGMPS-若丹明缀合物的竞争性取代。这HEK293细胞的K i值为3.6±0.3 nM(IC 50 = 2.3 nM),而MLTC-1细胞的K i值为10±1.0 nM(IC 50 = 3.9 nM)。通过生物发光共振能量转移对cAMP和cGMP的监测可以利用PDE5i对类固醇生成的影响,这表明西地那非以cAMP无关的方式增强了促性腺激素诱导的孕酮向睾丸激素的转化。
更新日期:2018-12-11
中文翻译:

用细胞内FRET / BRET联合方法探讨西地那非对孕酮和睾丸激素产生的影响
最近,基于Forster共振能量转移(FRET)的生物传感器已应用于生物学途径的研究。在这项研究中,首次在活HEK293和类固醇生成的MLTC-1细胞系中验证了一种新的生物传感器,以研究PDE5抑制剂对hCG / LH诱导的类固醇生成途径的影响。该传感器改善了供体(D),可以共价结合与PDE5催化结构域融合的四半胱氨酸基序的荧光素样二砷探针和受体(A)之间的FRET,受体(A)与缀合在伪底物cGMPS上的罗丹明探针之间。用HEK293和MLTC-1细胞获得的亲和常数(K d)值分别为5.6±3.2和13.7±0.8μM。该检测基于西地那非对cGMPS-若丹明缀合物的竞争性取代。这HEK293细胞的K i值为3.6±0.3 nM(IC 50 = 2.3 nM),而MLTC-1细胞的K i值为10±1.0 nM(IC 50 = 3.9 nM)。通过生物发光共振能量转移对cAMP和cGMP的监测可以利用PDE5i对类固醇生成的影响,这表明西地那非以cAMP无关的方式增强了促性腺激素诱导的孕酮向睾丸激素的转化。