Sensing Cyclic Adenosine Monophosphate and Guanine Nucleotide Exchange Factor Communication Through Rluc-Epac-Citrine2 BRET Sensor in Live GBM Cells

IF 2.3 Q2 ENGINEERING, ELECTRICAL & ELECTRONIC

IEEE Transactions on Molecular, Biological, and Multi-Scale Communications Pub Date : 2025-04-29 DOI:10.1109/TMBMC.2025.3565137

Elif Dilek;Vivash Naidoo;Bobin George Abraham;Saravanan Konda Mani;Kasim S. Abass;Sandhanasamy Devanesan;Mohamad S. AlSalhi;Sureka Chandrabose;Olli Yli-Harja;Akshaya Murugesan;Meenakshisundaram Kandhavelu

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引用次数: 0

Abstract

Cyclic adenosine 3’,5’-monophosphate (cAMP) is a versatile secondary messenger that communicates with Guanine Nucleotide Exchange Factor (EPAC) to transfer cellular signaling and regulates numerous physiological conditions. Early studies focused on measuring this communication is considered as crucial in GPCR ligand-mediated EPAC activation, where bioluminescence resonance energy transfer (BRET) sensor has been widely used to study the cAMP level in living cells. However, a BRET sensor pairing with the best brightness and photostability for detecting low levels of cAMP in single and whole-cell populations has yet to be developed. Here, we constructed a novel BRET-based cAMP biosensor with Rluc-Epac-Citrine2. A molecular communication study revealed a significant change of over 100° in the phi value for the residues Thr253, Val259, and Thr260 in the presence of cAMP, leading to strong cAMP-Epac-induced dynamics in the ternary complex. Spectrum scanning, luminescence, and fluorescence emission studies on glioblastoma multiforme (GBM) cells demonstrated closer proximity between donor and acceptor, ensuring the cAMP sensor’s activity. This sensor detects changes in endogenous cAMP levels, and the observed BRET signal can be enhanced by increasing the concentration of the substrate, coelenterazine. The sensor also efficiently detects the communication between cAMP and EPAC in live GBM cells over time. We used this sensor to assess the activation of GPR17, a potential biomarker for GBM. The activation of MDL 29,951, a GPR17 agonist, supports the sensor’s ability to detect Gi-coupled protein activation. This study also shows the feasibility of sensor readouts using inexpensive instrumentation such as plate readers and image systems. Overall, this study sheds new light on detecting cAMP communication with EPAC and GPR17 ligand-mediated EPAC in GBM cells, potentially aiding the development of precision therapies.

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通过Rluc-Epac-Citrine2 BRET传感器感知活GBM细胞中环腺苷单磷酸和鸟嘌呤核苷酸交换因子的通讯

环腺苷3 ',5 ' -单磷酸（cAMP）是一种多功能的次级信使，与鸟嘌呤核苷酸交换因子（EPAC）交流，传递细胞信号并调节多种生理条件。早期的研究集中在测量这种通讯被认为是GPCR配体介导的EPAC激活的关键，其中生物发光共振能量转移（BRET）传感器已被广泛用于研究活细胞中的cAMP水平。然而，一种具有最佳亮度和光稳定性的传感器配对用于检测单细胞和全细胞群体中的低水平cAMP尚未开发。本研究以rucc - epac - citrine2为原料，构建了一种新型的基于bret的cAMP生物传感器。一项分子通讯研究表明，在cAMP存在下，Thr253、Val259和Thr260残基的phi值发生了超过100°的显著变化，导致三元配合物中cAMP- epac诱导的强烈动力学。多形性胶质母细胞瘤（GBM）细胞的光谱扫描、发光和荧光发射研究表明，供体和受体之间的距离更近，确保了cAMP传感器的活性。该传感器检测内源性cAMP水平的变化，并且可以通过增加底物coelenterazine的浓度来增强观察到的BRET信号。该传感器还可以有效地检测活GBM细胞中cAMP和EPAC之间的通信。我们使用该传感器来评估GPR17的激活，GPR17是GBM的潜在生物标志物。MDL 29,951（一种GPR17激动剂）的激活支持该传感器检测gi偶联蛋白激活的能力。这项研究也显示了传感器读出的可行性，使用廉价的仪器，如板阅读器和图像系统。总的来说，这项研究揭示了在GBM细胞中检测cAMP与EPAC和GPR17配体介导的EPAC的通信，可能有助于精确治疗的发展。

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来源期刊

IEEE Transactions on Molecular, Biological, and Multi-Scale Communications Mathematics-Modeling and Simulation

CiteScore

3.90

自引率

13.60%

发文量

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