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Unimolecular FRET sensors: Simple linker designs and properties
摘要： Protein activation and deactivation is central to a variety of biological mechanisms, including cellular signaling and transport. Unimolecular fluorescent resonance energy transfer (FRET) probes are a class of fusion protein sensors that allow biologists to visualize using an optical microscope whether specific proteins are activated due to the presence nearby of small drug-like signaling molecules, ligands or analytes. Often such probes comprise a donor fluorescent protein attached to a ligand binding domain, a sensor or reporter domain attached to the acceptor fluorescent protein, with these ligand binding and sensor domains connected by a protein linker. Various choices of linker type are possible ranging from highly flexible proteins to hinge-like proteins. It is also possible to select donor and acceptor pairs according to their corresponding F¨oster radius, or even to mutate binding and sensor domains so as to change their binding energy in the activated or inactivated states. The focus of the present work is the exploration through simulation of the impact of such choices on sensor performance.
关键词： FRET Microscopy,Fusion Proteins,Diagnostics,Monte Carlo Simulation,Coarse Graining,Cellular Signaling
更新于2025-09-23 15:21:01
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Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue
摘要： Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX4 and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX4 – associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis.
关键词： ROS,endothelium,NADPH analogue,Angiogenesis,melanoma,cell proliferation,Cellular signaling
更新于2025-09-19 17:15:36