Use of BRET to Study Protein–Protein Interactions In Vitro and In Vivo

Dimri, Shalini; Basu, Soumya; De, Abhijit

doi:10.1007/978-1-4939-3724-0_5

Cited by 26 publications

(16 citation statements)

References 27 publications

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“…First, the emission wavelength of the donor and the excitation wavelength of the acceptor have to overlap. Second, the distance between the donor and the acceptor molecule should be less than 10 nm, and third the relative orientation of the donor and the acceptor should allow a non-radiative dipole-dipole coupling for sufficient energy transfer [36]. To address the first requirement, the spectral overlap for the eBRET2 pair Rluc8 and GFP2 was shown previously [37].…”

Section: Discussionmentioning

confidence: 99%

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF

et al. 2020

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In age-related macular degeneration (AMD) or diabetic retinopathy (DR), hypoxia and inflammatory processes lead to an upregulation of the vascular endothelial growth factor (VEGF) expression and thereby to pathological neovascularisation with incorrectly formed vessels prone to damage, thus increasing the vascular permeability and the risk of bleeding and oedema in the retina. State of the art treatment is the repeated intraocular injection of anti-VEGF molecules. For developing improved individualized treatment approaches, a minimally invasive, repeatable method for in vivo quantification of VEGF in the eye is necessary. Therefore, we designed single molecule eBRET2 VEGF biosensors by directly fusing a Renilla luciferase mutant (Rluc8) N-terminal and a green fluorescent protein (GFP2) C-terminal to a VEGF binding domain. In total, 10 different VEGF biosensors (Re01-Re10) were generated based on either single domains or full length of VEGF receptor 1 or 2 extracellular regions as VEGF binding domains. Full length expression of the biosensors in HEK293-T cells was verified via Western Blot employing an anti-Rluc8-IgG. Expression of alternative splice variants was eliminated through the deletion of the donor splice site by introduction of a silent point mutation. In all ten biosensors the energy transfer from the Rluc8 to the GFP2 occurs and generates a measurable eBRET2 ratio. Four biosensors show a relevant change of the BRET ratio (ΔBR) after VEGF binding. Furthermore, each biosensor shows a unique detection range for VEGF quantification and especially Re06 and Re07 have a high sensitivity in the range of in vivo VEGF concentrations in the eye, previously measured by invasive methods. In conclusion, we generated several eBRET2 biosensors that are suitable for VEGF quantification in vitro and could identify two eBRET2 biosensors, which may be suitable for non-invasive in vivo VEGF quantification with an implantable device.

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Section: Discussionmentioning

confidence: 99%

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF

et al. 2020

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“…The fluorophore on the acceptor will be activated and emit its characteristic light signal. 64,65 BRET has been applied to probe intracellular PPIs, such as studies investigating the interactions of circadian clock proteins in Escherichia coli 66 and the dimerization of G protein-coupled receptors (GPCRs) in mammalian cells. 67 Additionally, there are various optimized versions of BRET, such as BRET n (NanoLuc luciferase-based bioluminescence resonance energy transfer), which utilizes the highly sensitive NanoLuc luciferase as the donor and enables improved sensitivity and efficiency in PPI detection.…”

Section: Experimental Methodsmentioning

confidence: 99%

“…Upon PPI, the energy produced by the reaction between the donor and its substrate can be transferred to the acceptor due to the close proximity of the PPI partners. The fluorophore on the acceptor will be activated and emit its characteristic light signal . BRET has been applied to probe intracellular PPIs, such as studies investigating the interactions of circadian clock proteins in Escherichia coli and the dimerization of G protein‐coupled receptors (GPCRs) in mammalian cells .…”

Section: Modulation Of Ppismentioning

confidence: 99%

Emerging roles of allosteric modulators in the regulation of protein‐protein interactions (PPIs): A new paradigm for PPI drug discovery

Zhang

2019

Medicinal Research Reviews

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Protein‐protein interactions (PPIs) are closely implicated in various types of cellular activities and are thus pivotal to health and disease states. Given their fundamental roles in a wide range of biological processes, the modulation of PPIs has enormous potential in drug discovery. However, owing to the general properties of large, flat, and featureless interfaces of PPIs, previous attempts have demonstrated that the generation of therapeutic agents targeting PPI interfaces is challenging, rendering them almost “undruggable” for decades. To date, rapid progress in chemical and structural biology techniques has promoted the exploitation of allostery as a novel approach in drug discovery. By attaching to allosteric sites that are topologically and spatially distinct from PPI interfaces, allosteric modulators can achieve improved physiochemical properties. Thus, allosteric modulators may represent an alternative strategy to target intractable PPIs and have attracted intense pharmaceutical interest. In this review, we first briefly introduce the characteristics of PPIs and then present different approaches for investigating PPIs, as well as the latest methods for modulating PPIs. Importantly, we comprehensively review the recent progress in the development of allosteric modulators to inhibit or stabilize PPIs. Finally, we conclude with future perspectives on the discovery of allosteric PPI modulators, especially the application of computational methods to aid in allosteric PPI drug discovery.

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“…The bioluminescence resonance energy transfer (BRET) assay ( Figure 5 ) involving the use of RLuc has become popular since the late 1990s for measurements of protein–protein interactions and conformational rearrangements in live cells [ 138 , 139 , 140 ], for non-invasive bioimaging [ 141 ], and as probes for biosensing [ 123 , 142 ]. The sensitivity of BRET assays has recently been improved by introducing new BRET components: RLuc2 and RLuc8 with improved quantum yields, stability, and brightness [ 143 ] as well as a great variety of acceptors (GFP2, YFP, Venus, mOrange, TagRFP, TurboFP, semiconductor quantum dots or carbon-dots) [ 144 , 145 , 146 ].…”

Section: Ctz-dependent Luciferase Analytical Applicationmentioning

confidence: 99%

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

Krasitskaya

Bashmakova

Frank

2020

IJMS

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: The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate—coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization—Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme–substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.

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Use of BRET to Study Protein–Protein Interactions In Vitro and In Vivo

Cited by 26 publications

References 27 publications

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF

Creation of different bioluminescence resonance energy transfer based biosensors with high affinity to VEGF

Emerging roles of allosteric modulators in the regulation of protein‐protein interactions (PPIs): A new paradigm for PPI drug discovery

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

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