Buffer
NaCl concentration regulates Renilla
luciferase activity and ligand-induced conformational changes in the
BRET-based PDE5 sensor

Biswas, Kabir H.; Visweswariah, Sandhya S.

doi:10.19185/matters.201702000015

Cited by 13 publications

(20 citation statements)

References 32 publications

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“…Here, we have explored the possibility of using NanoLuc for sensing ionic strength. Contrary to the increase in the bioluminescence of Rluc reported previously [ 7 ], increased buffer ionic strength negatively impacted the bioluminescence of a recombinantly purified NanoLuc protein. Furthermore, the reversibility of the effect alludes to the possibility of its use as an ionic strength sensor in live cells.…”

Section: Introductioncontrasting

confidence: 97%

“…In the current study, we have attempted to develop an ionic strength sensing strategy using the NanoLuc protein. This was premised on the observation of increased bioluminescence that we had reported for the Rluc protein in cell lysates previously [ 7 ]. Towards this, we characterized the bioluminescence of a recombinantly purified NanoLuc in vitro using the substrate furimazine ( Supplementary Figure S1A ).…”

Section: Resultsmentioning

confidence: 99%

“…Cell lysate and live cell experiments were performed using human embryonic kidney (HEK) 293T cells (ATCC, Manassas, VA, USA) transfected with a plasmid construct expressing a fusion of the mNeonGreen and the NanoLuc protein (mNeonGreen–DEVD–NanoLuc) (Addgene: 98287; DEVD is the caspase 3 cleavage site) [ 55 ]. For assays with cell lysates, cells were washed in chilled Dulbecco’s phosphate-buffered saline (DPBS), harvested using a 2 mM ethylenediaminetetraacetic acid (EDTA) containing DPBS, and lysed after 48 h of transfection by sonication in a lysis buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol (DTT), 1× protease inhibitor cocktail (ThermoFisher Scientific, Massachusetts, USA) and 10% glycerol [ 7 , 10 ]. Sonicated cell lysates were centrifuged at 4 °C for 1 h at 20,817 relative centrifugal force (RCF) and the supernatant was collected.…”

Section: Methodsmentioning

confidence: 99%

“…Ionic strength determined by the concentrations of different ionic species in a medium, both cations and anions, fundamentally influences electrostatic interactions in and among biomolecules and, therefore, regulates a plethora of cellular processes [ 1 , 2 , 3 , 4 ]. For instance, ionic strength affects the conformational stability and folding/unfolding behavior of highly charged proteins through charge screening, as detected by changes in fluorescence resonance energy transfer (FRET) efficiency [ 5 ] or a ligand-induced conformational change in a multidomain protein as determined from intramolecular bioluminescence resonance energy transfer (BRET) assays [ 6 , 7 , 8 , 9 , 10 ]. Furthermore, ionic strength is also known to affect enzymatic activity [ 11 ], aggregation and gel formation upon protein unfolding [ 12 ], the assembly of disease-causing amyloids [ 13 , 14 ], or the binding affinity and fluorescence intensity of amyloid-specific dye [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

“…Unlike fluorescence-based methods, bioluminescence systems have a broad dynamic range, high sensitivity, and operational simplicity [ 28 , 29 , 30 , 31 ]. We have previously found that the bioluminescence of Renilla luciferase (Rluc) is affected by the ionic strength [ 7 ], and hypothesized that this ionic strength effect can be extrapolated to other luciferases and used for ionic strength sensing. Rluc has a relatively short bioluminescence half-life and low thermal stability compared to Nano Luciferase (NanoLuc).…”

Section: Introductionmentioning

confidence: 99%

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Intracellular Ionic Strength Sensing Using NanoLuc

Altamash

Ahmed

Rasool

et al. 2021

IJMS

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Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

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Section: Introductioncontrasting

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Intracellular Ionic Strength Sensing Using NanoLuc

Altamash

Ahmed

Rasool

et al. 2021

IJMS

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Engineering β-catenin-derived peptides for α-catenin binding

Uddin,

Rasool,

Geethakumari

et al. 2024

emergent mater.

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The complex formed by the β-catenin and α-catenin adaptor proteins acts as a molecular bridge that enables E-cadherin-based cell–cell adhesion assembly and maintenance in the epithelial tissue. This occurs through the interaction between the intracellular domain of E-cadherin and β-catenin on the one hand and between F-actin and α-catenin on the other hand. In addition to its role in cell–cell adhesion formation, it has been reported that E-cadherin mediates breast cancer cell metastasis to distant organs. Therefore, development of biomaterials such as peptides with ability to modulate the interaction between β-catenin and α-catenin presents an opportunity to modulate cell–cell adhesion. Here, we have performed computational and experimental analysis to develop β-catenin-derived peptides with the ability to bind α-catenin. Specifically, we analyzed the available β- and α-catenin complex structure and identified residues on β-catenin having potential to form new interactions upon mutation. We tested the wild-type (WT) and mutant β-catenin-derived peptides for their binding to α-catenin using conventional and steered molecular dynamics simulations, revealing an increased interaction of P128E and M131E mutant peptides. We then designed a Bioluminescence Resonance Energy Transfer (BRET)-based assay to monitor binding of the β-catenin-derived peptides with α-catenin, which revealed similar binding affinities of the WT and mutant β-catenin-derived peptides. Further, expression of the WT and the M131E mutant peptide resulted in a change in the aspect ratio of the cells suggestive of their ability to affect cell–cell adhesion. We envisage that the β-catenin-derived peptides engineered here will find application in blocking the interaction between β-catenin and α-catenin and, thus, modulate E-cadherin adhesion, which may lead to potential therapeutic avenue in abrogating E-cadherin-mediated metastasis of invasive breast cancer cells.

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