scholarly journals A Novel Multiple-Read Screen for Metabolically Active Compounds Based on a Genetically Encoded FRET Sensor for ATP

2018 ◽  
Vol 23 (9) ◽  
pp. 907-918 ◽  
Author(s):  
Ziyan Zhao ◽  
Rahul Rajagopalan ◽  
Adam Zweifach
Keyword(s):  
Oxidative Phosphorylation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cancer Therapeutics ◽  
K562 Cells ◽  
Initial Step ◽  
Term Treatment ◽  
Short Term Treatment ◽  
Atp Production ◽  
Glycolysis Inhibitors

Both glycolysis and mitochondrial energetics are targets of interest for developing antiproliferative cancer therapeutics. We developed a novel multiple-read assay based on long-term expression in K562 cells of a genetically encoded intramolecular Förster resonance energy transfer sensor for adenosine triphosphate (ATP). The assay, conducted in a fluorescent plate reader, can identify compounds that inhibit oxidative phosphorylation-dependent ATP production, glycolysis, or both after short-term treatment. We screened a National Cancer Institute (NCI) compound library, identifying inhibitors of oxidative phosphorylation-dependent ATP production and glycolysis. Three glycolysis inhibitors blocked hexokinase activity, demonstrating that our assay can serve as the initial step in a workflow to identify compounds that inhibit glycolysis via a defined desired mechanism. Finally, upon reviewing the literature, we found surprisingly little evidence that inhibiting glycolysis with small molecules is antiproliferative. Using NCI data on proliferation of K562 cells, we found that inhibitors of oxidative phosphorylation-dependent ATP production were no more antiproliferative than the overall library, whereas all glycolysis inhibitors were in the top third of most effective antiproliferative compounds. Our results thus present a powerful new way to screen for compounds that affect cellular metabolism and also provide important support for the idea that blocking glycosis is antiproliferative.

scholarly journals Ca2+ signalling in K562 human erythroleukaemia cells: effect of dimethyl sulphoxide and role of G-proteins in thrombin- and thromboxane A2-activated pathways

1995 ◽  
Vol 312 (1) ◽  
pp. 151-158 ◽  
Author(s):  
C P Thomas ◽  
M J Dunn ◽  
R Mattera
Keyword(s):  
G Proteins ◽  
Pcr Amplification ◽  
Thromboxane A2 ◽  
K562 Cells ◽  
Protein S ◽  
Term Treatment ◽  
Dependent Manner ◽  
Short Term Treatment ◽  
Concentration Dependent Manner ◽  
Prostaglandin F2 Alpha

The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In these cells, thrombin and U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha), a thromboxane A2 analogue, increased intracellular Ca2+ in a rapid and concentration-dependent manner. The peak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, suggestive of activation of phospholipase C. Short-term treatment with leupeptin abolished the calcium response to thrombin, but did not alter that to U46619. Both pertussis toxin (PT) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membranes from K562 cells revealed the presence of G12 alpha and G13 alpha; the other known PT-substrates, Gi1 alpha and G0 alpha, were not detected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with human erythrocyte G12 alpha and G13 alpha. An antibody raised against Gq/11 alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplification of reverse-transcribed K562 RNA followed by DNA sequencing showed that these cells express messages for both Gq alpha and G11 alpha. Treatment of K562 cells with DMSO reduced the levels of thrombin receptor mRNA, without simultaneous changes in the expression of G12 alpha and G13 alpha. We have thus identified Ca(2+)-mobilizing agonists and related G-proteins in K562 cells, together with changes induced by DMSO in this signalling pathway.

scholarly journals A Perspective of Epigenetic Regulation in Radiotherapy

2021 ◽  
Vol 9 ◽  
Author(s):  
Qin Peng ◽  
Kegui Weng ◽  
Shitian Li ◽  
Richard Xu ◽  
Yingxiao Wang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Epigenetic Regulation ◽  
Hdac Inhibitors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cancer Therapeutics ◽  
The United States ◽  
Epigenetic Drugs ◽  
Epigenetic Remodeling ◽  
Dna Break

Radiation therapy (RT) has been employed as a tumoricidal modality for more than 100 years and on 470,000 patients each year in the United States. The ionizing radiation causes genetic changes and results in cell death. However, since the biological mechanism of radiation remains unclear, there is a pressing need to understand this mechanism to improve the killing effect on tumors and reduce the side effects on normal cells. DNA break and epigenetic remodeling can be induced by radiotherapy. Hence the modulation of histone modification enzymes may tune the radiosensitivity of cancer cells. For instance, histone deacetylase (HDAC) inhibitors sensitize irradiated cancer cells by amplifying the DNA damage signaling and inhibiting double-strand DNA break repair to influence the irradiated cells’ survival. However, the combination of epigenetic drugs and radiotherapy has only been evaluated in several ongoing clinical trials for limited cancer types, partly due to a lack of knowledge on the potential mechanisms on how radiation induces epigenetic regulation and chromatin remodeling. Here, we review recent advances of radiotherapy and radiotherapy-induced epigenetic remodeling and introduce related technologies for epigenetic monitoring. Particularly, we exploit the application of fluorescence resonance energy transfer (FRET) biosensors to visualize dynamic epigenetic regulations in single living cells and tissue upon radiotherapy and drug treatment. We aim to bridge FRET biosensor, epigenetics, and radiotherapy, providing a perspective of using FRET to assess epigenetics and provide guidance for radiotherapy to improve cancer treatment. In the end, we discuss the feasibility of a combination of epigenetic drugs and radiotherapy as new approaches for cancer therapeutics.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Quantum Dot Bioconjugates as Energy Donors in Fluorescence Resonance Energy Transfer Assays

2003 ◽  
Vol 773 ◽  
Author(s):  
Aaron R. Clapp ◽  
Igor L. Medintz ◽  
J. Matthew Mauro ◽  
Hedi Mattoussi
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Molar Ratio ◽  
Binding Assays ◽  
Specific Sequence ◽  
Donor Acceptor

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

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