Development of cGAMP-Luc, a sensitive and precise coupled enzyme assay to measure cGAMP in complex biological samples.
J Biol Chem
; 295(15): 4881-4892, 2020 04 10.
Article
em En
| MEDLINE
| ID: mdl-32127400
ABSTRACT
2',5'/3',5'-cGMP-AMP (cGAMP) is a second messenger produced in response to cytosolic dsDNA that activates the stimulator of interferon genes (STING) pathway. We recently discovered that cGAMP is exported by cancer cells and that this extracellular signal is an immunotransmitter key to tumor detection and elimination by the innate immune system. The enhancement of extracellular cGAMP levels therefore holds great promise for managing cancer. However, there is still much more to understand about the basic biology of cGAMP before its full therapeutic potential can be realized. To answer these questions, we must be able to detect and quantitate cGAMP with an assay that is high-throughput, sensitive, and precise. Existing assays fall short of these needs. Here, we describe the development of cGAMP-Luc, a coupled enzyme assay that relies on the degradation of cGAMP to AMP by ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and an optimized assay for the detection of AMP by luciferase. We also developed STING-CAP, a STING-mediated method to concentrate and purify cGAMP from any type of biological sample. We conclude that cGAMP-Luc is an economical high-throughput assay that matches the accuracy of and surpasses the detection limit of MS, the current gold standard of cGAMP quantitation. We propose that cGAMP-Luc is a powerful tool that may enable discoveries that advance insights into extracellular cGAMP levels in healthy and diseased tissues, such as cancer.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pirofosfatases
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Diester Fosfórico Hidrolases
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Ensaios Enzimáticos
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Luciferases
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Proteínas de Membrana
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Neoplasias
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Nucleotídeos Cíclicos
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2020
Tipo de documento:
Article