Dataset Information

Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog.

ABSTRACT: Guanine deaminase (GDA) deaminates guanine to xanthine. Despite its significance, the study of human GDA remains limited compared to other metabolic deaminases. As a result, its substrate and inhibitor repertoire are limited, and effective real-time activity, inhibitory, and discovery assays are missing. Herein, we explore two emissive heterocyclic cores, based on thieno[3,4-d]pyrimidine (^thN) and isothiazole[4,3-d]pyrimidine (^tzN), as surrogate GDA substrates. We demonstrate that, unlike the thieno analog, ^thG_N, the isothiazolo guanine surrogate, ^tzG_N, does undergo effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog, ^tzX_N. Further, we showcase the potential of this fluorescent nucleobase surrogate to provide a visible spectral window for a real-time study of GDA and its inhibition.

SUBMITTER: Bucardo MS

PROVIDER: S-EPMC9109600 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog.

Bucardo Marcela S MS Wu You Y Ludford Paul T PT Li Yao Y Fin Andrea A Tor Yitzhak Y

ACS chemical biology 20210630 7

Guanine deaminase (GDA) deaminates guanine to xanthine. Despite its significance, the study of human GDA remains limited compared to other metabolic deaminases. As a result, its substrate and inhibitor repertoire are limited, and effective real-time activity, inhibitory, and discovery assays are missing. Herein, we explore two emissive heterocyclic cores, based on thieno[3,4-d]pyrimidine (thN) and isothiazole[4,3-d]pyrimidine (tzN), as surrogate ...[more]

PMID: 34190533

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Project description:BackgroundPrecise control of gene expression is essential to synchronize plant development with the environment. In perennial plants, transcriptional regulation remains poorly understood, mainly due to the long time required to perform functional studies. Transcriptional reporters based on luciferase have been useful to study circadian and diurnal regulation of gene expression, both by transcription factors and chromatin remodelers. The high mobility group proteins are considered transcriptional chaperones that also modify the chromatin architecture. They have been found in several species, presenting in some cases a circadian expression of their mRNA or protein.ResultsTransactivation experiments have been shown as a powerful and fast method to obtain information about the potential role of transcription factors upon a certain reporter. We designed and validated a luciferase transcriptional reporter using the 5' sequence upstream ATG of Populus tremula × alba LHY2 gene. We showed the robustness of this reporter line under long day and continuous light conditions. Moreover, we confirmed that pPtaLHY2::LUC activity reproduces the accumulation of PtaLHY2 mRNA. We performed transactivation studies by transient expression, using the reporter line as a genetic background, unraveling a new function of a high mobility group protein in poplar, which can activate the PtaLHY2 promoter in a gate-dependent manner. We also showed PtaHMGB2/3 needs darkness to produce that activation and exhibits an active degradation after dawn, mediated by the 26S proteasome.ConclusionsWe generated a stable luciferase reporter poplar line based on the circadian clock gene PtaLHY2, which can be used to investigate transcriptional regulation and signal transduction pathway. Using this reporter line as a genetic background, we established a methodology to rapidly assess potential regulators of diurnal and circadian rhythms. This tool allowed us to demonstrate that PtaHMGB2/3 promotes the transcriptional activation of our reporter in a gate-dependent manner. Moreover, we added new information about the PtaHMGB2/3 protein regulation along the day. This methodology can be easily adapted to other transcription factors and reporters.

Dataset Information

Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog.

Publications

Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog.

Similar Datasets

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