Protocol for elucidating metabolite binding and regulation of TET2 dioxygenase

Epigenetic enzyme activity is coupled to cellular metabolism through their reliance on metabolic cofactors and substrates. Here, we describe steps for combining biochemical assays and saturation transfer difference (STD) NMR spectroscopy to experimentally validate metabolite binding and assess the e...

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Vydáno v:STAR protocols Ročník 6; číslo 3; s. 104015
Hlavní autoři: Zhang, Shuyuan, Cheng, Zhou-Li, Ye, Dan
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States Elsevier Inc 19.09.2025
Elsevier
Témata:
Cell Biology
Dioxygenases - metabolism
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Epigenesis, Genetic
Humans
Magnetic Resonance Spectroscopy - methods
Molecular Biology
NMR
Protein Binding
Protein Biochemistry
Protein expression and purification
Proto-Oncogene Proteins - metabolism
Protocol
Protein Biochemistry
NMR
Molecular Biology
Protein expression and purification
Cell Biology
ISSN:2666-1667, 2666-1667
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Shrnutí:Epigenetic enzyme activity is coupled to cellular metabolism through their reliance on metabolic cofactors and substrates. Here, we describe steps for combining biochemical assays and saturation transfer difference (STD) NMR spectroscopy to experimentally validate metabolite binding and assess the effect on TET2 activity. This protocol enables the identification of both TET2 activators and inhibitors, providing a framework for studying the interplay between metabolism and epigenetic regulation. For complete details on the use and execution of this protocol, please refer to Cheng et al.1 [Display omitted] •Instructions for the purification of highly active, tag-free human TET2CD protein•Procedures for flow cytometry-based detection of TET2 activity in vitro•Guidance on the simultaneous screening of TET2 regulatory metabolites•Steps for STD NMR-based detection of metabolite-TET2 interactions Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Epigenetic enzyme activity is coupled to cellular metabolism through their reliance on metabolic cofactors and substrates. Here, we describe steps for combining biochemical assays and saturation transfer difference (STD) NMR spectroscopy to experimentally validate metabolite binding and assess the effect on TET2 activity. This protocol enables the identification of both TET2 activators and inhibitors, providing a framework for studying the interplay between metabolism and epigenetic regulation.
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ISSN:2666-1667
2666-1667
DOI:10.1016/j.xpro.2025.104015