Continuous Histone Deacylase Activity Assays.
Matthes ZessinMarat MeleshinWolfgang SipplMike SchutkowskiPublished in: Methods in molecular biology (Clifton, N.J.) (2022)
Protein lysine acylation represents one of the most common post-translational modifications. Obviously, highly reactive metabolic intermediates, like thioesters and mixed anhydrides between phosphoric acid and organic acids, modify lysine residues spontaneously. Additionally, enzymes using acyl-CoAs as co-substrates transfer the acyl residue specifically to defined sequences within proteins. The counteracting enzymes are called histone deacetylases (HDACs), releasing the free lysine side chain. Such enzymatic activities are involved in different cellular processes like tumor progression, immune response, regulation of metabolism, and aging. Modulators of such enzymatic activities represent valuable tools in drug discovery. Therefore, direct and continuous assays to monitor enzymatic activity of HDACs are needed. Here we describe different assay formats allowing both monitoring of Zn<sup>2+</sup>-dependent HDACs via UV-Vis-spectroscopy and NAD<sup>+</sup>-dependent HDACs (sirtuins) by fluorescence-based assay formats. Additionally, we describe methods enabling efficient screening of HDAC-inhibitors via fluorescence displacement assays.
Keyphrases
- high throughput
- drug discovery
- single molecule
- hydrogen peroxide
- amino acid
- immune response
- dna methylation
- fatty acid
- single cell
- energy transfer
- small molecule
- high resolution
- poor prognosis
- heavy metals
- nitric oxide
- dendritic cells
- toll like receptor
- risk assessment
- binding protein
- inflammatory response
- quantum dots