Abstract
Post-translational modifications (PTMs) of proteins provide an important mechanism for cell signal transduction control. Impaired PTM control is a key feature in multiple different disease states, and thus the enzyme-controlling PTMs have drawn attention as highly promising drug targets. Due to the importance of PTMs, various methods to monitor PTM enzyme activity have been developed, but universal high-throughput screening (HTS), a compatible method for different PTMs, remains elusive. Here, we present a homogeneous single-label thermal dissociation assay for the detection of enzymatic PTM removal. The developed method allows the use of micromolar concentration of substrate peptide, which is expected to be beneficial when monitoring enzymes with low activity and peptide binding affinity. We prove the thermal dissociation concept functionality using peptides for dephosphorylation, deacetylation, and demethylation and demonstrate the HTS-compatible flash isothermal method for PTM enzyme activity monitoring. Using specific inhibitors, we detected literature-comparable IC50 values and Z′ factors from 0.61 to 0.72, proving the HTS compatibility of the thermal peptide-break technology.
Highlights
Protein post-translational modifications (PTMs) are covalent additions or proteolytic cleavage events which represent an essential regulatory mechanism involved in cellular signal transduction processes
We have developed an antibody-free PTM monitoring method called the peptide-break technology which allows the detection of a variety of PTMs in a single platform.[27−29] The peptide-break technology is based on the peptide dimerization monitored using the single-label quenching resonance energy transfer (QRET) technology demonstrated previously for the detection of various PTM targets.[30,31]
We have previously developed the universal single-label peptide-break technology for the detection of enzymatic PTM reactions using leucine zippers or charge-based peptides.[27−29] These assays were optimized for detection at room temperature using low nanomolar peptide concentrations
Summary
Protein post-translational modifications (PTMs) are covalent additions or proteolytic cleavage events which represent an essential regulatory mechanism involved in cellular signal transduction processes. In a kinase activity assay, the phosphate group located in the vicinity of the coordinating fluorophore promotes the metal-ion binding, producing an increase in the fluorescence signal.[26] the methods are antibody-free, the nature of the approaches has limited their applicability to phosphorylation. We have developed an antibody-free PTM monitoring method called the peptide-break technology which allows the detection of a variety of PTMs in a single platform.[27−29] The peptide-break technology is based on the peptide dimerization monitored using the single-label quenching resonance energy transfer (QRET) technology demonstrated previously for the detection of various PTM targets.[30,31] Peptide dimer is formed between the Eu3+-labeled detection peptide and the selected substrate peptide designed for the studied PTM enzyme. The proposed antibody-free HTS method enables the use of high peptide concentrations, which is essential for some low-affinity or -activity enzymes.[32] The fluorescent thermal dissociation assay is demonstrated with methylation, dephosphorylation, and deacetylation assays
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