Abstract
Post-translational modifications (PTMs) are one of the most important regulatory mechanisms in cells, and they play key roles in cell signaling both in health and disease. PTM catalyzing enzymes have become significant drug targets, and therefore, tremendous interest has been focused on the development of broad-scale assays to monitor several different PTMs with a single detection platform. Most of the current methodologies suffer from low throughput or rely on antibody recognition, increasing the assay costs, and decreasing the multifunctionality of the assay. Thus, we have developed a sensitive time-resolved Förster resonance energy transfer (TR-FRET) detection method for PTMs of cysteine residues using a single-peptide approach performed in a 384-well format. In the developed assay, the enzyme-specific biotinylated substrate peptide is post-translationally modified at the cysteine residue, preventing the subsequent thiol coupling with a reactive AlexaFluor 680 acceptor dye. In the absence of enzymatic activity, increase in the TR-FRET signal between the biotin-bound Eu(III)-labeled streptavidin donor and the cysteine-coupled AlexaFluor 680 acceptor dye is observed. We demonstrate the detection concept with cysteine modifying S-nitrosylation and ADP-ribosylation reactions using a chemical nitric oxide donor S-nitrosoglutathione and enzymatic ADP-ribosyltransferase PtxS1-subunit of pertussis toxin, respectively. As a proof of concept, three peptide substrates derived from the small GTPase K-Ras and the inhibitory α-subunit of the heterotrimeric G-protein Gαi showed expected functionality in both chemical and enzymatic assays. Measurements yielded signal-to-background ratios of 28.7, 33.0, and 8.7 between the modified and the nonmodified substrates for the three peptides in the S-nitrosylation assay, 5.8 in the NAD+ hydrolysis assay, and 6.8 in the enzymatic ADP-ribosyltransferase inhibitor dose–response assay. The developed antibody-free assay for cysteine-modifying enzymes provides a detection platform with low nanomolar peptide substrate consumption, and the assay is potentially applicable to investigate various cysteine-modifying enzymes in a high throughput compatible format.
Highlights
Post-translational protein-modifying enzymes have become one of the most studied drug targets.[1,2] The main task of the post-translational modifications (PTMs) is to increase the protein diversity in cells by the addition or cleavage of chemical groups, such as phosphate, ADP-ribose, or acetyl.[1]
We report a homogeneous single-peptide technology for cysteine PTM detection based on time-resolved Förster resonance energy transfer (TR-FRET)
Upon chemical or enzymatic modification of the cysteine residue, the AlexaFluor 680 conjugation is prevented and, the label remains free in the solution leading to a low TR-FRET
Summary
Post-translational protein-modifying enzymes have become one of the most studied drug targets.[1,2] The main task of the post-translational modifications (PTMs) is to increase the protein diversity in cells by the addition or cleavage of chemical groups, such as phosphate, ADP-ribose, or acetyl.[1]. Glycosylation and acetylation are of high importance because of their prevalence.[5,6] Cysteine is less frequently modified than, for example, lysine, as free reactive cysteines are often buried inside the folded protein structure or cysteine residues are bound to adjacent cysteine residues.[7,8] cysteine modifications are highly important for cellular functions and cysteine is modified by many targets with a number of different types of modifications, such as nitrosylation, sulfhydrylation, glutathionylation, prenylation, palmitoylation, Michael adducts, and ADP-ribosylation, occur in cysteine residues.[7−9] This abundance and variety of the PTMs sets challenges for the detection methods available today
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