Chromatin-modifying enzymes are of great significance for the epigenetic regulation of numerous DNA-based processes, such as DNA transcription, replication, and damage repair.1
Protein arginine methyltransferase (PRMT) is one type of such an important chromatin-modifying enzyme that transfers the methyl group from S-adenosyl-L-methionine (AdoMet, SAM) to their protein substrates, including nucleosomal core histones, and releases S-adenosyl-L-homocysteine (AdoHcy, SAH) as a side product. Among PRMTs, type I PRMTs (e.g., PRMT1) catalyze asymmetric dimethylation of arginine residues, and type II PRMTs (e.g., PRMT5) catalyze symmetric dimethylation. On the chromatin template, both PRMT1 and PRMT5 target arginine 3 of histone H4 (i.e., H4R3) and alter the status of transcription at target genes.2,3
Several reports show that the expression levels of PRMTs are deregulated in certain diseases. PRMT1 is upregulated in breast cancer concomitantly with a change in substrate methylation.4
PRMT1 variant 2 has been regarded as a marker of unfavorable prognosis in colon cancer patients.5
Furthermore, PRMT1 was found to be an essential element in the oncogenic mixed-lineage leukemia fusion complexes and confers an aberrant transcriptional activation property critical for the induction of leukemia.6
PRMT5, a representative type II enzyme, was also shown to play strong oncogenic roles.7
Overexpression of PRMT5 increases transformation of NIH3T3 cells, suggesting its function in promoting tumor progression.3
PRMT5 protein expression has been induced in a wide variety of human lymphoid cancer cell lines, including patient-derived chronic myelogenous leukemia (MCL) cell lines as well as MCL clinical samples.8
The multiple lines of evidence point toward that targeting PRMT1 and PRMT5 activities with small-molecule inhibitors may be an attractive therapeutic strategy in cancer chemotherapy.
Discovery of PRMT inhibitors requires efficient enzymatic assays. Currently, several methods have been reported to measure methyltransferase activities. For example, radioisotope-labeled assays have been widely used to study the enzyme kinetics of PRMTs,9
but the assay protocols have to involve postreaction separation procedures (e.g., chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis [SDS-PAGE], filter binding), which are labor cumbersome and restrict their application in a high-throughput format. Several groups have developed protocols to monitor the side product AdoHcy as a way of measuring the activities of PRMTs and the other types of methyltransferases.10,11
In particular, AdoHcy can be converted to homocysteine by AdoHcy hydrolases, releasing its free thiol group that can react with sulfhydryl-sensitive dyes for fluorometric or colorimetric detection.10,12,13
The methyltransferase activity of PRMTs can also be detected by the competitive fluorescence polarization immunoassay14
and enzyme-coupled luciferase assay.15
In general, these nonradioactive coupled assays overcome certain shortcomings of the radioisotope labeled methods, but the involvement of additional components could complicate the assay conditions and may produce false positives in inhibitor screening.
We are particularly interested in developing methyltransferase assay strategies involving minimal ingredients to avoid potential complexity and ambiguity. For example, we recently designed a single-step fluorescent assay for sensitive measurement of PRMT1-mediated methylation.16
Herein, we describe a scintillation proximity assay (SPA) for the detection of PRMT activities with the aim of obtaining improved assay efficacy and throughput. In this strategy, a biotin-labeled peptide was used as the substrate of PRMT1 and PRMT5. PRMTs catalyze the transfer of the 3
H-methyl group from 3
H-SAM to the biotin–peptide. The methylated product is caught by streptavidin-coated SPA beads that contain scintillator molecules. The interaction between the product and the SPA beads results in the transfer of energy from tritium to scintillator, which leads to emission of luminescent signals that can be measured and quantified on a MicroBeta counter (PerkinElmer, Waltham, MA). We characterized in detail the efficacy and robustness of this method for PRMT activity measurement. The results suggest that this protocol has great application for high-throughput screening (HTS) of PRMT inhibitors.