Formulating a fluorogenic assay to evaluate S-adenosyl-L-methionine analogues as protein methyltransferase cofactors Journal Article
| Authors: | Wang, R.; Ibáñez, G.; Islam, K.; Zheng, W.; Blum, G.; Sengelaub, C.; Luo, M. |
| Article Title: | Formulating a fluorogenic assay to evaluate S-adenosyl-L-methionine analogues as protein methyltransferase cofactors |
| Abstract: | Protein methyltransferases (PMTs) catalyze arginine and lysine methylation of diverse histone and nonhistone targets. These posttranslational modifications play essential roles in regulating multiple cellular events in an epigenetic manner. In the recent process of defining PMT targets, S-adenosyl-L-methionine (SAM) analogues have emerged as powerful small molecule probes to label and profile PMT targets. To examine efficiently the reactivity of PMTs and their variants on SAM analogues, we transformed a fluorogenic PMT assay into a ready high throughput screening (HTS) format. The reformulated fluorogenic assay is featured by its uncoupled but more robust character with the first step of accumulation of the commonly-shared reaction byproduct S-adenosyl-L-homocysteine (SAH), followed by SAH-hydrolase-mediated fluorogenic quantification. The HTS readiness and robustness of the assay were demonstrated by its excellent Z′ values of 0.83-0.95 for the so-far-examined 8 human PMTs with SAM as a cofactor (PRMT1, PRMT3, CARM1, SUV39H2, SET7/9, SET8, G9a and GLP1). The fluorogenic assay was further implemented to screen the PMTs against five SAM analogues (allyl-SAM, propargyl-SAM, (E)-pent-2-en-4-ynyl-SAM (EnYn-SAM), (E)-hex-2-en-5-ynyl-SAM (Hey-SAM) and 4-propargyloxy-but-2-enyl-SAM (Pob-SAM)). Among the examined 8 × 5 pairs of PMTs and SAM analogues, native SUV39H2, G9a and GLP1 showed promiscuous activity on allyl-SAM. In contrast, the bulky SAM analogues, such as EnYn-SAM, Hey-SAM and Pob-SAM, are inert toward the panel of human PMTs. These findings therefore provide the useful structure-activity guidance to further evolve PMTs and SAM analogues for substrate labeling. The current assay format is ready to screen methyltransferase variants on structurally-diverse SAM analogues. This journal is © The Royal Society of Chemistry. |
| Journal Title: | Molecular BioSystems |
| Volume: | 7 |
| Issue: | 11 |
| ISSN: | 1742-206X |
| Publisher: | Royal Society of Chemistry |
| Date Published: | 2011-11-01 |
| Start Page: | 2970 |
| End Page: | 2981 |
| Language: | English |
| DOI: | 10.1039/c1mb05230f |
| PROVIDER: | scopus |
| PUBMED: | 21866297 |
| PMCID: | PMC3575546 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 2 November 2011" - "Source: Scopus" |
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