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Evidence-based Complementary and Alternative Medicine : eCAM (1)
Journal of Biomolecular Screening (1)
Feng, You (2)
Xie, Nan (2)
Deng, Jiang (1)
Gong, Qi-Hai (1)
Huang, Jing (1)
Huang, Xie-Nan (1)
Jin, Miyeong (1)
Li, Li-Sheng (1)
Stahley, Mary R. (1)
Stivers, James T. (1)
Wu, Jiang (1)
Yang, Dan-Li (1)
Zheng, Y. George (1)
Zheng, Yujun George (1)
Year of Publication
A Transient Kinetic Analysis of PRMT1 Catalysis
Stahley, Mary R.
Stivers, James T.
Zheng, Yujun George
Posttranslational modifications (PTMs) are important strategies used by eukaryotic organisms to modulate their phenotypes. One of the well studied PTMs, arginine methylation, is catalyzed by protein arginine methyltransferases (PRMTs) with SAM as the methyl donor. The functions of PRMTs have been broadly studied in different biological processes and diseased states, but the molecular basis for arginine methylation is not well defined. In this study, we report the transient-state kinetic analysis of PRMT1 catalysis. The fast association and dissociation rates suggest that PRMT1 catalysis of histone H4 methylation follows a rapid equilibrium sequential kinetic mechanism. The data give direct evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, from the stopped-flow fluorescence measurements, we have identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. These results provide new insights into the mechanism of arginine methylation and the rational design of PRMT inhibitors.
PRMT1; arginine methylation; transient-state kinetics; conformational transition; fluorescent probe; stopped flow
Inhibitory Effect of Ginsenoside Rg1 on Vascular Smooth Muscle Cell Proliferation Induced by PDGF-BB Is Involved in Nitric Oxide Formation
Evidence-based Complementary and Alternative Medicine : eCAM
Ginsenoside Rg1 (Rg1) has been reported to suppress the proliferation of vascular smooth muscle cells (VSMCs). This study aimed to observe the role of nitric oxide (NO) in Rg1-antiproliferative effect. VSMCs from the thoracic aorta of SD rats were cultured by tissue explant method, and the effect of Rg1 (20 mg·L−1, 60 mg·L−1, and 180 mg·L−1) on platelet-derived growth factor-BB (PDGF-BB)-induced proliferation was evaluated by MTT assay. The cell cycle was analyzed by flow cytometry. For probing the mechanisms, the content of NO in supernatant and cGMP level in VSMCs was measured by nitric oxide kit and cGMP radio-immunity kit, respectively; the expressions of protooncogene c-fos and endothelial NO synthase (eNOS) mRNA in the VSMCs were detected by real-time RT-PCR; the intracellular free calcium concentration ([Ca2+]i) was detected with Fura-2/AM-loaded VSMCs. Comparing with that in normal group, Rg1 180 mg·L−1 did not change the absorbance of MTT and cell percent of G0/G1, G2/M, and S phase in normal cells (P > 0.05). Contrarily, PDGF-BB could increase the absorbance of MTT (P < 0.01) and the percent of the S phase cells but decrease the G0/G1 phase cell percent in the cell cycle, accompanied with an upregulating c-fos mRNA expression (P < 0.01), which was reversed by additions of Rg1(20 mg·L−1, 60 mg·L−1, and 180 mg·L−1). Rg1 administration could also significantly increase the NO content in supernatant and the cGMP level in VSMCs, as well as the eNOS mRNA expression in the cells, in comparison of that in the group treated with PDGF-BB alone (P < 0.01). Furthermore, Rg1 caused a further increase in the elevated [Ca2+]i induced by PDGF-BB. It was concluded that Rg1 could inhibit the VSMC proliferation induced by PDGF-BB through restricting the G0/G1 phase to S-phase progression in cell cycle. The mechanisms may be related to the upregulation of eNOS mRNA and the increase of the formation of NO and cGMP.
Scintillation Proximity Assay of Arginine Methylation
Zheng, Y. George
Journal of Biomolecular Screening
Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein post-translational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, 3H-AdoMet was used as methyl donor, and biotin-modified histone H4 peptide served as a methylation substrate. Following the methylation reaction catalyzed by PRMTs, streptavidin-coated SPA beads were added to the reaction solution, and SPA signals were detected by a MicroBeta scintillation counter. No separation step is needed, which simplifies the assay procedure and greatly enhances the assay speed. Particularly, the miniaturization and robustness suggest that this method is suited for HTS of PRMT inhibitors.
protein arginine methyltransferases; PRMT; scintillation proximity assay; SPA; high-throughput screening; HTS
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