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Epigenetic control via allosteric regulation of mammalian protein arginine methyltransferases - PubMed

  • ️Sun Jan 01 2017

Epigenetic control via allosteric regulation of mammalian protein arginine methyltransferases

Kanishk Jain et al. Proc Natl Acad Sci U S A. 2017.

Abstract

Arginine methylation on histones is a central player in epigenetics and in gene activation and repression. Protein arginine methyltransferase (PRMT) activity has been implicated in stem cell pluripotency, cancer metastasis, and tumorigenesis. The expression of one of the nine mammalian PRMTs, PRMT5, affects the levels of symmetric dimethylarginine (SDMA) at Arg-3 on histone H4, leading to the repression of genes which are related to disease progression in lymphoma and leukemia. Another PRMT, PRMT7, also affects SDMA levels at the same site despite its unique monomethylating activity and the lack of any evidence for PRMT7-catalyzed histone H4 Arg-3 methylation. We present evidence that PRMT7-mediated monomethylation of histone H4 Arg-17 regulates PRMT5 activity at Arg-3 in the same protein. We analyzed the kinetics of PRMT5 over a wide range of substrate concentrations. Significantly, we discovered that PRMT5 displays positive cooperativity in vitro, suggesting that this enzyme may be allosterically regulated in vivo as well. Most interestingly, monomethylation at Arg-17 in histone H4 not only raised the general activity of PRMT5 with this substrate, but also ameliorated the low activity of PRMT5 at low substrate concentrations. These kinetic studies suggest a biochemical explanation for the interplay between PRMT5- and PRMT7-mediated methylation of the same substrate at different residues and also suggest a general model for regulation of PRMTs. Elucidating the exact relationship between these two enzymes when they methylate two distinct sites of the same substrate may aid in developing therapeutics aimed at reducing PRMT5/7 activity in cancer and other diseases.

Keywords: PRMT5; PRMT7; allosteric regulation; epigenetics; histone methylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Analysis of methylarginine production by HsPRMT5/MEP50 on histone H4 (1-21) peptide. (A) A representative cation exchange chromatogram (n = 3) for hydrolysates of reactions with H4 (1-21) WT (blue) and H4 (1-21) R17MMA (red) as substrates with [methyl-3H]-AdoMet. Fractions (1 min) were collected and analyzed for the presence of nonradioactive methylarginine standards and radioactivity. The black line indicates the retention profile of the standards as determined by ninhydrin assays (47, 48). The colored lines represent radioactive methylarginine derivatives which elute about 1 min before the nonradioactive standards due to the isotope effect (50). For details of the reaction and chromatography conditions, see Methods. (B) An expanded view of A to emphasize the differences in SDMA levels. (C) Data from three replicate experiments were used to show changes in 3H-MMA and 3H-SDMA produced with H4 (1-21) WT (blue bars) or its R17MMA derivative (red bars); the P values were determined through two-tailed t tests. The error bars represent SDs. (D) The SDMA/MMA ratio was calculated from the data in C. The P value was determined as for C, and the error bars represent SDs.

Fig. 2.
Fig. 2.

Monomethylation of H4 R17 affects the positive cooperativity exhibited by HsPRMT5/MEP50. Initial kinetic measurements were made, and the data were fit to the Hill equation (38). (A) Enzyme activity of HsPRMT5/MEP50 with H4 (1-21) WT (blue), H4 (1-21) R17MMA (brown), and H4 (1-21) R3K (red) is shown for triplicate assays (error bars represent SD). (B) An expanded view of A at the low substrate concentrations. Best fit curves are shown for K0.5, kcat, and Hill coefficient values for the H4 (1-21) WT substrate of 0.39 μM, 5.63 h−1, and 2.83, respectively. For H4 (1-21) R17MMA, the parameters were 0.13 μM, 9.25 h−1, and 1.3, respectively. For details about reaction conditions and concentrations, see Methods. Statistical analysis of K0.5 values (C), statistical analysis of kcat values (D), and statistical analysis of the Hill coefficient values (E). The dashed line represents a Hill coefficient of 4. Data were taken from the triplicate assays shown in A and B; error bars represent SD. The P values were calculated using a one-way ANOVA test with a Dunnett test for multiple comparisons using the GraphPad Prism 6.0 software.

Fig. S1.
Fig. S1.

Binding affinity of AdoMet with HsPRMT5/MEP50 and HsPRMT1. Initial kinetic measurements were made, and the data were fit to the Michaelis–Menten equation. (A) Enzyme activity of HsPRMT5/MEP50 with varying [AdoMet] and 10 μM H4 (1-21) WT peptide in triplicate (error bars represent SD). The solid line was best fit to the Michaelis–Menten equation with the following parameters: a Km of 1.66 ± 0.37 μM and a kcat of 1.72 ± 0.14 h−1. (B) Enzyme activity of HsPRMT1 with varying [AdoMet] and 10 μM H4 (1-21) WT peptide in triplicate (error bars represent SD). The solid line was best fit to the Michaelis–Menten equation with the following parameters: a Km of 2.41 ± 0.35 μM and a kcat of 3.11 ± 0.27 h−1. For details about reaction conditions and concentrations, see Methods.

Fig. S2.
Fig. S2.

HsPRMT5/MEP50 and HsPRMT1 exhibit positive cooperativity as a function of modifications on the substrate H4 peptide. Initial kinetic measurements were made, and the data were fit to the Hill equation (37). (A) Enzyme activity of HsPRMT5/MEP50 with H4 (1-21) R17A (red), H4 (1-21) R17K (green), H4 (1-21) R19A (purple), and H4 (1-21) R19K (orange) in triplicate assays (error bars represent SD). (B) A closeup of the graph from A to make differences at low [substrate] clearer. (C) Enzyme activity of HsPRMT1 with H4 (1-21) R17A (red), H4 (1-21) R17K (green), H4 (1-21) R19A (purple), and H4 (1-21) R19K (orange) in triplicate assays (error bars represent SD). (D) A close-up of the graph from C to make differences at low [substrate] clearer. For details about reaction conditions and concentrations, see Methods.

Fig. 3.
Fig. 3.

HsPRMT1 exhibits positive cooperativity. Initial kinetic measurements were made, and the data were fit to the Hill equation (38). (A) Enzyme activity of HsPRMT1 with H4 (1-21) WT (blue), H4 (1-21) R17MMA (brown), and H4 (1-21) R3K (red) is shown for triplicate assays (error bars represent SD). (B) An expanded view of A at the low substrate concentrations. Best fit curves are shown for K0.5, kcat, and Hill coefficient values for the H4 (1-21) WT substrate of 0.58 μM, 4.90 h−1, and 1.54, respectively. For H4 (1-21) R17MMA, the parameters were 0.38 μM, 7.85 h−1, and 1.98, respectively. For details about reaction conditions and concentrations, see Methods. Statistical analysis of K0.5 values (C), statistical analysis of kcat values (D), and statistical analysis of the Hill coefficient values (E). The dashed line represents a Hill coefficient of 2. Data were taken from the triplicate assays shown in A and B; error bars represent SD. The P values were calculated using a one-way ANOVA test with a Dunnett test for multiple comparisons using the GraphPad Prism 6.0 software.

Fig. 4.
Fig. 4.

Model for allosteric regulation of PRMT5/MEP50 activity by PRMT7. The green blocks represent residue R3 on histone H4 (1-21), while the red blocks represent residue R17. PRMT5/MEP50 is shown in purple.

Fig. S3.
Fig. S3.

Electrostatic potential map of HsPRMT5/MEP50 protomer reveals potential allosteric sites. (A) The protomer of HsPRMT5/MEP50 (PDB ID code: 4GQB) is shown; the beige subunit represents PRMT5, while the brown subunit represents MEP50. The active site is highlighted by a solid black enclosure. (B) Dashed black enclosures on the opposite face of the structure in A indicate negatively charged cavities as potential allosteric binding sites on the electrostatic potential map, generated using APBS in PyMOL (red to blue corresponds to −5 kT/e to 5 kT/e).

Fig. S4.
Fig. S4.

Electrostatic potential map of HsPRMT1 homodimer reveals potential allosteric sites. The protomer of HsPRMT1 (PDB ID code: 1ORI) is shown. (A) The cartoon representation and electrostatic map for the PRMT1 dimer with the active site (solid black enclosure) facing forward; the dashed black enclosure indicates a negatively charged cavity as potential allosteric binding site on the electrostatic potential map. (B) A 180° rotation of the molecules in A about the y axis shows an additional putative allosteric site. Electrostatic potentials were generated using APBS in PyMOL (red to blue corresponds to −5 kT/e to 5 kT/e).

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References

    1. Walsh G, Jefferis R. Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol. 2006;24:1241–1252. - PubMed
    1. Fuhrmann J, Clancy KW, Thompson PR. Chemical biology of protein arginine modifications in epigenetic regulation. Chem Rev. 2015;115:5413–5461. - PMC - PubMed
    1. Bedford MT, Clarke SG. Protein arginine methylation in mammals: Who, what, and why. Mol Cell. 2009;33:1–13. - PMC - PubMed
    1. Blanc RS, Richard S. Arginine methylation: The coming of age. Mol Cell. 2017;65:8–24. - PubMed
    1. Wang Y-C, Peterson SE, Loring JF. Protein post-translational modifications and regulation of pluripotency in human stem cells. Cell Res. 2014;24:143–160. - PMC - PubMed

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