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. 2023 Apr;597(7):933-946.
doi: 10.1002/1873-3468.14586. Epub 2023 Feb 7.

The catalytic domains of all human KDM5 JmjC demethylases catalyse N-methyl arginine demethylation

Joanna Bonnici  1   2 , Razanne Oueini  1 , Eidarus Salah  1 , Catrine Johansson  1   3 , Christopher J Schofield  1 , Akane Kawamura  1   2
Affiliations

The catalytic domains of all human KDM5 JmjC demethylases catalyse N-methyl arginine demethylation

Joanna Bonnici et al. FEBS Lett. 2023 Apr.

Abstract

The demethylation of Nε -methyllysine residues on histones by Jumonji-C lysine demethylases (JmjC-KDMs) has been established. A subset of JmjC-KDMs has also been reported to have Nω -methylarginine residue demethylase (RDM) activity. Here, we describe biochemical screening studies, showing that the catalytic domains of all human KDM5s (KDM5A-KDM5D), KDM4E and, to a lesser extent, KDM4A/D, have both KDM and RDM activities with histone peptides. Ras GTPase-activating protein-binding protein 1 peptides were shown to be RDM substrates for KDM5C/D. No RDM activity was observed with KDM1A and the other JmjC-KDMs tested. The results highlight the potential of JmjC-KDMs to catalyse reactions other than Nε -methyllysine demethylation. Although our study is limited to peptide fragments, the results should help guide biological studies investigating JmjC functions.

Keywords: 2-oxoglutarate non-heme oxygenase; JmjC-KDM; epigenetics; histone N-methyl arginine/lysine demethylase; post translational modification.

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Figures

Fig. 1
Fig. 1
JmjC‐catalysed demethylation of N‐methylated lysines and arginines. Demethylation of N‐methyllysine (top) and N‐methylarginine (bottom) residues involves oxidative decarboxylation of 2OG to form succinate, carbon dioxide, and an unstable hemiaminal, which fragments, releasing formaldehyde and the demethylated product.
Fig. 2
Fig. 2
All KDM5 subfamily members demethylate N‐methyl‐arginines. (A) MALDI–TOF MS analyses reveal −14 Da peaks from H3(1–21)R2me2a corresponding to removal of methyl groups following incubation with KDM5A‐D (90 min, red) at 37 °C. Charge state of labelled ions: [MH]+. *: low‐level demethylation (< 10% relative abundance). (B) Comparison of KDM5A‐D‐catalysed demethylation of H3(1–21)R2me2a and H3(1–21)K4me3 under the same assay conditions (37 °C, 90 min). Average demethylation (%) ± SD (n = 3 independent assays) are shown for each substrate/enzyme combination. See Table S3 for assay conditions.
Fig. 3
Fig. 3
Some KDM4 subfamily members demethylate N‐methyl‐arginines. (A) MALDI–TOF MS analyses reveal −14 Da peaks corresponding to the removal of methyl groups of H3(1–21)K4me3R9me2a following incubation (60 min) with KDM4A and KDM4E. Charge state of labelled ions: [MH]+. (B) Comparison of KDM4A‐E catalysed demethylation of H3(1–21)R2me2a (cyan), H3(1–21)R2me2aK4me3 (purple), H3(1–21)R9me2a (red), H3(1–21)K4me3R9me2a (blue), H3(1–21)K9me3 (green), and H3(1–21)K4me3K9me3 (orange) under the same assay conditions (37 °C, 60 min). Average demethylation (%) ± SD (n = 3 independent assays) are shown for each substrate/enzyme combination. See Table S3 for assay conditions. (C) Representative deconvoluted LC–MS for 16‐h incubation of KDM4E with calf histone H4 showing a −28 Da mass shift consistent with removal of two methyl groups. n = 3 (independent assays).
Fig. 4
Fig. 4
KDM5 demethylate N‐methyl‐arginines of non‐histone proteins. Representative MALDI–TOF MS following incubation of KDM5C (left) and KDM5D (right) with G3BP1(444–465)R447me2a (top), and G3BP1(444–465)R460me2a (bottom) showing −14 Da decreases in mass corresponding to partial demethylation (n = 2, technical duplicates). (*) low‐level demethylation (< 10% relative abundance). Y‐axis: relative abundance (%). Refer to Table S3 for assay conditions.
Fig. 5
Fig. 5
Summary of RDM activities with histone H3 and H4 peptides and non‐histone peptides. RDM activity of H3R2me2a has been observed previously with KDM4A(1–1064) [14], but was not observed with differently prepared KDM4A(1–1064) or KDM4A(1–359) used in this work. KDM5C RDM activity with H3R8me1 and H4R3me1 was observed here with a 20‐mer peptide, but not in a previous study with a 15‐mer peptide [14]. Additional observations from this work: RDM activity with KDM4A and H3R9me2a (in the presence of H3K4me3), KDM4C and H3R9me2a, KDM4D and H3R2me2a, H4R3me2a, H4R3me1; KDM5A and KDM5B and H3R2me2a; KDM5C and G3BP1R447me2a and G3BP1R460me2a; and KDM5D and H3R2me2a, G3BP1R447me2a and G3BP1R460me2a. RDM activity observed either in this study or previously or in both is in blue. Low‐level RDM activity is indicated with dashed blue (< 10% product formation). All methylated arginine histone substrates have been reported [31] except for H3R9me2a which is a variant (underlined) of the natural histone H3K9me3 substrate of the KDM4s. Note that not all putative methylated arginine histone substrates tested are shown – only those yielding positive results. Enzymes in bold were part of initial screens, and the rest were used in follow‐up investigations.
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References

    1. Allis CD and Jenuwein T (2016) The molecular hallmarks of epigenetic control. Nat Rev Genet 17, 487–500. - PubMed
    1. Paik WK and Kim S (1973) Enzymatic demethylation of calf thymus histones. Biochem Biophys Res Commun 51, 781–788. - PubMed
    1. Markolovic S, Leissing TM, Chowdhury R, Wilkins SE, Lu X and Schofield CJ (2016) Structure‐function relationships of human JmjC oxygenases‐demethylases versus hydroxylases. Curr Opin Struct Biol 41, 62–72. - PubMed
    1. Forneris F, Binda C, Vanoni MA, Battaglioli E and Mattevi A (2005) Human histone demethylase LSD1 reads the histone code. J Biol Chem 280, 41360–41365. - PubMed
    1. Hopkinson RJ, Hamed RB, Rose NR, Claridge TD and Schofield CJ (2010) Monitoring the activity of 2‐oxoglutarate dependent histone demethylases by NMR spectroscopy: direct observation of formaldehyde. Chembiochem 11, 506–510. - PubMed

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