Abstract
JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor α, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6Δ344–403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.
Highlights
Chang et al [15] assigned JMJD6 as an histone N-methyl arginyl demethylase (RDM) acting on mono- and symmetric/ asymmetric di-methyl forms of arginine residues as observed in studies with isolated enzyme (Fig. 1B)
We describe biochemical and biophysical studies on isolated JMJD6 using multiple substrates reported as targets for JMJD6-catalyzed lysine-residue hydroxylation and N-methyl arginyl demethylation
This assignment is consistent with crystallographic studies of JMJD6 with its natural ferrous iron cofactor and 2OG cosubstrate; these results imply that the JMJD6 active site is more similar to the JmjC hydroxylases, such as FIH, rather than the JmjC demethylases
Summary
Chang et al [15] assigned JMJD6 as an histone N-methyl arginyl demethylase (RDM) acting on mono- and symmetric/ asymmetric di-methyl forms of arginine residues as observed in studies with isolated enzyme (Fig. 1B). Subsequent to its assignment as an RDM, JMJD6 was reported to catalyze lysine C-5 hydroxylation (Fig. 1C) of the splicing regulatory (SR) proteins luc-7–like 2 (LUC7L2), cisplatin resistance-associated overexpressed protein (CROP), and u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65) [17]. We describe biochemical and biophysical studies on isolated JMJD6 using multiple substrates reported as targets for JMJD6-catalyzed lysine-residue hydroxylation and N-methyl arginyl demethylation. The combined results support the assignment of purified JMJD6 as a lysyl C-5 hydroxylase rather than an N-methyl arginyl demethylase. We intend that the results will aid in the accurate assignment of JMJD6 substrates in the cellular context and help to define its physiological roles
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