Biochemical investigations using mass spectrometry to monitor JMJD6-catalysed hydroxylation of multi-lysine containing bromodomain-derived substrates.

Abstract

Jumonji-C domain-containing protein 6 (JMJD6) is a human 2-oxoglutarate (2OG)/Fe(ii)-dependent oxygenase catalysing post-translational C5 hydroxylation of multiple lysine residues, including in the bromodomain-containing proteins BRD2, BRD3 and BRD4. The role(s) of JMJD6-catalysed substrate hydroxylation are unclear. JMJD6 is important in development and JMJD6 catalysis may promote cancer. We report solid-phase extraction coupled to mass spectrometry assays monitoring JMJD6-catalysed hydroxylation of BRD2-4 derived oligopeptides containing multiple lysyl residues. The assays enabled determination of apparent steady-state kinetic parameters for 2OG, Fe(ii), l-ascorbate, O₂ and BRD substrates. The JMJD6 K ^app _m for O₂ was comparable to that reported for the structurally related 2OG oxygenase factor inhibiting hypoxia-inducible factor-α (FIH), suggesting potential for limitation of JMJD6 activity by O₂ availability in cells, as proposed for FIH and some other 2OG oxygenases. The new assays will help development of small-molecule JMJD6 inhibitors for functional assignment studies and as potential cancer therapeutics.