Title: Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6
Journal: Proceedings of the National Academy of Sciences of the United States of America
Published: 2022
Background
Protein hydroxylation is a post-translational modification (PTM) catalyzed by 2-oxoglutarate-dependent dioxygenases that can influence protein structure, interactions, and activity. While proline and asparagine hydroxylation in hypoxia signaling (e.g., HIF regulation) is well characterized, the extent and roles of other hydroxylation events in cells remain underexplored. JMJD6, a member of the Jumonji C (JmjC) domain family of dioxygenases, has been linked to diverse cellular functions including transcription, RNA splicing, and development. Early studies proposed roles for JMJD6 in arginine demethylation, but recent evidence indicates that its primary enzymatic activity may be lysine hydroxylation. Despite this, global identification of JMJD6-dependent hydroxylation events in cells has been limited due to technical challenges in detecting low-abundance and context-specific modifications.
This study therefore aims to systematically profile JMJD6-catalyzed lysine hydroxylation across the proteome, to better understand the prevalence and biological contexts of this modification.
Materials & Methods
Peptide Synthesis and Biotin Conjugation to JQ1.
The biotinylated JMJD6 peptide was synthesized at the Peptide Chemistry Facility, Francis Crick Institute. In brief, solid-phase peptide synthesis was performed on an auto mated peptide synthesizer using a Rink Amide AM resin LL (0.05 mmol) and N(α)-Fmoc amino acids, including Fmoc-6-aminohexanoic acid, which served as a linker for the biotin group. The peptide was biotinylated on the synthesizer using d-biotin dissolved in 1:1 dimethyl sulfoxide (DMSO):N-methyl-2-pyrrolidone. Peptides were cleaved from the resin, precipitated by addition of diethyl ether, and purified by high pressure liquid chromatography. Conjugation of biotin-PEG11-amine to JQ1 was performed using established methods.
Sample Preparation for Mass Spectrometry
Lysine derivatization with propionic anhydride was performed by combining standard in-gel trypsinolysis workflows and histone derivatization methods. In brief, acrylamide gel pieces were destained by addition of 50% methanol/5% acetic acid solution and either reduced and alkylated by addition of 10 mM dithiothreitol and 50 mM iodocaetamide or derivatized without prior reduction/alkylation [FLAG-JMJD6 and JMJD6 peptide experiments]. Gel pieces were dehydrated by addition of 100% acetonitrile (MeCN) prior to two rounds of propionylation, which was performed in small batches to enable precise timing of the reaction. Gel pieces were rehydrated by addition of four volumes of 100 mM ammonium bicarbonate directly followed by one volume of propionylation reagent (propionic anhydride and anhydrous MeCN in a 1:3 vol/vol ratio). Samples were mixed and the pH was immediately adjusted to pH 8 by addition of ammonium hydroxide. The reaction was incubated at 51 °C for 20 min with constant mixing. A second round of derivatization was performed prior to two exchanges (dehydration and rehydration) in 100% MeCN and 100 mM ammonium bicarbonate to remove residual propionylation reagent prior to in-gel digestion with trypsin. For the JMJD6 interactome experiments, FLAG-affinity eluates were precipitated by methanol/chloroform and resuspended in urea buffer (6 M urea, 100 mM Tris pH 7.8) prior to reduction/alkylation and trypsinolysis, which was performed in solution under denaturing (1 M urea) conditions. Where appropriate, peptide preparations were desalted by solid-phase extraction in accordance with the manufacturer's instructions, and resuspended in aqueous 2% (vol/vol) MeCN, 0.1% (vol/vol) formic acid for mass spectrometric analysis.
Results
Figure 1. The BIDs of bromodomain proteins are polyhydroxylated by JMJD6. (A) Schematic of affinity enrichment and chemical derivatization: #1 Bromodomain proteins are enriched by JQ1 pulldown; #2 lysine/hydroxylysine residues are derivatized by addition of propionic anhydride; #3 derivatization blocks trypsinolysis to yield lysine-rich peptides for MS analysis. (B) Summary of BRD4 hydroxylations; extensive hydroxylation (14 sites) was observed in the BID, which is both lysine rich and predicted to be structurally disordered. (C) Stoichiometry of hydroxylation on the indicated residues of BRD4 (Upper), BRD3 (Middle), and BRD2 (Lower) in HEK293 cells.
Figure 2. (A) MSMS analysis illustrating polyhydroxylation of a lysine-rich region in NKAP. Fragment ions are from a peptide corresponding to residues 246 to 253, obtained following derivatization with propionic anhydride and digestion with trypsin. (B) Stoichiometry of JMJD6-dependent hydroxylation identified by JMJD6(81 to 96) peptide affinity purification from wild-type and JMJD6-defective HeLa cells. (C) MSMS mirror plot of FACT complex subunit SPT16 (SUPT16H, residues 1037 to 1046) peptide in doubly hydroxylated (Upper) and unmodified (Lower) form derived from wild-type and JMJD6-defective HeLa cells, respectively. (D) Summary of hydroxylysine assignments from assembled screens. Columns depict the number of hydroxylation sites assigned to JMJD6 targets. (E) Bubble plot showing the relationship between JMJD6-catalyzed hydroxylation (red bubble; scaled by stoichiometry hydroxylation at the target site), the probability of disorderedness (determined by the IUPred2A prediction tool), and calculated charge in the vicinity (−5 to +5 residues) of the hydroxylation site.
Conclusions
This study demonstrates that JMJD6 catalyzes widespread lysine hydroxylation across numerous cellular proteins, particularly targeting intrinsically disordered, lysine-rich regions. The identified hydroxylation events are not restricted to classical hypoxia signaling pathways but are broadly distributed among proteins involved in transcriptional regulation, RNA processing, and protein–protein interactions. The findings establish lysine hydroxylation as a prevalent and previously underappreciated post-translational modification in cells. Importantly, the study highlights the functional relevance of JMJD6-mediated hydroxylation in modulating protein interactions and cellular organization rather than acting as a simple on–off regulatory switch. Overall, this work expands the biological scope of protein hydroxylation and underscores the need for sensitive mass spectrometry–based strategies to systematically profile hydroxylation events and elucidate their roles in complex cellular processes.
Reference
- Cockman, M. E., Sugimoto, Y., Pegg, H. B., Masson, N., Salah, E., Tumber, A., Flynn, H. R., Kirkpatrick, J. M., Schofield, C. J., & Ratcliffe, P. J. (2022). Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6. Proceedings of the National Academy of Sciences of the United States of America, 119(32), e2201483119. https://doi.org/10.1073/pnas.2201483119
