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| Item Type: | Preprint |
|---|---|
| Title: | Quantitative profiling of JMJD6-catalysed lysine hydroxylation reveals residuedependent oxygen sensitivity |
| Creators: |
Kesavan, Pallavi  ORCID: https://orcid.org/0000-0002-5587-0105, Stuermer, Sophie M. ORCID: https://orcid.org/0009-0001-9319-3600, Räbel, Katrin, Popp, Oliver ORCID: https://orcid.org/0000-0001-6240-4666, Mertins, Philipp ORCID: https://orcid.org/0000-0002-2245-528X, Cockman, Matthew E. ORCID: https://orcid.org/0000-0002-3310-4821 and Sugimoto, Yoichiro ORCID: https://orcid.org/0000-0003-1646-7679
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| Abstract: | Lysine hydroxylation is increasingly recognised as a widespread post-translational modification in human cells, with more than 100 modified sites identified to date. Jumonji domain-containing protein 6 (JMJD6) catalyses hydroxylation at multiple lysines within lysine-rich regions and is a major contributor to this modification across the proteome. As JMJD6 requires oxygen as a co-substrate, lysine hydroxylation has been proposed to couple oxygen availability to cellular function. However, the biological significance of this modification remains incompletely understood, in part due to technical challenges associated with the detection of hydroxylation within lysine-rich regions by mass spectrometry. To address limitations of conventional approaches, we systematically evaluated key steps in the analysis of proteomic data from lysine-derivatised samples and developed a workflow for comprehensive, accurate, and quantitative analysis of lysine hydroxylation. Methodological improvements included optimisation of database search strategies to increase peptide coverage in lysine-rich regions and incorporation of immonium ion signatures to substantially improve confidence in hydroxylysine identification. We further demonstrated that stoichiometry derived from peptide precursor ion intensity faithfully captures hypoxia-responsive changes in lysine hydroxylation at amino acid resolution. Application of this workflow to bromodomain (BRD) proteins – epigenetic readers containing lysine-rich regions extensively hydroxylated by JMJD6 – revealed marked heterogeneity in the apparent kinetics of hydroxylation among target lysines, with evidence of interdependence between neighbouring sites. Hypoxia suppressed hydroxylation in a site-dependent manner, with greater suppression observed at sites displaying slower rates of hydroxylation. Together, the development and application of this workflow establish a methodological and biological framework for understanding how oxygen availability regulates protein function through lysine hydroxylation. |
| Source: | bioRxiv |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Article Number: | 2026.06.08.730680 |
| Date: | 8 June 2026 |
| Official Publication: | https://doi.org/10.64898/2026.06.08.730680 |
| Related to: |
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