Post-translational modifications (PTMs) are essential regulators of cellular processes, influencing gene expression, protein stability, and protein-protein interactions.1 Among these, lysine and arginine modifications such as acetylation, methylation, citrullination and other acylation variants are key players in epigenetic regulation.2,3 However, the development of assay systems that can adapt to a wide range of PTMs remains a challenge. Here, we present a generalized fluorescent turn-on platform that utilizes simple peptide substrates to study the installation and removal of a diverse set of lysine and arginine PTMs, with a focus on epigenetically relevant ones.
In addition to synthetic installation of native post-translationally modified residues in peptides, we employed thialysine and thiaarginine analogs to mimic modified lysine and arginine residues, enabling facile introduction of functional PTM mimetics using simple cysteine chemistry.4,5 We utilize the cleavage of peptidyl lysine and arginine bonds by trypsin, which are only removed when these residues are in their unmodified state. Conversely, in their post-translationally modified state, the peptides remain intact leading to internal fluorescent quenching, making the system adaptable to studies of both writer and eraser enzymes.
Model PTMs that have been studied are removal of lysine acetylation, lactylation and β-hydroxybutyrylation by SIRT3, removal of methylated lysine variants by KDM3A and KDM4A as well as arginine citrullination by PAD4, highlighting the versatility of this approach. By integrating modularity and fluorogenic detection, this system provides an accessible, flexible, efficient, and adaptable tool for PTM studies. Its broad applicability offers significant potential for exploring enzymatic mechanisms, PTM crosstalk, and protein regulation across diverse biological contexts.
References
1. B. S. Sharma, V. Prabhakaran, A. P. Desai, J. Bajpai, R. J. Verma and P. K. Swain, Oncogen, 2019, 2, 12.
2. J. Fuhrmann, K. W. Clancy and P. R. Thompson, Chem. Rev., 2015, 115, 5413-5461.
3. A. H. Shukri, V. Lukinović, F. Charih and K. K. Biggar, Biochim. Biophys. Acta Gene Regul. Mech., 2023, 194990.
4. J. C. J. Hintzen and J. Mecinović, Tetrahedron Lett., 2023, 124, 154602.
5. S. Ofori, H. S. Desai, F. Shikwana, L. M. Boatner, E. R. Dominguez Iii, J. O. Castellón and K. M. Backus, Chem. Commun., 2024, 60, 8856-8859.