The TRIM33 Bromodomain\nRecognizes Histone Lysine Lactylation

Histone lysine lactylation (Kla) regulates inflammatory\ngene expression\nin activated macrophages and mediates the polarization of inflammatory\n(M1) to reparative (M2) macrophages. However, the molecular mechanisms\nand key protein players involved in Kla-mediated transcriptional changes\nare unknown. As Kla is structurally similar to lysine acetylation\n(Kac), which is bound by bromodomains, we hypothesized that bromodomain-containing\nproteins bind histone Kla. Here, we screened 28 recombinantly expressed\nbromodomains for binding to histone Kla peptides via AlphaScreen assays.\nTRIM33 was the sole bromodomain tested that bound histone Kla peptides.\nTRIM33 attenuates inflammatory genes during late-stage macrophage\nactivation; thus, TRIM33 provides a potential link between histone\nKla and macrophage polarization. Orthogonal biophysical techniques,\nincluding isothermal titration calorimetry and protein-detected nuclear\nmagnetic resonance, confirmed the submicromolar binding affinity of\nthe TRIM33 bromodomain to both Kla and Kac histone post-translational\nmodifications. Sequence alignments of human bromodomains revealed\na unique glutamic acid residue within the TRIM33 binding pocket that\nwe found confers TRIM33 specificity for binding Kla compared with\nother bromodomains. Molecular modeling of interactions of Kla with\nthe TRIM33 bromodomain binding pocket and site-directed mutagenesis\nof glutamic acid confirmed the critical role of this residue in the\nselective recognition of Kla by TRIM33. Collectively, our findings\nimplicate TRIM33, a bromodomain-containing protein, as a novel reader\nof histone Kla, potentially bridging the gap between histone Kla and\nmacrophage polarization. This study enhances our understanding of\nthe regulatory role of histone Kla in macrophage-mediated inflammation\nand offers insights into the underlying structural and biophysical\nmechanisms.