ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory...

Many cellular proteins form homo- or hetero-oligomeric complexes through dimerization, and ligand oligomerization is crucial for inducing receptor oligomerization. Intermolecular disulfide bond formation is critical for protein oligomerization that regulates biological functions. HMGB1 is a nuclear protein that acts as a DAMP when secreted. HMGB1 is redox-sensitive, contains three cysteines: Cys²³, Cys⁴⁵, and Cys¹⁰⁶, and its function varies depending on the redox state of the extracellular space. However, the homo-dimerization of extracellular HMGB1 and its immunological significance have not been identified. In this study, we investigated the immunological significance of Cys¹⁰⁶-mediated HMGB1 homo-dimerization. In the extracellular environment, LPS and LTA induced HMGB1 self-association leading to H₂O₂ anchoring Cys¹⁰⁶-Cys¹⁰⁶-mediated HMGB1 intermolecular disulfide bond formation. Despite treatment with H₂O₂, LPS, or LTA, HMGB1 dimerization was blocked in presence of Cys¹⁰⁶ residue mutation, the ROS scavenger NAC, and the thiol-reducing agent DTT. Inflammatory stimulation induced the secretion of monomeric HMGB1 but not dimeric HMGB1. HMGB1 dimerization was promoted by PAMPs and H₂O₂ in the extracellular environment. Compared to monomeric HMGB1, Cys¹⁰⁶-Cys¹⁰⁶-linked dimeric HMGB1 significantly enhanced intracellular NF-κB signaling and cytokine production through increased direct binding affinity for TLR2 and TLR4 and effective HMGB1-mediated delivery of PAMPs to their receptors. Therefore, we have demonstrated that dimeric HMGB1 enhances its effect on pro-inflammatory signaling.