ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory signaling
Man Sup Kwak, Myeonggil Han, Yong Joon Lee, Seoyeon Choi, Jeong Hwa Kim, In Ho Park, Jeon‐Soo Shin
Abstract
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: Cys 23 , Cys 45 , and Cys 106 , 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 Cys 106 -mediated HMGB1 homo-dimerization. In the extracellular environment, LPS and LTA induced HMGB1 self-association leading to H 2 O 2 anchoring Cys 106 –Cys 106 -mediated HMGB1 intermolecular disulfide bond formation. Despite treatment with H 2 O 2 , LPS, or LTA, HMGB1 dimerization was blocked in presence of Cys 106 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 H 2 O 2 in the extracellular environment. Compared to monomeric HMGB1, Cys 106 –Cys 106 -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. • HMGB1 is secreted extracellularly in its monomeric form and subsequently dimerized by ROS and PAMPs. • Cys 106 is crucial for antiparallel dimerization of HMGB1 via disulfide bond formation. • Di-HMGB1 increases binding affinity for TLR2 and TLR4, thereby enhancing pro-inflammatory signaling.