Kwak Man Sup, Han Myeonggil, Lee Yong Joon, Choi Seoyeon, Kim Jeonghwa, Park In Ho, Shin Jeon-Soo
Department of Microbiology, Yonsei University College of Medicine, Seoul, 03722, South Korea; Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul, 03722, South Korea.
Department of Microbiology, Yonsei University College of Medicine, Seoul, 03722, South Korea; Brain Korea 21 FOUR Project for Medical Science, Yonsei University College of Medicine, Seoul, 03722, South Korea.
Redox Biol. 2025 Mar;80:103521. doi: 10.1016/j.redox.2025.103521. Epub 2025 Jan 31.
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, Cys, and Cys, 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-mediated HMGB1 homo-dimerization. In the extracellular environment, LPS and LTA induced HMGB1 self-association leading to HO anchoring Cys-Cys-mediated HMGB1 intermolecular disulfide bond formation. Despite treatment with HO, LPS, or LTA, HMGB1 dimerization was blocked in presence of Cys 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 HO in the extracellular environment. Compared to monomeric HMGB1, Cys-Cys-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.
许多细胞蛋白通过二聚化形成同源或异源寡聚复合物，配体寡聚化对于诱导受体寡聚化至关重要。分子间二硫键的形成对于调节生物学功能的蛋白质寡聚化至关重要。HMGB1是一种核蛋白，分泌时作为一种损伤相关分子模式（DAMP）起作用。HMGB1对氧化还原敏感，含有三个半胱氨酸：Cys、Cys和Cys，其功能取决于细胞外空间的氧化还原状态。然而，细胞外HMGB1的同源二聚化及其免疫学意义尚未明确。在本研究中，我们研究了半胱氨酸介导的HMGB1同源二聚化的免疫学意义。在细胞外环境中，脂多糖（LPS）和脂磷壁酸（LTA）诱导HMGB1自缔合，导致HO锚定半胱氨酸-半胱氨酸介导的HMGB1分子间二硫键形成。尽管用HO、LPS或LTA处理，但在存在半胱氨酸残基突变、活性氧（ROS）清除剂N-乙酰半胱氨酸（NAC）和硫醇还原剂二硫苏糖醇（DTT）的情况下，HMGB1二聚化被阻断。炎症刺激诱导单体HMGB1的分泌，但不诱导二聚体HMGB1的分泌。在细胞外环境中，病原体相关分子模式（PAMPs）和HO促进HMGB1二聚化。与单体HMGB1相比，半胱氨酸-半胱氨酸连接的二聚体HMGB1通过增加对Toll样受体2（TLR2）和Toll样受体4（TLR4）的直接结合亲和力以及有效介导HMGB1将PAMPs递送至其受体，显著增强细胞内核因子κB（NF-κB）信号传导和细胞因子产生。因此，我们证明了二聚体HMGB1增强了其对促炎信号传导的作用。
