Mapping the intracellular HMGB1 interactome and alterations induced by Toll-like receptor 4 activation

The evolutionary conserved, mammalian protein HMGB1 is involved in chromatin-related mechanisms, autophagy, and sensing of immunogenic DNA. HMGB1 translocates from the nucleus to the cytosol in stressed myeloid cells, implicating functional changes. However, the role of intracellular HMGB1 in homeostatic conditions and alterations caused by stress remains largely unexplored. To better understand the intracellular roles of HMGB1, we defined the HMGB1 interactome in resting and LPS-stressed monocytic THP-1 cells using BioID-based proximity proteomics. We identified >100 proteins as parts of the HMGB1 interactome, where the majority were previously unknown interactors. Eight proteins significantly differed between resting and LPS-stressed cells: CACTIN, EIF4G3, GNA12, HSPB1, KOW domain-containing protein, MTHFD11, TADA2B and TPD52L2. Selected HMGB1 interactors were computationally docked to HMGB1, and interactions were confirmed in vitro by proximity ligation assays. Several proteins have implications in cell migration, which was verified experimentally in HMGB1 knockout and shRNA-mediated knockdown cells. HMGB1 deficiency led to an increase in migration compared to wild-type cells or scrambled shRNA control. Furthermore, larger cell size and dysregulated actin polymerization were evident in these cells. In conclusion, we have for the first time identified the intracellular HMGB1 interactome and could identify several HMGB1-protein interactions. Our results reveal previously undescribed homeostatic intracellular roles of HMGB1 in addition to changes caused by cell stress. Our study forms a gateway for future research on HMGB1 functions, both as a pivotal protein for mammalian cell homeostasis and in cellular stress responses.