Yoshiyuki Kioi

Cytokine release syndrome (CRS) is a life-threatening complication induced by systemic inflammatory responses to infections, including bacteria and chimeric antigen receptor T cell therapy. There are currently no immunotherapies with proven clinical efficacy and understanding of the molecular mechanisms of CRS pathogenesis is limited. Here, we found that patients diagnosed with CRS from sepsis, acute respiratory distress syndrome (ARDS), or burns showed common manifestations: strikingly elevated levels of the four proinflammatory cytokines interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and IL-10 and the coagulation cascade activator plasminogen activator inhibitor-1 (PAI-1). Our in vitro data indicate that endothelial IL-6 trans-signaling formed an inflammation circuit for robust IL-6, IL-8, and MCP-1 production and promoted PAI-1 production; additionally, an IL-6 signaling blockade by the human monoclonal antibody tocilizumab blunted endothelial cell activation. Plasma from severe COVID-19 patients similarly exhibited increased IL-6, IL-10, and MCP-1 levels, but these levels were not as high as those in patients with CRS from other causes. In contrast, the PAI-1 levels in COVID-19 patients were as highly elevated as those in patients with bacterial sepsis or ARDS. Tocilizumab treatment decreased the PAI-1 levels and alleviated critical illness in severe COVID-19 patients. Our findings suggest that distinct levels of cytokine production are associated with CRS induced by bacterial infection and COVID-19, but both CRS types are accompanied by endotheliopathy through IL-6 trans-signaling. Thus, the present study highlights the crucial role of IL-6 signaling in endothelial dysfunction during bacterial infection and COVID-19.

Introducing synthetic fluorophores into specific endogenous proteins and analyzing their function in living cells are a great challenge in chemical biology. Toward this end, we demonstrate the target-selective and site-specific fluorescent labeling of native FKBP12 (FK506-binding protein 12) in vitro and in living cells using ligand-directed tosyl (LDT) chemistry. The LDT-mediated labeling yielded a semisynthetic FKBP12 containing the Oregon green (OG) dye near the catalytic pocket. The OG-labeled FKBP12 (OG-FKBP12) acted as a fluorescent reporter that allows monitoring of its interaction with rapamycin and FRB (FKBP-rapamycin-binding domain) in vitro. We also successfully demonstrated the visualization of the rapamycin-mediated complexation of the OG-FKBP12 and FRB inside of living cells by the combined use with fluorescent protein-tag technology and Förster resonance energy-transfer imaging.

Here we describe how a (19)F-probe incorporated into an endogenous protein by a chemical biology method revealed protein dynamics. By explicit determination of ligand-bound and unbound structures with X-ray crystallography, the quantitative comparison of the protein's dynamics in live cells and in vitro is presented. These results clearly demonstrated the greater conformational fluctuations of the intracellular protein, partially due to macromolecular crowding effects.

Caged enzymes whose activities can be controlled by light represent a powerful tool for various biological analyses. However, limited methods are available for the construction of caged proteins and enzymes. We recently developed a novel protein labeling method termed ligand-directed acyl imidazole (LDAI) chemistry, which allows us to selectively modify natural dihydrofolate reductase and folate receptor in a test tube and in live cell contexts. In this work, we have examined in detail the reaction characteristics of the LDAI chemistry using carbonic anhydrase I (CAI) as a model enzyme. In addition to modifying Lys residues with a carbamate bond, the LDAI method modified Ser and Tyr residues with a carbonate bond. Owing to the relatively labile carbonate bond formed, the LDAI chemistry was demonstrated to be applicable for a rational one-step construction of caged enzymes. This method is simple and based on the transient tethering of an inhibitor with moderate activity that is directed to the active site on an enzyme surface. We successfully showed that the activity of the caged CAI was almost completely suppressed by LDAI-based labeling and fully recovered by photoirradiation in the crude conditions (such as cell lysates) as well as in test tube settings.