title: Real-time kinetics of high-mobility group box 1 (HMGB1) oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.
abstract: Some extracellular proteins are initially secreted in reduced forms via a non-canonical pathway bypassing the endoplasmic reticulum and become oxidized in the extracellular space. One such protein is HMGB1 (high-mobility group box 1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation. Using a unique NMR-based approach, we have investigated the kinetics of HMGB1 oxidation and the half-lives of all-thiol and disulfide HMGB1 species in serum, saliva, and cell culture medium. In this approach, salt-free lyophilized (15)N-labeled all-thiol HMGB1 was dissolved in actual extracellular fluids, and the oxidation and clearance kinetics were monitored in situ by recording a series of heteronuclear (1)H-(15)N correlation spectra. We found that the half-life depends significantly on the extracellular environment. For example, the half-life of all-thiol HMGB1 ranged from ~17 min (in human serum and saliva) to 3 h (in prostate cancer cell culture medium). Furthermore, the binding of ligands (glycyrrhizin and heparin) to HMGB1 significantly modulated the oxidation kinetics. Thus, the balance between the roles of all-thiol and disulfide HMGB1 proteins depends significantly on the extracellular environment and can also be artificially modulated by ligands. This is important because extracellular HMGB1 has been suggested as a therapeutic target for inflammatory diseases and cancer. Our work demonstrates that the in situ protein NMR approach is powerful for investigating the behavior of proteins in actual extracellular fluids containing an enormous number of different molecules.
authors: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1068, USA.
authors: Levani
authors: L
authors: Zandarashvili
authors: Debashish
authors: D
authors: Sahu
authors: Kwanbok
authors: K
authors: Lee
authors: Yong Sun
authors: YS
authors: Lee
authors: Pomila
authors: P
authors: Singh
authors: Krishna
authors: K
authors: Rajarathnam
authors: Junji
authors: J
authors: Iwahara
mesh_terms: D000328:Adult; D000368:Aged; D045744:Cell Line, Tumor; D024243:HMGB1 Protein / Q000097:blood*; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008297:Male; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D012463:Saliva / Q000378:metabolism*
chemical_list: D024243:HMGB1 Protein