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首页> 美国卫生研究院文献>The Journal of Biological Chemistry >Real-time Kinetics of High-mobility Group Box 1 (HMGB1) Oxidation in Extracellular Fluids Studied by in Situ Protein NMR Spectroscopy
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Real-time Kinetics of High-mobility Group Box 1 (HMGB1) Oxidation in Extracellular Fluids Studied by in Situ Protein NMR Spectroscopy

机译：原位蛋白NMR光谱研究细胞外液中高迁移率基团1（HMGB1）氧化的实时动力学。

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摘要

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 15N-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 1H-15N 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.
机译：一些细胞外蛋白最初通过绕过内质网的非经典途径以还原形式分泌，并在细胞外空间被氧化。一种这样的蛋白是HMGB1（高迁移率族框1）。细胞外HMGB1具有不同的氧化还原状态，在炎症中起不同的作用。使用基于NMR的独特方法，我们研究了HMGB1氧化的动力学以及全硫醇和二硫键HMGB1在血清，唾液和细胞培养基中的半衰期。用这种方法，将无盐的冻干的 15 N标记的全硫醇HMGB1溶解在实际的细胞外液中，并通过记录一系列异核 1来原位监测氧化和清除动力学。 H- 15 N相关光谱。我们发现半衰期在很大程度上取决于细胞外环境。例如，全硫醇HMGB1的半衰期约为17分钟（在人血清和唾液中）至3小时（在前列腺癌细胞培养基中）。此外，配体（甘草甜素和肝素）与HMGB1的结合显着调节了氧化动力学。因此，全硫醇和二硫键HMGB1蛋白的作用之间的平衡在很大程度上取决于细胞外环境，也可以由配体人工调节。这很重要，因为已建议将细胞外HMGB1作为炎症性疾病和癌症的治疗靶标。我们的工作表明，原位蛋白质NMR方法对于研究蛋白质在包含大量不同分子的实际细胞外液中的行为非常有效。

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