Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.
- Keywords
- HL-60 cells, NADPH oxidase, NOX4, U-937 cells, macrophages, phorbol 12-myristate 13-acetate, protein-centered radicals,
- MeSH
- Acetophenones pharmacology MeSH
- Staining and Labeling MeSH
- Cell Differentiation * drug effects MeSH
- Electron Spin Resonance Spectroscopy MeSH
- HL-60 Cells MeSH
- Hydroxyl Radical MeSH
- Humans MeSH
- Monocytes cytology drug effects metabolism MeSH
- NADP metabolism MeSH
- Cell Proliferation drug effects MeSH
- Proteins metabolism MeSH
- Tetradecanoylphorbol Acetate pharmacology MeSH
- Cell Shape drug effects MeSH
- U937 Cells MeSH
- Cell Survival drug effects MeSH
- Free Radicals metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Acetophenones MeSH
- acetovanillone MeSH Browser
- Hydroxyl Radical MeSH
- NADP MeSH
- Proteins MeSH
- Tetradecanoylphorbol Acetate MeSH
- Free Radicals MeSH
Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2•-) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2•- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2•- and HO•, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid-water interface. This helps to prevent oxidative modification of the amino acid's hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.
- Keywords
- EPR, mass spectrometry, photosystem II, reactive oxygen species, tocopherol,
- MeSH
- alpha-Tocopherol chemistry metabolism MeSH
- Amino Acids chemistry metabolism MeSH
- Arabidopsis enzymology genetics radiation effects MeSH
- Photosynthesis physiology radiation effects MeSH
- Photosystem II Protein Complex chemistry genetics metabolism MeSH
- Hydroxyl Radical chemistry metabolism MeSH
- Protein Interaction Domains and Motifs MeSH
- Intramolecular Transferases chemistry genetics metabolism MeSH
- Protein Conformation, alpha-Helical MeSH
- Protein Conformation, beta-Strand MeSH
- Oxygen chemistry metabolism MeSH
- Models, Molecular MeSH
- Mutation MeSH
- Oxidation-Reduction MeSH
- Superoxides chemistry metabolism MeSH
- Light MeSH
- Thermodynamics MeSH
- Thermosynechococcus enzymology genetics radiation effects MeSH
- Thylakoids enzymology genetics radiation effects MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Iron chemistry metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Names of Substances
- alpha-Tocopherol MeSH
- Amino Acids MeSH
- Photosystem II Protein Complex MeSH
- Hydroxyl Radical MeSH
- Intramolecular Transferases MeSH
- Oxygen MeSH
- Superoxides MeSH
- tocopherol cyclase MeSH Browser
- Iron MeSH
