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Protein o,o’-Dityrosine Cross-Linking Disrupts Cystic Fibrosis Mucus Viscoelastic Dynamics
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A3881 - Protein o,o’-Dityrosine Cross-Linking Disrupts Cystic Fibrosis Mucus Viscoelastic Dynamics
Author Block: M. L. Locy, C. Fernandez, M. Johnson, A. Lenzie, S. M. Rowe, V. J. Thannickal; Medicine, University of Alabama at Birmingham, Birmingham, AL, United States.
Rationale: Cystic fibrosis (CF) is a multi-organ disease with lung morbidity being the primary cause of early mortality. Cardinal features of CF airway disease include increased viscosity and decreased clearance of neutrophil-laden mucus. Although oxidative stress has been implicated in CF pathogenesis, its role in mucus viscoelasticity are not well understood. Oxidative tyrosine modifications, including o,o’-dityrosine, have been shown to be increased in sputum of CF patients. o,o’-Dityrosine is a stable, covalent, irreversible modification that is catalyzed by hydrogen peroxide (H2O2)-mediated oxidation of a heme peroxidase (hPx), such as myeloperoxidase (MPO) produced by neutrophils and macrophages. In this study, we explored the mechanisms by which protein-associated o,o’-dityrosine contributes to disease pathogenesis.
Methods: Patient-derived primary human bronchial epithelial (HBE) cells were grown in an air-liquid interface. H2O2 measurements were performed utilizing the hPx-dependent homovanillic acid assay. Micro-resolution Optical Coherence Tomography was used to evaluate mucociliary transport (MCT). Data were analyzed by student t test (mean ± SEM, statistical significance was defined as p≤0.05).
Results: We observed that CF donor-derived HBE cells (F508del homozygous) produce increased extracellular H2O2 when compared to non-CF control cells (n=3-4 donors; 2.56 ± 0.87 vs. 0.64 ± 0.15 pmol/min; p≤0.05). MPO treatment decreased MCT by 2.6-fold in CF HBE cells when compared to vehicle treated cells (n=3 donors; p≤0.05), and co-treatment with L-tyrosine, which competitively inhibits protein o,o’-dityrosine cross-linking, abrogated the effect of MPO on MCT. MPO had no effect on MCT in non-CF donor-derived HBE cells, which lack the H2O2 hyperproduction observed in CF HBE cells, a necessary reactant to induce o,o’-dityrosine.
Conclusions: These studies suggest that o,o’-dityrosine cross-linking delays MCT in CF, and may contribute to the elevated viscoelastic properties of CF mucus. Delineating the source of H2O2 production, the proteins modified by o,o’-dityrosine cross-linking, and how this modification changes mucus dynamics within the lung could lead to the development of novel therapies targeting o,o’-dityrosine in CF patients.
Author Block: M. L. Locy, C. Fernandez, M. Johnson, A. Lenzie, S. M. Rowe, V. J. Thannickal; Medicine, University of Alabama at Birmingham, Birmingham, AL, United States.
Rationale: Cystic fibrosis (CF) is a multi-organ disease with lung morbidity being the primary cause of early mortality. Cardinal features of CF airway disease include increased viscosity and decreased clearance of neutrophil-laden mucus. Although oxidative stress has been implicated in CF pathogenesis, its role in mucus viscoelasticity are not well understood. Oxidative tyrosine modifications, including o,o’-dityrosine, have been shown to be increased in sputum of CF patients. o,o’-Dityrosine is a stable, covalent, irreversible modification that is catalyzed by hydrogen peroxide (H2O2)-mediated oxidation of a heme peroxidase (hPx), such as myeloperoxidase (MPO) produced by neutrophils and macrophages. In this study, we explored the mechanisms by which protein-associated o,o’-dityrosine contributes to disease pathogenesis.
Methods: Patient-derived primary human bronchial epithelial (HBE) cells were grown in an air-liquid interface. H2O2 measurements were performed utilizing the hPx-dependent homovanillic acid assay. Micro-resolution Optical Coherence Tomography was used to evaluate mucociliary transport (MCT). Data were analyzed by student t test (mean ± SEM, statistical significance was defined as p≤0.05).
Results: We observed that CF donor-derived HBE cells (F508del homozygous) produce increased extracellular H2O2 when compared to non-CF control cells (n=3-4 donors; 2.56 ± 0.87 vs. 0.64 ± 0.15 pmol/min; p≤0.05). MPO treatment decreased MCT by 2.6-fold in CF HBE cells when compared to vehicle treated cells (n=3 donors; p≤0.05), and co-treatment with L-tyrosine, which competitively inhibits protein o,o’-dityrosine cross-linking, abrogated the effect of MPO on MCT. MPO had no effect on MCT in non-CF donor-derived HBE cells, which lack the H2O2 hyperproduction observed in CF HBE cells, a necessary reactant to induce o,o’-dityrosine.
Conclusions: These studies suggest that o,o’-dityrosine cross-linking delays MCT in CF, and may contribute to the elevated viscoelastic properties of CF mucus. Delineating the source of H2O2 production, the proteins modified by o,o’-dityrosine cross-linking, and how this modification changes mucus dynamics within the lung could lead to the development of novel therapies targeting o,o’-dityrosine in CF patients.
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