S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells
Abstract
:1. Introduction
2. Results
2.1. Intracellular Cellular NADP+/NADPH and GSH/GSSG Ratios
2.2. Effect of ZDON on Extracellular Acidification Rate (ECAR) upon IS Exposure
2.3. Effects of ZDON on the Expression of eNOS and Phospho-eNOS upon IS Exposure
2.4. In Situ TGase Assay to Identify Potential TG2/TGase Substrates
2.5. SNO-RAC on Purified TG2, Total SNO Proteins and S-nitrosylation of TG2/TGase Substrate Proteins during IS-Induced Injury
2.6. Time Courses Studies on the Expression of TG2/TGase Substrates, Redox, and Inflammatory Proteins upon IS Exposure Either in the Presence or Absence of ZDON
3. Discussion
4. Materials and Methods
4.1. Chemicals
4.2. Antibodies
4.3. SDS-PAGE and Immunoblotting
4.4. Cells
4.5. Cell Treatments and Total Lysates Preparation
4.6. Cellular GSH/GSSG Assay
4.7. Cellular NADP+/NADPH Assay
4.8. XFe24 Seahorse Glycolysis Stress Assays
4.9. Resin Assisted Capture for S-nitrosothiols (SNO-RAC)
4.10. SNO-RAC of Purified TG2
4.11. Total SNO Proteins Content
4.12. In Situ TG2/TGase Assay
4.13. Intracellular ROS Generation Assay
4.14. Molecular Modeling
4.15. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Ligand | PDB Code | Cysteine Number |
---|---|---|
GDP | 1KV3 | 98, 545, 554 |
GTP | 4PYG | 10, 98, 269, 545, 554 |
Ac-P(DON)LPF-NH2 | 2Q3Z | 10, 98, 269, 545, 554 |
Z-DON-VPL-Ome (ZDON) | 3S3J | 10, 98, 545 |
Ca2+ | 6KZB | 10, 27, 98, 269, 545, 554 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
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Lin, C.-J.; Chiu, C.Y.; Liao, E.-C.; Wu, C.-J.; Chung, C.-H.; Greenberg, C.S.; Lai, T.-S. S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells. Int. J. Mol. Sci. 2023, 24, 10935. https://doi.org/10.3390/ijms241310935
Lin C-J, Chiu CY, Liao E-C, Wu C-J, Chung C-H, Greenberg CS, Lai T-S. S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells. International Journal of Molecular Sciences. 2023; 24(13):10935. https://doi.org/10.3390/ijms241310935
Chicago/Turabian StyleLin, Cheng-Jui, Chun Yu Chiu, En-Chih Liao, Chih-Jen Wu, Ching-Hu Chung, Charles S. Greenberg, and Thung-S. Lai. 2023. "S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells" International Journal of Molecular Sciences 24, no. 13: 10935. https://doi.org/10.3390/ijms241310935
APA StyleLin, C. -J., Chiu, C. Y., Liao, E. -C., Wu, C. -J., Chung, C. -H., Greenberg, C. S., & Lai, T. -S. (2023). S-Nitrosylation of Tissue Transglutaminase in Modulating Glycolysis, Oxidative Stress, and Inflammatory Responses in Normal and Indoxyl-Sulfate-Induced Endothelial Cells. International Journal of Molecular Sciences, 24(13), 10935. https://doi.org/10.3390/ijms241310935