Unbiased proteomics identifies plasminogen activator inhibitor-1 as a negative regulator of endothelial nitric oxide synthase
| Autor: | Mohammad Mahfuzul Haque, Brant E. Isakson, Mauro Siragusa, Katherine R. Heberlein, William C. Sessa, Dennis J. Stuehr, Jonathan V. Pascale, Victor Garcia, Eon Joo Park, Florian Fröhlich |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Nitric Oxide Synthase Type III Biological Availability Proximity ligation assay 030204 cardiovascular system & hematology Nitric Oxide Gene Expression Regulation Enzymologic Piperazines Umbilical vein Cell Line Nitric oxide 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Enos Stable isotope labeling by amino acids in cell culture Plasminogen Activator Inhibitor 1 Human Umbilical Vein Endothelial Cells para-Aminobenzoates Humans Multidisciplinary biology Chemistry Biological Sciences biology.organism_classification Cell biology Vasodilation Endothelial stem cell 030104 developmental biology Plasminogen activator inhibitor-1 Plasminogen activator Protein Binding |
| Zdroj: | Proc Natl Acad Sci U S A |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1918761117 |
| Popis: | Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein-protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein-protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS-PAI-1 binding promote increases in NO production and enhance vasodilation in vivo. |
| Databáze: | OpenAIRE |
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