Unbiased proteomics identifies plasminogen activator inhibitor-1 as a negative regulator of endothelial nitric oxide synthase
- 27 April 2020
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 117 (17), 9497-9507
- https://doi.org/10.1073/pnas.1918761117
Abstract
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 post-translational 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.This publication has 66 references indexed in Scilit:
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