Dynamic S-nitrosylation of endothelial nitric oxide synthase in vascular endothelial cells.

摘要

The endothelial isoform of nitric oxide synthase (eNOS) is a key signaling protein expressed by vascular endothelial cells. Following activation of cell surface receptors, eNOS activity is dynamically regulated by numerous post-translational modifications and protein-protein interactions. Nitric oxide (NO) produced by eNOS is a key determinant of vascular homeostasis and in its canonical mechanism activates soluble guanylate cyclase in target cells. This dissertation examines the regulation of eNOS activity by S-nitrosylation, the covalent adduction of NO-derived nitrosyl groups to the reduced cysteine thiols of proteins, and explores the dynamic modulation of eNOS S -nitrosylation by cell surface receptors.; Chapter 2 shows that eNOS is constitutively S-nitrosylated by eNOS-derived NO in cultured endothelial cells. Following agonist stimulation, eNOS is rapidly denitrosylated concomitant with enzyme activation and translocation from plasma membrane caveolae to internal membranes; eNOS is thereafter progressively renitrosylated as it returns to basal activity levels. I show that S-nitrosylation of purified eNOS inhibits enzyme activity and that eNOS inhibition is reversed by denitrosylation. I also document that eNOS targeting to the plasma membrane is necessary for eNOS S-nitrosylation and that eNOS is endogenously S-nitrosylated at the cysteines comprising the zinc-tetrathiolate structure of the eNOS dimer interface.; Chapter 3 uses mass spectrometry to confirm that eNOS S-nitrosylation occurs at its zinc-tetrathiolate. I then analyze the S-nitrosylation patterns of recombinant eNOS constructs in transfected cells and show that subcellular targeting is a critical determinant of eNOS S-nitrosylation. I document that S-nitrosylation is restricted to membrane-targeted eNOS, and show that eNOS translocation from caveolae to internal membranes is necessary for agonist-modulated denitrosylation. Finally, my findings are extended from cultured endothelial cells to establish that agonist-modulated eNOS S-nitrosylation can be detected in individual arterial preparations isolated from mice.; The studies in this dissertation document the dynamic receptor-regulated S-nitrosylation of a key cardiovascular signaling protein. Understanding the physiologic and pathologic mechanisms by which eNOS S-nitrosylation is regulated may lead to the identification of novel mechanisms whereby NO-dependent signaling pathways are regulated in the vascular wall.