Abstract
Nitric oxide (NO) is a highly reactive signaling molecule that plays diverse and important bioregulatory roles. In endothelial cells, NO is synthesized by eNOS, a member of the NO synthase (NOS) family, whose activity is tightly regulated at the posttranslational level by acylation, phosphorylation, S-nitrosylation and protein-protein interactions. The biological actions of NO arise as a direct consequence of chemical reactions between NO, or NO-derived species, and protein targets. S-nitrosylation of specific cysteine residues in target proteins is emerging as an important mechanism of NO signaling. Although eNOS is known to undergo a complex pattern of trafficking within endothelial cells, the distinct activities, modes of regulation, and protein targets of NO at eNOS-containing organelles are largely unexplored. We hypothesize that eNOS is differentially regulated at alternative subcellular loci and NO is preferentially transferred to protein acceptors that neighbor NO synthases. To test these hypotheses, we sought to develop a conditional heterodimerization approach that can target eNOS, on-demand, to discrete subcellular loci (via specific targeting sequences e.g. nuclear, mitochondrial, Golgi and cell membrane) and assess the consequences for eNOS activity, regulation and protein S-nitrosylation patterns. Furthermore, we sought to devise a proteomic strategy for unbiased identification of S-nitrosylation sites on proteins that mediate NO bioactivities. Use of these new tools should enable the resolution of controversies regarding the role of diffusion in NO biology, as well as the identification of novel protein targets and mechanism that regulate NO biosynthesis at distinct loci within endothelial cells. In this thesis research, I describe the development and application of "eNOS CID" and "SNOSID" as powerful new tools to address the impact of eNOS compartmentalization on NO signalling in endothelial cells.