Tetrahydrobiopterin (BH4) is a required cofactor for nitric oxide syntheses (NOS). NO is a reactive signaling molecule that plays key physiological and path physiological roles in mammalian systems. When produced in blood vessels at an appropriate rate and tempo, NO maintains vessel patency, matches blood flow to tissue needs, and maintains an anti-inflammatory state. Notably, proinflammatory cytokines and immunostimulants (e.g., (LPS) induce NO overproduction that can lead to circulatory shock, multi-organ failure and cardiovascular collapse. While upregulation of the gene encoding iNOS is essential for initiation of high-output NO synthesis, it is insufficient. Immunostimulants, additionally upregulate expression of GTP cyclohydrolase (GTPCH), the rate-limiting enzyme for de novo BH4 synthesis. A major goal of this research is to define fundamental postranslational mechanisms that determine the activity of GTPCH in immunoactivated vascular cells and tissues. Preliminary findings have revealed previously unrecognized chemical modifications and protein-protein interactions that are hypothesized to mediate GTPCH activity by LPS - this information could provide a rationale for future therapies that are intended to limit NO-mediated toxicities in inflammatory conditions. We also seek to elucidate how BH4 availability impacts on the SNO-proteome, i.e., the subset of proteins that undergo reversible S-nitrosylation. Notably, actions of NO are mediated by covalent modifications of target proteins, key among these is the reversible addition NO to sulfur in Cys residues, a process termed S-nitrosylation. BH4 insufficiency promotes the formation of peroxynitrite, predictably resulting in S-nitrosylation of a different population of protein Cys residues than those modified by NO when BH4 levels are replete. Despite the great apparent importance of S-nitrosylation for regulating protein functions, lability of the S-NO bond has precluded prior attempts to broadly identify S-nitrosylated proteins in cells and tissues. Proposed studies are enabled by our use of a novel and unbiased proteomic method for high-throughput affinity-capture of SNO-peptides and analysis of sites of Cys modification and protein of origin. This approach will enable the first unambiguous identification for of the """"""""immuno-SNO-proteome"""""""", in mouse tissues and allow identification of fundamental determinants. Bioinformatic approaches will be used to reveal contextual features of the specific Cys residues in these peptides that promote SNO-protein formation. Goals of these proteomic investigations are: (i) to identify yet unrecognized protein targets for signaling by NO-derived species in cells and tissues, (ii) elucidate the relevance of Cys context within a protein as a determinant of NO targeting to specific proteins and sites, (iii) determine how BH4 levels and cellular oxidant stress impacts on protein S-nitrosylation in vascular cells and tissues. Together, the proposed studies will elucidate new and important posttranslational mechanisms the regulate GTPCH activity and define how changing levels of BH4 shape cell signaling by NO in the setting of vascular inflammation.
