Protein S-nitrosylation is a ubiquitous, post-translational effector mechanism in which a Cys residue is modified by NO. Dysregulated S-nitrosylation is associated with diverse inflammatory diseases and pathological condi- tions. A concept is emerging in which site-specific modification of disease-related proteins, rather than a global nitrosative activity, contributes to disease initiation and progression. However, molecular mechanisms directing site selectivity are not understood. Our long-term goal is to elucidate mechanisms underlying selective S- nitrosylation, and its pathophysiological role in cardiovascular disease (CVD). We recently observed a remark- able S-nitrosylation-mediated inactivation of the GAIT (IFN-Gamma-Activated Inhibitor of Translation) transla- tional control system, discovered in our laboratory. Interferon (IFN)-?, a prototypic activator of myeloid cells, induces assembly of the GAIT complex that binds RNA elements in the 3?UTR of select target mRNAs, e.g., vascular endothelial growth factor-A, and inhibits their translation. The GAIT complex consists of four ?house- keeping? proteins including ribosomal protein L13a and GAPDH. We recently reported that low density lipopro- tein (LDL) oxidized by the physiological myeloperoxidase (MPO)-H2O2-NO2- system (LDLox) inactivates the GAIT system, thereby increasing expression of VEGF-A and other GAIT targets. LDLox induces S-nitrosylation of GAPDH, inactivating the chaperone-like activity by which GAPDH protects L13a, resulting in degradation of nearly the entire cell complement of L13a. An unprecedented site-specific S-nitrosylation of GAPDH at Cys247 is essential for loss of shielding activity. Recently, we found that LDLox plus IFN-? markedly induce iNOS (inducible nitric oxide synthase) in human monocytes and is required for S-nitrosylation of GAPDH at Cys247. We also showed that a heterotrimeric complex of S100A8, S100A9, and iNOS is cotranslationally assembled, and specifically S-nitrosylates GAPDH at Cys247. Serum and LDL from CVD subjects induce Cys247 GAPDH S- nitrosylation, L13a degradation, and VEGF-A expression in monocytes, supporting the physiological signifi- cance of these events. To further investigate the function of GAPDH S-nitrosylation in vivo, we have engineered Cys245 (mouse analog of human Cys247)-to-Ala knock-in mice. We have begun to investigate the role of S100A8/9 on global S-nitrosylation of macrophage proteins, and on macrophage function. The iNOS- S100A8/9 complex induces S-nitrosylation of a cohort of ~100 proteins, with likely involvement in macrophage gene expression, lipid metabolism, and foam cell formation. In this Project we will test the following hypothesis: LDLox, in the presence of IFN-? induces iNOS and subsequent cotranslational assembly of a ternary iNOS- S100A8/A9 nitrosylase complex that directs nitrosylation of Cys247 on GAPDH and other selected targets, causing dysregulation of the GAIT system and contributing to accelerated atherosclerotic lesion progression. Our results can lead to new biomarkers with strong predictive power, and potentially to new therapeutic targets that disrupt site-selective S-nitrosylation.
Project 2 (P2). Project Narrative Protein S-nitrosylation is a ubiquitous, post-translational effector mechanism in which a Cys residue is modified by NO, and is associated with diverse inflammatory diseases and pathological conditions. A concept is emerging in which site-specific modification of disease-related proteins, rather than a global nitrosative activity, contributes to disease initiation and progression. Our long-term goal is to elucidate mechanisms underlying selective S-nitrosylation and its pathophysiological role in cardiovascular disease.
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