Cellular regulation of nitric oxide synthase expression and S-nitrosylation

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Nitric oxide (NO) is a bioactive gas that controls a wide array of cell biological processes. It exerts its biological effects mainly by causing targeted protein S-nitrosylation, which is the covalent addition of a nitroso moiety onto target cysteines and is an enzymatically-controlled process in cells. S-nitrosylation controls protein function and has been proposed to be of similar importance to cell biology as protein phosphorylation. However, its regulation and biological effects are relatively understudied. Because of the biological importance of NO, the long-term goal of my research program is to understand the biological effects and cellular regulation of this gas. In order to fully understand the biology of NO, it is important to also study the mechanisms by which its production is regulated. NO is synthesized by enzymes called nitric oxide synthases (NOSs), of which there are three isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Cytokines modulate the expression of all NOSs, and we have studied the cytokine regulation of iNOS and eNOS expression. With regard to iNOS, we have shown that NO initiates a positive feedback loop that amplifies iNOS expression by inducing Ras S-nitrosylation. We also know that NO-mediated amplification of iNOS expression occurs through increased mRNA translation. However, we do not know how Ras S-nitrosylation is regulated in cells and how iNOS mRNA translation is controlled by the NO-mediated feedback pathway that we have identified. These questions need to be investigated. In addition to iNOS, we have also determined that the cytokine IL-17 up-regulates eNOS expression through an unknown post-translational mechanism. Based on our findings, the short-term objectives of this Discovery Grant are to define the cellular regulation of Ras S-nitrosylation and post-transcriptional regulation of iNOS and eNOS expression. The work proposed will advance our understanding of the cell biological effects of NO and of NOS regulation. The three aims are:Aim 1: Determine the mechanism controlling Ras S-nitrosylation and its effect on Ras function. We have determined that iNOS-derived NO leads to Ras S-nitrosylation but do not know the mechanism by which this occurs. Much of the S-nitrosylation that occurs in cells is enzymatically-regulated by nitrosylases, which catalyze the reaction, and by de-nitrosylases, which reverse it. We will identify the nitrosylases and de-nitrosylases that target Ras. The effect of S-nitrosylation on Ras function will also be determined by examining the effect of this protein modification on the association of Ras with signaling molecules and on Ras localization. Aim 2: Determine the mechanism by which NO amplifies iNOS mRNA translation . We have characterized a new NO-mediated signaling pathway that amplifies iNOS mRNA translation but do not know the specific translational mechanisms involved. We will determine this by identifying iNOS mRNA binding proteins that are affected by NO. The function of candidate proteins will then be determined by inhibiting their expression with siRNA and by mutating their RNA binding motifs in iNOS mRNA sequences. Aim 3: Determine the post-translational mechanism by which IL-17 increases eNOS protein levels. We have shown that IL-17 increases eNOS protein levels through a post-translational process but do not know the mechanism. The effect of IL-17 on eNOS post-translational modifications that are known to affect protein stability, such as phosphorylation and ubiqutinylation, will be determined by proteomic analysis. The role of candidate protein modifications will then be determined by mutating the modification site(s).