NO-Modified Biomolecules and Pulmonary Signaling

描述(申请人提供)：一氧化氮(NO)在肺生理学中的核心作用被它所发挥的许多功能所强调，包括维持血管紧张性、血管紧张性、炎症，甚至肺的生长和发育。除了这些重要的生理作用外，NO还与许多肺部疾病有关，包括ARDS、哮喘和囊性纤维化。到目前为止，这种简单的双原子分子能够产生如此广泛的信号的分子机制尚不清楚，此外，NO代谢的中断如何在病理中发挥作用也不清楚。在炎症中，NO最重要的来源是诱导型iNOS酶。诱导型一氧化氮合酶的一个关键下游作用是由硫醇残基的S亚硝化生成S亚硝硫醇(SNO)。我们假设，通过对不同靶蛋白的SNO修饰，iNOS来源的NO既可以调节损伤的促炎反应，也可以调节对损伤的溶解反应。我们已经建立了一个模型，在这个模型中，在驻留巨噬细胞内的炎症反应早期产生的NO服务于S-亚硝酸酯细胞外靶标，如表面活性蛋白-D(SP-D)；而在反应的后期，随着NO通量的增加和其他氧化剂的产生，细胞内靶标的S-亚硝化，如核因子-β，促进了分解和修复。我们计划研究驻留和招募的巨噬细胞中iNOS和SNO降解酶GSNOR的存在如何改变博莱霉素介导的肺损伤的结果。我们之所以选择这个损伤模型，是因为它既有炎症阶段，也有消退/修复阶段，因此是检验我们的假设的理想选择。在第一个目标中，将通过采用过继转移来实现这些平衡S-亚硝化反应的酶的差异表达。我们将在分子、细胞和器官功能水平上确定驻留和招募的巨噬细胞内iNOS和GSNOR丢失的影响。在第二个目标中，我们将研究如何利用特定SNO靶蛋白SP-D的信号机制的知识来加重或加重博莱霉素介导的肺损伤。这些研究使用最先进的技术来确定NO是如何通过不同靶蛋白的S亚硝化来传递信号的，并可能为治疗设计提供新的途径。

DESCRIPTION (provided by applicant): The central role that Nitric Oxide (NO) plays within pulmonary physiology is highlighted by the number of functions in which it plays a role including the maintenace of ariway tone, blood vessel tone, inflammation, and even lung growth and development. In addition to these important physiological roles, NO has also been implicated in a number of pulmonary diseases including ARDS, Asthma, and cystic fibrosis. As yet the molecular mechanisms by which this simple diatomic molecule can produce such a wide range of signals is unclear, furthermore, it is unclear how disruption of NO metabolism may play a role in pathology. In inflammation, the most important source of NO is the inducible form of the enzyme iNOS. A key downstream effect of iNOS-derived NO is S-nitrosylation of thiol residues to form S-nitrosothiol (SNO). We hypothesize that, by SNO modification of different target proteins, iNOS-derived NO can regulate both the pro-inflammatory and the resolution responses to injury. We have constructed a model in which NO produced early in the inflammatory response within resident macrophages serves to S-nitrosylate extracellular targets, such as Surfactant Protein-D (SP-D); while later in the response, with increasing fluxes of NO and the generation of other oxidants, intracellular S-nitrosylation of targets, such as NF-?B, promotes resolution and repair. We plan to investigate how the presence of iNOS and the SNO-degrading enzyme, GSNOR, in resident and recruited macrophages alters the outcome of bleomycin-mediated lung injury. We have chosen this injury model as it has both an inflammatory and a resolution/repair phase and is therefore ideal for examining our hypothesis. In the first aim differential expression of these enzymes that balance the S-nitrosylation response will be achieved with the use of adoptive transfer. We will determine the effects of loss of iNOS and GSNOR within resident and recruited macrophages at the molecular, cellular, and organ function level. In the second aim, we will examine how we can use knowledge of the signaling mechanisms of a particular SNO target protein, SP-D, to either accentuate or exacerbate bleomycin-mediated lung injury. These studies use state of the art techniques to determine how NO can signal through S-nitrosylation of different target proteins and may provide novel avenues for therapeutic design.