Golgi-associated NO-cGMP Signaling Defect in Muscular Dystrophy

描述（由申请人提供）：肌营养不良蛋白缺失导致杜氏肌营养不良症的机制通常归因于细胞外基质和皮质肌动蛋白细胞骨架之间的连接中断导致的膜脆性。抗肌萎缩蛋白及其相关蛋白的这种结构作用得到了大量证据的支持。此外，肌营养不良蛋白复合物作为信号蛋白的肌膜支架，包括神经元一氧化氮合酶（nNOS 5，肌肉特异性亚型），几种激酶，水和离子通道和其他蛋白。在mdx小鼠肌肉中nNOS的转基因表达改善营养不良表型的证明已经将注意力集中在这种信号蛋白上。最近，我们已经发现，这种酶的第二种亚型，nNOS 2，是本地化的高尔基体，并在这个网站上的表达减少mdx肌肉，甚至在第一个营养不良的表型迹象是明显的。此外，我们发现可溶性鸟苷酸环化酶（sGC），这是激活NO，cGMP激活的蛋白激酶G（PKG）与nNOS 2共定位在高尔基体上。缺乏nNOS 5和nNOS 2（KN 2）的小鼠有许多缺陷，包括严重的肌病缺陷。基于这些初步结果，我们将在结构，生化和功能水平上表征KN 2小鼠的肌肉。此外，我们将测试骨骼肌中nNOS 2的转基因表达将拯救/改善KN 2和mdx表型的假设。nNOS 2在减弱1-肾上腺素能血管收缩收缩中的作用将提供关于这种酶在减轻功能性缺血（肌营养不良的一个组成部分）中的重要性的信息。最后，我们将确定NO/cGMP/PKG途径和直接蛋白质亚硝基化的高尔基体底物。最后，我们将鉴定调节NO/cGMP/PKG通路的磷酸二酯酶（PDE）。对这一信号通路的完整理解将使选择治疗靶点的方法更加合理，包括PDE抑制剂，其减缓肌肉变性的进展并改善营养不良表型。 公共卫生相关性：这里提出的研究将提供有关导致肌营养不良症肌肉变性机制的新信息。对高尔基体（一种调节蛋白质修饰和转运到表面膜的细胞器）的新型信号传导机制的关注，可能会揭示减缓肌肉变性进展的新治疗靶点。总的来说，这个项目产生的知识将与许多类型的肌肉疾病有关。

DESCRIPTION (provided by applicant): The mechanism by which the loss of dystrophin leads to Duchenne Muscular Dystrophy is generally attributed to membrane fragility due to disruption of the linkage between the extracellular matrix and the cortical actin cytoskeleton. This structural role for dystrophin and its associated proteins is supported by substantial evidence. In addition, the dystrophin complex serves as a sarcolemmal scaffold for signaling proteins, including neuronal nitric oxide synthase (nNOS5, the muscle-specific isoform), several kinases, water and ion channels and other proteins. The demonstration that transgenic expression of nNOS in mdx mouse muscle ameliorates the dystrophic phenotype has focused attention on this signaling protein. Recently, we have found that a second isoform of this enzyme, nNOS2, is localized on the Golgi, and that its expression at this site is reduced in mdx muscle, even before the first phenotypic signs of dystrophy are evident. Furthermore, we find that soluble guanylate cyclase (sGC), which is activated by NO, and cGMP-activated protein kinase G (PKG) are colocalized with nNOS2 on the Golgi. Mice lacking both nNOS5 and nNOS2 (KN2) have numerous deficiencies, including a severe myopathic defect. Based on these preliminary results, we will characterize muscle from KN2 mice at the structural, biochemical and functional levels. In addition, we will test the hypothesis that transgenic expression of nNOS2 in skeletal muscle will rescue/ameliorate the KN2 and mdx phenotypes. The role of nNOS2 in blunting 1-adrenergic vasoconstriction in contracting muscle will provide information on the importance of this enzyme in alleviating functional ischemia, a component of muscular dystrophy. Finally, we will determine the Golgi substrates of the NO/cGMP/PKG pathway and of direct protein nitrosylation. Finally, we will identify the phosphodiesterase(s) (PDEs) that regulate(s) the NO/cGMP/PKG pathway. A complete understanding of this signaling pathway will enable a more rationale approach to selecting therapeutic targets, including PDE inhibitors, which slow the progression of muscle degeneration and ameliorate the dystrophic phenotype. PUBLIC HEALTH RELEVANCE: The research proposed here will provide new information about the mechanisms that lead to muscle degeneration in muscular dystrophies. The focus on a novel signaling mechanism at the Golgi, a cellular organelle that regulates protein modification and trafficking to the surface membrane, may reveal new therapeutic targets for slowing the progress of muscle degeneration. In general, the knowledge generated by this project will be relevant to many types of muscle diseases.