Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine, 473 West 12th Avenue, Columbus, OH 43210, USA.
Biochem Biophys Res Commun. 2012 Sep 28;426(3):386-9. doi: 10.1016/j.bbrc.2012.08.099. Epub 2012 Aug 30.
Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.
一氧化氮（NO）由诱导型一氧化氮合酶（iNOS）产生，对炎症损伤和宿主防御至关重要。虽然以前认为 iNOS 是一种可溶性蛋白，但最近有报道称它在细胞内形成聚集物。但是，导致 iNOS 聚集物形成的原因尚不清楚。在这里，我们提供了证据表明，iNOS 聚集物的形成是由其自身产物 NO 介导的。暴露于炎症刺激物（脂多糖和干扰素-γ）会在小鼠巨噬细胞中诱导强烈的 iNOS 表达。虽然最初作为一种可溶性蛋白存在，但 iNOS 会随着时间的推移逐渐形成蛋白聚集体。聚集的 iNOS 没有活性。用 NOS 抑制剂 N-硝基-L-精氨酸甲酯（L-NAME）处理细胞会阻断 iNOS 产生 NO，而不影响 iNOS 表达。然而，L-NAME 可以阻止细胞中 iNOS 的聚集。通过荧光成像直接观察 GFP-iNOS 转染细胞，发现 NO 阻断对 iNOS 聚集物形成的抑制作用。此外，向 L-NAME 处理的细胞中添加外源性 NO 可以重新捕获 iNOS 聚集物形成。这些研究表明，iNOS 聚集物的形成是由 NO 引起的。NO 诱导 iNOS 聚集和失活的发现表明聚集物的形成是 iNOS 调节中的一种反馈抑制机制。
