Leclerc Patrick, Biarc Jordane, St-Onge Mireille, Gilbert Caroline, Dussault Andrée-Anne, Laflamme Cynthia, Pouliot Marc
Centre de Recherche en Rhumatologie et Immunologie and Department of Anatomy-Physiology, Faculty of Medicine, Laval University, Quebec City, Quebec, Canada.
PLoS One. 2008 May 21;3(5):e2229. doi: 10.1371/journal.pone.0002229.
The inducible cyclooxygenase isoform (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca(2+)-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE(2) biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE(2) biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2). Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2) biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.
诱导型环氧化酶同工型（COX-2）与炎症、肿瘤发生以及生理事件相关。尽管人们为了解这种多面性酶的生物学特性付出了努力，但仍有许多有待了解之处。核结合蛋白（Nuc）是一种普遍存在的钙结合蛋白，具有一个假定的COX结合结构域。在本研究中，我们调查了其在人中性粒细胞中的表达和亚细胞定位、其对COX-2的亲和力以及其对前列腺素E2（PGE2）生物合成的可能影响。包括氮空化结合Percoll分级分离、免疫荧光、共聚焦和电子显微镜在内的互补亚细胞定位方法共同表明，Nuc、COX-2以及所有参与类前列腺素合成的主要酶均存在于人中性粒细胞的高尔基体和内质网中。免疫沉淀实验表明Nuc与COX-2之间具有高亲和力。向纯化的人重组（hr）COX-2中添加hrNuc会导致对花生四烯酸的PGE2生物合成呈剂量依赖性增加。Nuc与表达COX-2的中性粒细胞裂解物共同孵育也增加了它们产生PGE2的能力。此外，用hrNuc对中性粒细胞进行转染可特异性增强PGE2生物合成。总之，这些结果鉴定出一种可能对类前列腺素生物合成有影响的COX-2相关蛋白。
