Functional Role of HSP90 Complexes with Endothelial Nitric-oxide Synthase (eNOS) and Calpain on Nitric Oxide Generation in Endothelial Cells
Functional Role of HSP90 Complexes with Endothelial Nitric-oxide Synthase (eNOS) and Calpain on Nitric Oxide Generation in Endothelial Cells
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DOI：
10.1074/jbc.m803638200
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发表时间：
2008-10-24
影响因子：
4.8
通讯作者：
Melloni, Edon
中科院分区：
文献类型：
--
作者：
Averna, Monica;Stifanese, Roberto;Melloni, Edon
虽然一些报道表明eNOS是一种高度敏感的钙蛋白酶底物，但在特定的直接实验中从未分析过伴随的合酶和蛋白酶的Ca 2（+）依赖性活化的发生。在这项研究中，我们已经探讨了在体内eNOS如何可以进行Ca 2（+）依赖性的移位和激活，防止被激活的钙蛋白酶降解。在这里，我们证明了短暂暴露于Ca 2（+）负载后，胞质eNOS-HSP 90复合物以一种形式募集钙蛋白酶，其中伴侣蛋白和合酶几乎完全抵抗蛋白酶的消化。此外，在HSP 90抑制剂格尔德霉素的存在下，NO产生显着减少，eNOS蛋白发生广泛降解，这表明从膜上解离并与伴侣蛋白结合与合酶的保护相关。使用分离的膜制备物的实验证实了HSP 90在eNOS从小窝解离中的主要作用。细胞长期暴露于Ca 2+负荷导致eNOS和HSP 90的广泛降解，伴随着NO产生的大抑制。我们建议，由热休克蛋白90对钙蛋白酶介导的eNOS降解的保护作用，代表了一种新的和关键的机制，确保了可逆的细胞内运输和激活的合成酶。
Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca2(+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca2(+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca2(+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca2+-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.