Chaperone-dependent regulation of endothelial nitric-oxide synthase intracellular trafficking by the co-chaperone/ubiquitin ligase CHIP
Chaperone-dependent regulation of endothelial nitric-oxide synthase intracellular trafficking by the co-chaperone/ubiquitin ligase CHIP
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DOI：
10.1074/jbc.m304738200
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发表时间：
2003-12-05
影响因子：
4.8
通讯作者：
Patterson, C
中科院分区：
文献类型：
--
作者：
Jiang, JH;Cyr, D;Patterson, C
内皮一氧化氮合酶（eNOS）是一种负责生成内皮一氧化氮的酶，受到严格而复杂的调控。适当的eNOS细胞定位对于细胞外刺激与NO产生的最佳耦合至关重要。此外，分子伴侣Hsp90与eNOS相互作用并积极调节eNOS活性。Hsp90是通过与其共同伴侣的物理相互作用来调节的。CHIP （hsp70相互作用蛋白的羧基末端）就是这样一种共伴侣，它重塑了Hsp90异复合体，并通过CHIP的泛素-蛋白异肽连接酶活性导致一些Hsp90底物的蛋白质降解。在这里，我们展示了集成到eNOS中的CHIP。在瞬时转染的COS细胞中，Hsp90复合物特异性降低可溶性eNOS水平。令人惊讶的是，与Hsp90抑制剂格尔达霉素（geldanamycin）诱导eNOS泛素化及其随后的蛋白质降解的作用相反，CHIP并没有靶向eNOS泛素化和蛋白酶体依赖性降解。相反，CHIP可能是通过其共伴侣活性将可溶性eNOS分割成不溶性和非活性的细胞区室。这种效应似乎是由于eNOS从高尔基体转移，否则eNOS运输到质膜和随后的激活是必需的。与过表达研究的观察结果一致，在缺乏CHIP的小鼠肺内皮细胞中，eNOS在膜上的定位和活性增加。综上所述，这些结果证明了一种新的协同伴侣依赖机制，通过这种机制调节eNOS的转运，并表明CHIP在通过高尔基腔的蛋白质转运中可能具有广泛的作用。
Endothelial nitric-oxide synthase (eNOS), the enzyme responsible for production of endothelial NO, is under tight and complex regulation. Proper cellular localization of eNOS is critical for optimal coupling of extracellular stimulation with NO production. In addition, the molecular chaperone Hsp90 interacts with eNOS and positively regulates eNOS activity. Hsp90 is modulated by physical interaction with its co-chaperones. CHIP ( carboxyl terminus of Hsp70-interacting protein) is such a co-chaperone that remodels the Hsp90 heterocomplex and causes protein degradation of some Hsp90 substrates through the ubiquitin-protein isopeptide ligase activity of CHIP. Here we show that CHIP incorporated into the eNOS . Hsp90 complex and specifically decreased soluble eNOS levels in transiently transfected COS cells. Surprisingly, in contrast to the effects of the Hsp90 inhibitor geldanamycin, which induces eNOS ubiquitylation and its subsequent protein degradation, CHIP did not target eNOS for ubiquitylation and proteasome-dependent degradation. Instead, CHIP partitioned soluble eNOS into an insoluble and inactive cellular compartment, presumably through its co-chaperone activity. This effect seems to be due to displacement of eNOS from the Golgi apparatus, which is otherwise required for trafficking of eNOS to the plasmalemma and subsequent activation. Consistent with observations from overexpression studies, eNOS localization to the membrane and activity were increased in mouse lung endothelial cells lacking CHIP. Taken together, these results demonstrate a novel co-chaperone-dependent mechanism through which eNOS trafficking is regulated and suggest a potentially generalized role for CHIP in protein trafficking through the Golgi compartment.