Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides
Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides
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DOI：
10.1073/pnas.0407224102
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发表时间：
2005-01-18
影响因子：
11.1
通讯作者：
Sessa, WC
中科院分区：
文献类型：
--
作者：
Bernatchez, PN;Bauer, PM;Sessa, WC
在内皮细胞中，NO是由内皮NO合成酶（eNOS）合成的，而内皮NO合成酶受微囊主要外壳蛋白Cav-1 （Cav-1）的负调控。我们发现，将Cav-1氨基酸82-101 （Cav）与来自Antennapedia （AP）的内化序列融合后，可在体外阻断NO释放，并在体内阻断炎症和肿瘤血管生成。为了表征AP-Cav肽和Cav-1介导eNOS抑制的分子机制，我们将AP-Cav的Cav部分细分为三个结构域（Cav- a、-B和-C），合成了5个重叠肽（AP-Cav- a、-AB、-B、-BC和-C），并测试了它们对eNOS依赖性活性的影响。含有Cav-B结构域的肽（氨基酸89-95）诱导培养内皮细胞中enos依赖性NO释放、NO依赖性耳内炎症和离体小静脉的水力传导的时间和剂量依赖性抑制。AP-Cav-B的丙氨酸扫描显示，Thr-90和-91 （T90,91）以及ph -92 （F92）对AP-Cav-B和ap - cav介导的eNOS抑制至关重要。与野生型Cav-1相比，Cav-1 cDNA中F92突变为A92导致eNOS抑制活性丧失。这些数据强调了氨基酸89-95，特别是F92在介导AP-Cav和Cav-1对eNOS的抑制中的重要性。
In endothelia, NO is synthesized by endothelial NO synthase (eNOS), which is negatively regulated by caveolin-1 (Cav-1), the primary coat protein of caveolae. We show that delivery of Cav-1 amino acids 82-101 (Cav)fused to an internalization sequence from Antennapedia (AP) blocks NO release in vitro and inflammation and tumor angiogenesis in vivo. To characterize the molecular mechanism by which the AP-Cav peptide and Cav-1 mediate eNOS inhibition, we subdivided the Cav portion of AP-Cav into three domains (Cav-A, -B, and -C), synthesized five overlapping peptides (AP-Cav-A, -AB, -B, -BC, and -C), and tested their effects on eNOS-dependent activities. Peptides containing the Cav-B domain (amino acids 89-95) induced time- and dose-dependent inhibition of eNOS-dependent NO release in cultured endothelial cells, NO-dependent inflammation in the ear, and hydraulic conductivity in isolated venules. Alanine scanning of AP-Cav-B revealed that Thr-90 and -91 (T90,91) and Phe-92 (F92) are crucial for AP-Cav-B and AP-Cav-mediated inhibition of eNOS. Mutation of F92 to A92 in the Cav-1 cDNA caused the loss of eNOS inhibitory activity compared with wild-type Cav-1. These data highlight the importance of amino acids 89-95 and particularly F92 in mediating eNOS inhibition by AP-Cav and Cav-1.