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Study for Ineractions between functional domains of neuronal nitric oxide synthase and cellular proteins

神经元一氧化氮合酶功能域与细胞蛋白相互作用的研究

基本信息

批准号：
14580640
负责人：
SAGAMI Ikuko
金额：
$ 2.3万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

In neuronal nitric-oxide synthase (nNOS), calmodulin (CaM) binding is thought to trigger electron transfer from the reductase domain to the heme domain, which is essential for O2 activation and NO formation. On the other hands, caveolin is known to down-regulate both neuronal (nNOS) and endothelial nitric-oxide synthase (eNOS). In the present study, direct interactions of recombinant caveolin-1 with both the oxygenase and reductase domains of nNOS were demonstrated by using in vitro binding assays. To elucidate the mechanism of nNOS regulation by caveolin, we examined the effects of a caveolin-1 scaffolding domain peptide (82-101:CaV1p1) on the catalytic activities of wild-type and mutant nNOSs. CaV1p1 inhibited NO formation activity and NADPH oxidation of wild-type nNOS in a dose-dependent manner with an 1C50 value of 1.8 []M. Mutations of F584 and W587 within a caveolin-binding consensus motif of the oxygenase domain did not result in the loss of CaV1p1 inhibition, indicating that an alternate region of nNOS mediates inhibition by caveolin. The addition of CaV1p1 also inhibited more than 90% of the cytochrome c reductase activity in the isolated reductase domain with or without the calmodulin (CaM) binding site, whereas CaV1p1 inhibited ferricyanide reductase activity by only 50%. These results suggest that there are significant differences m the mechanism of inhibition by caveolin for nNOS as compared to those previously reported for eNOS. Further analysis of the interaction through the use of several reductase domain deletion mutants revealed that the FMN domain was essential for successful interaction between caveolin-1 and nNOS reductase. Interestingly, CaV1p1 inhibited CaM-dependent, but not CaM-independent, NO formation activities of an autoinhibitory domain deletion mutant (delta40), and a C-terminal truncation mutant (deltaC33), suggesting that CaV1p1 inhibits interdomain electron-transfer induced by CaM from the reductase domain to the oxygenase domain.

在神经元型一氧化氮合酶（nNOS）中，钙调素（CaM）结合被认为触发从还原酶结构域到血红素结构域的电子转移，这对于O2活化和NO形成是必需的。另一方面，已知小窝蛋白下调神经元型（nNOS）和内皮型一氧化氮合酶（eNOS）。在本研究中，直接相互作用的重组小窝蛋白-1与nNOS的加氧酶和还原酶结构域证明通过使用体外结合试验。为了阐明小窝蛋白调节nNOS的机制，我们研究了小窝蛋白-1支架结构域肽（82-101：CaV 1 p1）对野生型和突变型nNOS催化活性的影响。CaV 1 p1以剂量依赖性方式抑制野生型nNOS的NO形成活性和NADPH氧化，1C 50值为1.8 []M。加氧酶结构域的小窝蛋白结合共有基序内的F584和W587突变并没有导致CaV 1 p1抑制的丧失，表明nNOS的替代区域介导小窝蛋白的抑制。CaV 1 p1的添加还抑制了具有或不具有钙调蛋白（CaM）结合位点的分离的还原酶结构域中超过90%的细胞色素c还原酶活性，而CaV 1 p1仅抑制了50%的铁氰化物还原酶活性。这些结果表明，有显着差异的抑制机制小窝蛋白的nNOS相比，以前报道的eNOS。通过使用几个还原酶结构域缺失突变体的相互作用的进一步分析表明，FMN结构域是至关重要的小窝蛋白-1和nNOS还原酶之间的成功相互作用。有趣的是，CaV 1 p1抑制依赖钙调素，但不是钙调素独立的，NO形成活动的自抑制结构域缺失突变体（delta 40），和C-末端截短突变体（deltaC 33），表明CaV 1 p1抑制域间电子转移诱导钙调素从还原酶域的加氧酶域。