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

在神经元一氧化氮合酶（nNOS）中，钙调素（CaM）结合被认为触发电子从还原酶结构域转移到血红素结构域，这是O2激活和NO形成所必需的。另一方面，已知小窝蛋白下调神经元（nNOS）和内皮一氧化氮合成酶（eNOS）。在本研究中，重组小窝蛋白-1与nNOS的加氧酶和还原酶结构域的直接相互作用通过体外结合实验得到证实。为了阐明小洞蛋白调控nNOS的机制，我们检测了小洞蛋白-1脚手架结构域肽（82-101:CaV1p1）对野生型和突变型nNOS催化活性的影响。CaV1p1抑制野生型nNOS的NO形成活性和NADPH氧化呈剂量依赖性，其1C50值为1.8 []M。在加氧酶结构域的小窝蛋白结合一致基序中，F584和W587的突变不会导致CaV1p1抑制的丧失，这表明nNOS的另一个区域介导了小窝蛋白的抑制作用。添加CaV1p1也抑制了90%以上的细胞色素c还原酶活性，无论是否含有钙调素（CaM）结合位点，而CaV1p1仅抑制了50%的铁氰化物还原酶活性。这些结果表明，与先前报道的eNOS相比，小窝蛋白对nNOS的抑制机制存在显著差异。通过使用几个还原酶结构域缺失突变体对相互作用的进一步分析表明，FMN结构域对于小窝蛋白-1和nNOS还原酶之间的成功相互作用至关重要。有趣的是，CaV1p1抑制了CaM依赖性而非CaM非依赖性的自抑制结构域缺失突变体（delta40）和c端截断突变体（deltaC33）的NO形成活性，这表明CaV1p1抑制了由CaM诱导的从还原酶结构域到加氧酶结构域的结构域间电子转移。

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.