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

在神经元型一氧化氮合酶(NNOS)中，钙调素(CaM)的结合被认为触发了从还原酶域到血红素域的电子转移，这是O2激活和NO形成所必需的。另一方面，小窝蛋白可以下调神经元(NNOS)和内皮型一氧化氮合酶(ENOS)的表达。在本研究中，利用体外结合实验证明了重组小窝蛋白-1与nNOS的加氧酶和还原酶结构域直接相互作用。为了阐明小窝蛋白对nNOS的调节机制，我们研究了小窝蛋白-1支架结构域肽(82-101：CaV1p1)对野生型和突变型nNOS催化活性的影响。CaV1p1以剂量依赖的方式抑制野生型nNOS的NO生成活性和NADPH氧化，其1C50值为1.8[]M。F584和W587在加氧酶结构域的一个小凹蛋白结合的共同基序内的突变不会导致CaV1p1抑制作用的丧失，这表明nNOS的一个交替区域介导了小窝蛋白的抑制作用。CaV1p1对含或不含钙调素(CaM)结合部位的细胞色素c还原酶活性均有90%以上的抑制作用，而CaV1p1对铁氰化物还原酶的抑制作用仅为50%。这些结果表明，小窝蛋白抑制nNOS的机制与以前报道的eNOS有显著不同。通过使用几个还原酶域缺失突变体进一步分析相互作用，发现FMN结构域是小窝蛋白-1与nNOS还原酶成功相互作用的关键。有趣的是，CaV1p1抑制了自抑制结构域缺失突变体(Delta40)和C末端截断突变体(DeltaC33)依赖于CaM的NO生成活性，但不抑制CaM非依赖于CaM的NO生成活性，这表明CaV1p1抑制了CaM诱导的从还原酶域到加氧酶域的域间电子转移。

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.