S-nitrosylation of endothelial nitric oxide synthase is associated with monomerization and decreased enzyme activity
S-nitrosylation of endothelial nitric oxide synthase is associated with monomerization and decreased enzyme activity
复制标题
DOI：
10.1073/pnas.0300464101
复制
发表时间：
2004-02-24
影响因子：
11.1
通讯作者：
Black, SM
中科院分区：
文献类型：
--
作者：
Ravi, K;Brennan, LA;Black, SM
内皮一氧化氮合酶 (eNOS) 仅以同型二聚体形式具有活性。最近的数据表明，外源性 NO 在完整动物和血管内皮细胞中都可以作为 eNOS 活性的抑制剂。然而，NO发挥其抑制作用的确切机制尚不清楚。我们在牛主动脉内皮细胞中的初步实验表明，外源性 NO 会降低 NOS 活性，并导致 eNOS 晚餐水平降低。然后我们进行了一系列研究来调查二聚体破坏的机制。将纯化的人 eNOS 蛋白暴露于 NO 供体或钙介导的酶激活导致 eNOS 从主要是二聚体酶转变为主要是单体酶。进一步的研究表明，内源性NOS活性或NO暴露引起eNOS的S-亚硝基化，并且硫氧还蛋白和硫氧还蛋白还原酶系统的存在可以显着保护eNOS二聚体水平并防止由此产生的单体化和活性丧失。此外，外源NO处理导致二聚体界面处的四硫醇锌簇破坏。为了进一步确定该区域内的 S-亚硝基化是否可以解释 NO 对 eNOS 的影响，我们纯化了缺乏四硫醇簇的 C99A eNOS 突变酶并分析了其寡聚状态。这种酶主要是单体的，表明四硫醇簇在晚餐维持和稳定性中发挥作用。因此，本研究通过半胱氨酸残基的 S-亚硝基化，将 NO 的抑制作用与二聚体界面四硫醇锌簇的破坏联系起来。
Endothelial nitric oxide synthase (eNOS) is active only as a homodimer. Recent data has demonstrated that exogenous NO can act as an inhibitor of eNOS activity both in intact animals and vascular endothelial cells. However, the exact mechanism by which NO exerts its inhibitory action is unclear. Our initial experiments in bovine aortic endothelial cells indicated that exogenous NO decreased NOS activity with an associated decrease in eNOS dinner levels. We then undertook a series of studies to investigate the mechanism of dimer disruption. Exposure of purified human eNOS protein to NO donors or calcium-mediated activation of the enzyme resulted in a shift in eNOS from a predominantly dimeric to a predominantly monomeric enzyme. Further studies indicated that endogenous NOS activity or NO exposure caused S-nitrosylation of eNOS and that the presence of the thioredoxin and thioredoxin reductase system could significantly protect eNOS dimer levels and prevent the resultant monomerization and loss of activity. Further, exogenous NO treatment caused zinc tetrathiolate cluster destruction at the dimer interface. To further determine whether S-nitrosylation within this region could explain the effect of NO on eNOS, we purified a C99A eNOS mutant enzyme lacking the tetrathiolate cluster and analyzed its oligomeric state. This enzyme was predominantly monomeric, implicating a role for the tetrathiolate cluster in dinner maintenance and stability. Therefore, this study links the inhibitory action of NO with the destruction of zinc tetrathiolate cluster at the dimeric interface through S-nitrosylation of the cysteine residues.