Song Y, Zweier J L, Xia Y
Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
Am J Physiol Cell Physiol. 2001 Dec;281(6):C1819-24. doi: 10.1152/ajpcell.2001.281.6.C1819.
Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased Ca(2+)/calmodulin (Ca(2+)/CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fluorescence-quenching measurements revealed that HSP90 markedly reduced the K(d) of CaM to nNOS (0.5 +/- 0.1 nM vs. 9.4 +/- 1.8 nM in the presence and absence of HSP90, P < 0.01). Ca(2+) ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS.
近期研究表明，热休克蛋白90（HSP90）可增强内皮型一氧化氮合酶和神经元型一氧化氮合酶（分别为eNOS和nNOS）生成一氧化氮（NO）的能力。然而，这些发现是基于间接的NO测量。此外，尽管我们之前的研究表明HSP90的作用涉及增加Ca(2+)/钙调蛋白（Ca(2+)/CaM）的结合，但一直缺乏对HSP90对CaM与nNOS结合影响的定量测量。通过电子顺磁共振光谱，我们直接测量了纯化的nNOS产生的NO信号。HSP90以剂量依赖的方式增加nNOS生成NO。色氨酸荧光猝灭测量显示，HSP90显著降低了CaM与nNOS的解离常数（存在和不存在HSP90时分别为0.5±0.1 nM和9.4±1.8 nM，P<0.01）。Ca(2+)离子载体引发了nNOS转染细胞强烈的NO生成，而HSP90抑制剂格尔德霉素可显著降低这种生成。因此，这些研究提供了直接证据，证明HSP90在体外和完整细胞中增强了nNOS的催化功能。HSP90的作用是通过增强CaM与nNOS的结合来介导的。
