hsp90 is required for heme binding and activation of apo-neuronal nitric-oxide synthase - Geldanamycin-mediated oxidant generation is unrelated to any action of hsp90
hsp90 is required for heme binding and activation of apo-neuronal nitric-oxide synthase - Geldanamycin-mediated oxidant generation is unrelated to any action of hsp90
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DOI：
10.1074/jbc.m201940200
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发表时间：
2002-06-07
影响因子：
4.8
通讯作者：
Osawa, Y
中科院分区：
文献类型：
--
作者：
Billecke, SS;Bender, AT;Osawa, Y
已经确定神经元NO合成酶（nNOS）与伴侣蛋白hsp90相关，尽管这种相互作用的功能作用尚未明确。我们已经发现，用根腐素或格尔达霉素抑制hsp90几乎可以阻止血红素介导的血红素缺陷apo-nNOS在昆虫细胞中的激活和组装。这种效果是浓度依赖性的，在20 pm时达到75%以上的抑制作用。当hsp90被抑制时，nNOS的羰基亚铁复合物不能形成，这表明功能性血红素插入被阻止。我们提出，基于hsp90的伴侣机制通过打开蛋白中的疏水血红素结合间隙，促进血红素进入载脂蛋白nnos。此前有报道称，hsp90抑制剂格尔达霉素可解除内皮NOS活性偶联，增加内皮NOS依赖性O-2（）。-)生产。格尔达霉素是一种安霉素苯醌，我们在这里表明，它可以使昆虫细胞中的nNOS以及纯化的蛋白质产生氧化剂。在20 μ m的浓度下，格尔达霉素使NADPH氧化和纯化的nNOS形成过氧化氢的能力增加3倍，而非醌类hsp90抑制剂radicicol则没有影响。因此，与已知的其他醌的倾向一致，格尔达霉素通过一个独立于hsp90的过程直接与nNOS氧化还原循环，警告不要在氧化还原活性系统中使用格尔达霉素作为hsp90的特异性抑制剂。
It is established that neuronal NO synthase (nNOS) is associated with the chaperone hsp90, although the functional role for this interaction has not been defined. We have discovered that inhibition of hsp90 by radicicol or geldanamycin nearly prevents the heme-mediated activation and assembly of heme-deficient apo-nNOS in insect cells. This effect is concentration-dependent with over 75% inhibition achieved at 20 pm radicicol. The ferrous carbonyl complex of nNOS is not formed when hsp90 is inhibited, indicating that functional heme insertion is prevented. We propose that the hsp90-based chaperone machinery facilitates functional heme entry into apo-nNOS by the opening of the hydrophobic heme-binding cleft in the protein. Previously, it has been reported that the hsp90 inhibitor geldanamycin uncouples endothelial NOS activity and increases endothelial NOS-dependent O-2(.-) production. Geldanamycin is an ansamycin benzoquinone, and we show here that it causes oxidant production from nNOS in insect cells as well as with the purified protein. At a concentration of 20 mum, geldanamycin causes a 3-fold increase in NADPH oxidation and hydrogen peroxide formation from purified nNOS, whereas the non-quinone hsp90 inhibitor radicicol had no effect. Thus, consistent with the known propensity of other quinones, geldanamycin directly redox cycles with nNOS by a process independent of any action on hsp90, cautioning against the use of geldanamycin as a specific inhibitor of hsp90 in redox-active systems.