Dynamic cycling with Hsp90 stabilizes neuronal nitric oxide synthase through calmodulin-dependent inhibition of ubiquitination.
Dynamic cycling with Hsp90 stabilizes neuronal nitric oxide synthase through calmodulin-dependent inhibition of ubiquitination.
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DOI：
10.1021/bi901058g
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发表时间：
2009-09-08
期刊：
影响因子：
2.9
通讯作者：
Osawa Y
中科院分区：
文献类型：
--
作者：
Peng HM;Morishima Y;Clapp KM;Lau M;Pratt WB;Osawa Y
由神经元型一氧化氮合酶（nNOS）产生的NO需要钙调蛋白，并且通过分子伴侣Hsp 90来增强，所述分子伴侣Hsp 90与酶动态循环。nNOS的蛋白酶体降解通过自杀失活和通过用Hsp 90抑制剂处理而增强，后者表明与Hsp 90的动态循环稳定nNOS。在这里，我们使用一个纯化的泛素化系统含有CHIP（热休克蛋白70相互作用蛋白的羧基末端）作为E3连接酶，以显示热休克蛋白90抑制CHIP依赖的nNOS泛素化。像建立的热休克蛋白90增强NO的合成，热休克蛋白90抑制nNOS泛素化是Ca 2 +/钙调素依赖性的，这表明热休克蛋白90与酶的相同的相互作用是负责增强nNOS活性和抑制泛素化。已经确定CHIP与Hsp 90和Hsp 70结合，但我们在这里显示这两种分子伴侣对nNOS泛素化具有相反的作用，Hsp 70刺激和Hsp 90抑制。我们已经使用了两种机制为基础的灭活剂，胍那苄和NG-氨基-L-精氨酸，改变血红素/底物结合裂缝，促进nNOS泛素化，可以抑制热休克蛋白90。我们设想，作为nNOS经历毒性损伤，血红素/底物结合裂缝打开暴露疏水残基作为展开的初始步骤。只要热休克蛋白90可以形成甚至短暂的复合物与开放的裂缝，泛素化的热休克蛋白70依赖性泛素E3连接酶，如CHIP，被抑制。当裂缝的解折叠进展到不能与Hsp 90循环的状态时，Hsp 70依赖的泛素化不受阻碍。以这种方式，Hsp 70/Hsp 90机制做出nNOS稳定与降解的质量控制决定。
NO production by neuronal nitric oxide synthase (nNOS) requires calmodulin and is enhanced by the chaperone Hsp90, which cycles dynamically with the enzyme. The proteasomal degradation of nNOS is enhanced by suicide inactivation and by treatment with Hsp90 inhibitors, the latter suggesting that dynamic cycling with Hsp90 stabilizes nNOS. Here, we use a purified ubiquitinating system containing CHIP (carboxyl terminus of Hsp70-interacting protein) as the E3 ligase to show that Hsp90 inhibits CHIP-dependent nNOS ubiquitination. Like the established Hsp90 enhancement of NO synthesis, Hsp90 inhibition of nNOS ubiquitination is Ca2+/calmodulin-dependent, suggesting that the same interaction of Hsp90 with the enzyme is responsible for both enhancement of nNOS activity and inhibition of ubiquitination. It is established that CHIP binds to Hsp90 as well as to Hsp70, but we show here the two chaperones have opposing actions on nNOS ubiquitination, with Hsp70 stimulating and Hsp90 inhibiting. We have used two mechanism-based inactivators, guanabenz and NG-amino-L-arginine, to alter the heme/substrate binding cleft and promote nNOS ubiquitination that can be inhibited by Hsp90. We envision that as nNOS undergoes toxic damage, the heme/substrate binding cleft opens exposing hydrophobic residues as the initial step in unfolding. As long as Hsp90 can form even transient complexes with the opening cleft, ubiquitination by Hsp70-dependent ubiquitin E3 ligases, like CHIP, is inhibited. When unfolding of the cleft progresses to a state that cannot cycle with Hsp90, Hsp70-dependent ubiquitination is unopposed. In this way, the Hsp70/Hsp90 machinery makes the quality control decision for stabilization versus degradation of nNOS.
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