Heat shock protein 90α increases superoxide generation from neuronal nitric oxide synthases.
Heat shock protein 90α increases superoxide generation from neuronal nitric oxide synthases.
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DOI：
10.1016/j.jinorgbio.2020.111298
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发表时间：
2021-01
影响因子：
3.9
通讯作者：
Feng C
中科院分区：
文献类型：
--
作者：
Zheng H;Weaver JM;Feng C
神经元型一氧化氮合酶（nNOS）会产生超氧化物，尤其是在左旋精氨酸（L - Arg）底物浓度不理想的情况下。据报道，热休克蛋白90（Hsp90）可抑制nNOS蛋白产生超氧化物。然而，所使用的来自牛脑组织的市售Hsp90产品，其Hsp90α和Hsp90β含量不明确，且Hsp90蛋白的寡聚状态也未确定。这两种Hsp90对NOS产生超氧化物可能具有相反的作用。重要的是，新出现的证据表明，nNOS剪接变体通过在氧化还原信号传导中发挥不同的作用而参与不同的生物学功能。在本研究中，采用纯化的天然二聚体状态的重组人Hsp90α进行电子顺磁共振（EPR）自旋捕集实验，以研究Hsp90α对nNOS剪接变体nNOSµ和nNOSα产生超氧化物的影响。研究发现，在L - Arg缺乏的条件下，人Hsp90α可显著增加nNOSµ和nNOSα蛋白产生超氧化物，并且Hsp90α对nNOSµ和nNOSα产生超氧化物的影响程度不同。咪唑抑制了自旋加合物信号，这表明在Hsp90α存在的情况下，超氧化物是在nNOS的血红素位点产生的，而补充L - Arg则会减少nNOS - Hsp90α产生的超氧化物。此外，NADPH消耗速率值呈现出与Hsp90α和L - Arg相关的类似趋势/差异。总之，这些EPR自旋捕集和NADPH氧化动力学结果表明，Hsp90α可显著增加nNOS产生超氧化物，并且Hsp90α对nNOSµ和nNOSα蛋白具有明显不同的作用。
先前的研究表明，热休克蛋白（Hsp）90与一氧化氮合酶（NOS）的血红素结构域结合。本文的电子顺磁共振（EPR）自旋捕集结果表明，二聚体人Hsp90α会增加神经元型NOS（nNOS）血红素产生超氧化物，并且这种增强作用在nNOSµ和nNOSα异构体之间存在差异。
Neuronal nitric oxide synthase (nNOS) generates superoxide, particularly at sub-optimal l-arginine (L-Arg) substrate concentrations. Heat shock protein 90 (Hsp90) was reported to inhibit superoxide generation from nNOS protein. However, commercially available Hsp90 product from bovine brain tissues with unspecified Hsp90α and Hsp90β contents and an undefined Hsp90 protein oligomeric state was utilized. These two Hsp90s can have opposite effect on superoxide production by NOS. Importantly, emerging evidence indicates that nNOS splice variants are involved in different biological functions by functioning distinctly in redox signaling. In the present work, purified recombinant human Hsp90α in its native dimeric state was used in electron paramagnetic resonance (EPR) spin trapping experiments to study the effects of Hsp90α on superoxide generation from nNOS splice variants nNOSµ and nNOSα. Human Hsp90α was found to significantly increase superoxide generation from nNOSµ and nNOSα proteins under l-Arg-depleted conditions and Hsp90α influenced superoxide production by nNOSµ and nNOSα at varying degrees. Imidazole suppressed the spin adduct signal, indicating that superoxide was produced at the heme site of nNOS in the presence of Hsp90α, whereas l-Arg repletion diminished superoxide production by the nNOS-Hsp90α. Moreover, NADPH consumption rate values exhibited a similar trend/difference as a function of Hsp90α and l-Arg. Together, these EPR spin trapping and NADPH oxidation kinetics results demonstrated noticeable Hsp90α-induced increases in superoxide production by nNOS and a distinguishable effect of Hsp90α on nNOSµ and nNOSα proteins. Previous studies showed that heat shock protein (Hsp) 90 binds to heme domain of nitric oxide synthase (NOS). Electron paramagnetic resonance (EPR) spin trapping results herein indicated that dimeric human Hsp90α increases superoxide generation from neuronal NOS (nNOS) heme, and that the enhancement effect differs between nNOSµ and nNOSα isoforms.
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