Dynamic cycling with a unique Hsp90/Hsp70-dependent chaperone machinery and GAPDH is needed for heme insertion and activation of neuronal NO synthase.
Dynamic cycling with a unique Hsp90/Hsp70-dependent chaperone machinery and GAPDH is needed for heme insertion and activation of neuronal NO synthase.
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具有独特的HSP90/HSP70依赖性伴侣机械和GAPDH的动态循环是血红素插入和激活神经元NO合酶的需要的动态循环。
DOI：
10.1016/j.jbc.2022.102856
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发表时间：
2023-02
影响因子：
4.8
通讯作者：
Osawa, Yoichi
中科院分区：
文献类型：
--
作者：
Morishima, Yoshihiro;Lau, Miranda;Pratt, William B.;Osawa, Yoichi
已知热休克蛋白90（Hsp 90）通过高度动态的相互作用介导细胞中血红素插入和血红素缺乏的神经元型一氧化氮（NO）合酶（apo-nNOS）的激活，这已经非常难以使用亚细胞系统进行机理研究。由于许多关键血红素蛋白的血红素含量受Hsp 90和血红素伴侣GAPDH的调节，因此开发用于研究这种伴侣介导的血红素调节的体外系统将是非常有用的。在这里，我们表明，使用抗体固定的载脂蛋白-nNOS导致不仅成功组装的伴侣复合物，但能够显示出明确的依赖于热休克蛋白90和GAPDH血红素介导的活化载脂蛋白-nNOS。Hsp 70和Hsp 90的结合动力学，ATP和K+依赖性，以及Hsp 90·apo-nNOS杂合物组装中对Hsp 70的绝对需求都指向与公认的调节类固醇激素受体的典型机器相似的伴侣机制。然而，与类固醇受体不同，使用含有Hsp 90、Hsp 70、Hsp 40、Hop和p23的纯化蛋白系统不能激活apo-nNOS。因此，血红素插入需要一个独特的热休克蛋白90-伴侣复合物。有了这个新开发的体外系统，它重现了需要GAPDH以及Hsp 90的细胞过程，进一步的机制研究现在可以更好地了解基于Hsp 90的伴侣系统的组成部分，以及这种异源复合物如何与GAPDH一起调节nNOS和可能的其他血红素蛋白。
Heat shock protein 90 (Hsp90) is known to mediate heme insertion and activation of heme-deficient neuronal nitric oxide (NO) synthase (apo-nNOS) in cells by a highly dynamic interaction that has been extremely difficult to study mechanistically with the use of subcellular systems. In that the heme content of many critical hemeproteins is regulated by Hsp90 and the heme chaperone GAPDH, the development of an in vitro system for the study of this chaperone-mediated heme regulation would be extremely useful. Here, we show that use of an antibody-immobilized apo-nNOS led not only to successful assembly of chaperone complexes but the ability to show a clear dependence on Hsp90 and GAPDH for heme-mediated activation of apo-nNOS. The kinetics of binding for Hsp70 and Hsp90, the ATP and K+ dependence, and the absolute requirement for Hsp70 in assembly of Hsp90•apo-nNOS heterocomplexes all point to a similar chaperone machinery to the well-established canonical machine regulating steroid hormone receptors. However, unlike steroid receptors, the use of a purified protein system containing Hsp90, Hsp70, Hsp40, Hop, and p23 is unable to activate apo-nNOS. Thus, heme insertion requires a unique Hsp90–chaperone complex. With this newly developed in vitro system, which recapitulates the cellular process requiring GAPDH as well as Hsp90, further mechanistic studies are now possible to better understand the components of the Hsp90-based chaperone system as well as how this heterocomplex works with GAPDH to regulate nNOS and possibly other hemeproteins.
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影响因子：
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作者：
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DOI：
10.1073/pnas.1205854109
发表时间：
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影响因子：
11.1
作者：
Ghosh, Arnab;Stuehr, Dennis J.
通讯作者：
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