Heat shock protein 90 regulates soluble guanylyl cyclase maturation by a dual mechanism
Heat shock protein 90 regulates soluble guanylyl cyclase maturation by a dual mechanism
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DOI：
10.1074/jbc.ra119.009016
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发表时间：
2019-08-30
影响因子：
4.8
通讯作者：
Stuehr, Dennis J.
中科院分区：
文献类型：
--
作者：
Dai, Yue;Schlanger, Simon;Stuehr, Dennis J.
可溶性鸟苷酸环化酶（sGC）是由α亚基和含血红素的β亚基组成的异二聚体。它通过响应一氧化氮（NO）产生cGMP参与信号传导。血红素插入sGC的β 1亚基（sGC β）对功能至关重要，热休克蛋白90（HSP 90）与不含血红素的sGC β（apo-sGC β）结合以驱动其血红素插入。在这里，我们通过构建预测与HSP 90相互作用受损的sGC β变体来测试模型化apo-sGC β-HSP 90复合物的准确性和相关性。利用定点突变、纯化的重组蛋白、哺乳动物细胞表达和荧光方法，我们发现（i）模型预测的apo-sGC β中的三个区域介导了体外和哺乳动物细胞中与HSP 90的直接复合物形成;（ii）这种HSP 90复合物的形成与血红素插入apo-sGC β的程度和环化酶活性直接相关;和（iii）具有HSP 90结合缺陷的apo-sGC β突变体反而与细胞中的sGC α结合并形成无活性、无血红素的sGC异二聚体。我们的研究结果揭示了细胞apo-sGC β-HSP 90复合物的分子特征，并揭示了其在sGC成熟过程中实现血红素插入同时防止非活性异二聚体形成的双重重要性。
The enzyme soluble guanylyl cyclase (sGC) is a heterodimer composed of an alpha subunit and a heme-containing beta subunit. It participates in signaling by generating cGMP in response to nitric oxide (NO). Heme insertion into the beta 1 subunit of sGC (sGC beta) is critical for function, and heat shock protein 90 (HSP90) associates with heme-free sGC beta (apo-sGC beta) to drive its heme insertion. Here, we tested the accuracy and relevance of a modeled apo-sGC beta-HSP90 complex by constructing sGC beta variants predicted to have an impaired interaction with HSP90. Using site-directed mutagenesis, purified recombinant proteins, mammalian cell expression, and fluorescence approaches, we found that (i) three regions in apo-sGC beta predicted by the model mediate direct complex formation with HSP90 both in vitro and in mammalian cells; (ii) such HSP90 complex formation directly correlates with the extent of heme insertion into apo-sGC beta and with cyclase activity; and (iii) apo-sGC beta mutants possessing an HSP90-binding defect instead bind to sGC alpha in cells and form inactive, heme-free sGC heterodimers. Our findings uncover the molecular features of the cellular apo-sGC beta-HSP90 complex and reveal its dual importance in enabling heme insertion while preventing inactive heterodimer formation during sGC maturation.