Selective cysteines oxidation in soluble guanylyl cyclase catalytic domain is involved in NO activation.
Selective cysteines oxidation in soluble guanylyl cyclase catalytic domain is involved in NO activation.
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DOI：
10.1016/j.freeradbiomed.2020.11.001
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发表时间：
2021-01
影响因子：
7.4
通讯作者：
Beuve A
中科院分区：
文献类型：
--
作者：
Alapa M;Cui C;Shu P;Li H;Kholodovych V;Beuve A
一氧化氮(NO)与可溶性鸟苷酸环化酶(Gc1)结合，刺激其催化活性生成cGMP。尽管NO-cGMP信号在心血管生理学中起着关键作用，但Gc1的激活机制仍不明确。Gc1中保守的半胱氨酸(Cys)被认为是通过硫醇-氧化还原修饰来调节酶的活性。我们发现Gc1的活性是通过蛋白质二硫键异构酶和硫氧还蛋白1的混合二硫键来调节的。在这里，我们研究了NO刺激的Gc1活性是由硫醇/二硫键开关介导的这一新概念，目的是定位参与其中的特定半胱氨酸。首先，我们证明了二硫醇还原剂三(2-羧乙基)膦降低了Gc1对NO的反应，表明半胱氨酸氧化在NO激活中的意义。其次，使用二溴联氨，当两个邻近的半胱氨酸硫醇交联时，我们发现与非刺激条件相比，在NO刺激的Gc1中荧光减弱。这表明NO刺激的Gc1含有更多的结合半胱氨酸，潜在的二硫键。第三，为了用质谱法鉴定NO调节的半胱氨酸氧化，我们比较了在胰蛋白酶多肽中发现的所有半胱氨酸的氧化还原状态，其中10个半胱氨酸在NO刺激的GC1中被氧化，2个半胱氨酸被还原。第四，我们利用计算模型来缩小可能涉及二硫键的Cys候选范围，并确定了Cys489和Cys571。第五，我们的突变研究表明，Cys489和Cys571参与了Gc1对NO的反应，可能是一个硫醇/二硫键开关。这些发现表明，GC1Cys对氧化还原环境的敏感性在心血管生理学中对NO信号转导起关键作用。
Nitric oxide (NO) binds to soluble guanylyl cyclase (GC1) and stimulates its catalytic activity to produce cGMP. Despite the key role of the NO-cGMP signaling in cardiovascular physiology, the mechanisms of GC1 activation remain ill-defined. It is believed that conserved cysteines (Cys) in GC1 modulate the enzyme’s activity through thiol-redox modifications. We showed that GC1 activity is modulated via mixed-disulfide bond by protein disulfide isomerase and thioredoxin 1. Herein we investigated the novel concept that NO-stimulated GC1 activity is mediated by thiol/disulfide switches and aimed to map the specific Cys that are involved. First, we showed that the dithiol reducing agent Tris (2-carboxyethyl)-phosphine reduces GC1 response to NO, indicating the significance of Cys oxidation in NO activation. Second, using dibromobimane, which fluoresces when crosslinking two vicinal Cys thiols, we demonstrated decreased fluorescence in NO-stimulated GC1 compared to unstimulated conditions. This suggested that NO-stimulated GC1 contained more bound Cys, potentially disulfide bonds. Third, to identify NO-regulated Cys oxidation using mass spectrometry, we compared the redox status of all Cys identified in tryptic peptides, among which, ten were oxidized and two were reduced in NO-stimulated GC1. Fourth, we resorted to computational modeling to narrow down the Cys candidates potentially involved in disulfide bond and identified Cys489 and Cys571. Fifth, our mutational studies showed that Cys489 and Cys571 were involved in GC1’response to NO, potentially as a thiol/disulfide switch. These findings imply that specific GC1 Cys sensitivity to redox environment is critical for NO signaling in cardiovascular physiology.
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DOI：
10.1073/pnas.76.1.219
发表时间：
1979-01-01
影响因子：
11.1
作者：
BRAUGHLER, JM;MITTAL, CK;MURAD, F
通讯作者：
MURAD, F
影响因子：
2.9
作者：
Fritz BG;Roberts SA;Ahmed A;Breci L;Li W;Weichsel A;Brailey JL;Wysocki VH;Tama F;Montfort WR
通讯作者：
Montfort WR
影响因子：
4.8
作者：
Bhattacharjee, H;Rosen, BP
通讯作者：
Rosen, BP
DOI：
10.1016/0005-2744(78)90121-3
发表时间：
1978-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
CRAVEN, PA;DERUBERTIS, FR
通讯作者：
DERUBERTIS, FR
影响因子：
4.8
作者：
Chang, FJ;Lemme, S;Beuve, A
通讯作者：
Beuve, A