Garbán Hermes J, Márquez-Garbán Diana C, Pietras Richard J, Ignarro Louis J
Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2632-6. doi: 10.1073/pnas.0409854102. Epub 2005 Feb 7.
Nitric oxide (NO) and estrogen receptor (ER) are both important mediators of signal transduction in cardiovascular and reproductive tissues. In this study, we evaluated NO-mediated S-nitrosylation of ER and assessed the effect of this structural modification on transcription-related functions of ER. We have found selective inhibitory effects of NO on specific binding of ER to specific estrogen-responsive elements (ERE) that can be reversed in the presence of the reducing agent, DTT, thus suggesting that S-nitrosylation of thiolate-zinc centers may occur within the ER molecule. Furthermore, we examined inhibitory effects of NO on ER-dependent transcriptional activity by using an ERE-driven reporter gene system. By monitoring biophysical changes in the structure of NO-treated or untreated human recombinant ERalpha,we obtained evidence for the formation of S-nitrosothiols in the ER molecule. In addition, we have detected specific S-nitrosylation of cysteine residues within the ER molecule by immunodetection of S-nitrosocysteine moieties in ER. Collectively, these findings suggest an important physiological role for NO in modification of human ER structure by S-nitrosylation, an effect that leads, in turn, to impaired DNA-binding activity of ER and subsequent blockade of estrogen-dependent gene transcription. Thus, NO-induced S-nitrosylation of ER can occur at cysteine residues that coordinate Zn2+ within the two major DNA-binding Zn-finger domains of ER, resulting in selective inhibition of DNA-binding at specific ERE. This cross-communication between NO and ER may favor activation of rapid (nongenomic) signaling pathways and subsequent modulation of downstream genomic activity.
一氧化氮（NO）和雌激素受体（ER）都是心血管和生殖组织中信号转导的重要介质。在本研究中，我们评估了NO介导的ER的S-亚硝基化，并评估了这种结构修饰对ER转录相关功能的影响。我们发现NO对ER与特定雌激素反应元件（ERE）的特异性结合具有选择性抑制作用，在还原剂二硫苏糖醇（DTT）存在下这种抑制作用可被逆转，这表明硫醇锌中心的S-亚硝基化可能发生在ER分子内。此外，我们通过使用ERE驱动的报告基因系统研究了NO对ER依赖性转录活性的抑制作用。通过监测经NO处理或未处理的人重组ERα结构的生物物理变化，我们获得了ER分子中形成S-亚硝基硫醇的证据。此外，我们通过免疫检测ER中的S-亚硝基半胱氨酸部分，检测到了ER分子内半胱氨酸残基的特异性S-亚硝基化。总体而言，这些发现表明NO在通过S-亚硝基化修饰人ER结构中具有重要的生理作用，这种作用反过来会导致ER的DNA结合活性受损以及随后雌激素依赖性基因转录的阻断。因此，NO诱导的ER的S-亚硝基化可发生在ER两个主要DNA结合锌指结构域内与Zn2+配位的半胱氨酸残基上，导致对特定ERE处DNA结合的选择性抑制。NO与ER之间的这种相互作用可能有利于快速（非基因组）信号通路的激活以及随后对下游基因组活性的调节。
