Department of Obstetrics and Gynecology, University of California-Irvine, Irvine, California 92697, USA.
J Cell Physiol. 2012 Jan;227(1):146-59. doi: 10.1002/jcp.22712.
Covalent adduction of a NO moiety to cysteines (S-nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol-17β (E2) rapidly stimulated protein SNO that maximized within 10-30 min post-E2 exposure. E2-bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2-induced protein SNO was attenuated by selective ERβ, but not ERα, antagonists. A specific ERβ but not ERα agonist was able to induce protein SNO. Overexpression of ERβ, but not ERα, significantly enhanced E2-induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2-stimulated SNO. E2-induced SNO was inhibited by N-nitro-L-arginine-methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen-induced SNO is mediated by endogenous NO via eNOS and mainly ERβ in UAEC. We further analyzed the nitroso-proteomes by CyDye switch technique combined with two-dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the 54 identified, nine were novel SNO-proteins, 32 were increased, eight were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen-responsive nitroso-proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation.
NO 部分与半胱氨酸的共价结合（S-亚硝基化或 SNO）是 NO 直接调节蛋白质功能的主要途径。在子宫动脉内皮细胞（UAEC）中，雌二醇-17β（E2）迅速刺激蛋白质 SNO，E2 暴露后 10-30 分钟内达到最大值。E2-牛血清白蛋白也能刺激蛋白质 SNO。ICI 182,780 阻断了两者对 SNO 的刺激，这表明与定位于质膜上的特定雌激素受体（ER）有关的机制。E2 诱导的蛋白质 SNO 被选择性 ERβ，但不是 ERα，拮抗剂所抑制。特定的 ERβ而不是 ERα 激动剂能够诱导蛋白质 SNO。ERβ 的过表达，但不是 ERα，显著增强了 E2 诱导的 SNO。ERβ 的过表达，而不是 ERα，显著增加了基础 SNO，但没有进一步增强 E2 刺激的 SNO。E2 诱导的 SNO 被 N-硝基-L-精氨酸甲酯和特定的内皮型一氧化氮合酶（eNOS）siRNA 抑制。因此，雌激素诱导的 SNO 是通过内源性 NO 通过 eNOS 介导的，主要是在 UAEC 中的 ERβ。我们进一步通过 CyDye 切换技术结合二维（2D）荧光差异凝胶电泳分析了硝基蛋白组。在 2D 凝胶上可以看到许多硝基蛋白（斑点）。选择了 60 个斑点，并进行基质辅助激光解吸/电离-飞行时间质谱分析。在鉴定的 54 个中，有 9 个是新的 SNO 蛋白，32 个是增加的，8 个是减少的，其余的在 E2 作用下没有变化。串联 MS 鉴定出 Cys139 是 GAPDH 中 SNO 的特定位点。基础和雌激素反应性硝基蛋白组的途径分析表明，SNO 调节多种蛋白质功能，直接暗示 SNO 是雌激素调节子宫内皮功能和子宫血管舒张的一种新机制。
