Developmental Toxicology Laboratory, Department of Environmental Health Sciences, 1420 Washington Heights, University of Michigan, Ann Arbor, MI 48109-2029, USA.
Free Radic Biol Med. 2013 Oct;63:325-37. doi: 10.1016/j.freeradbiomed.2013.05.040. Epub 2013 Jun 2.
Developmental signals that control growth and differentiation are regulated by environmental factors that generate reactive oxygen species (ROS) and alter steady-state redox environments in tissues and fluids. Protein thiols are selectively oxidized and reduced in distinct spatial and temporal patterns in conjunction with changes in glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox potentials (E(h)) to regulate developmental signaling. The purpose of this study was to measure compartment-specific thiol redox status in cultured organogenesis-stage rat conceptuses and to evaluate the impact of thiol oxidation on the redox proteome. The visceral yolk sac (VYS) has the highest initial (0 h) total intracellular GSH (GSH+2GSSG) concentration (5.5 mM) and the lowest Eh (-223 mV) as determined by HPLC analysis. Total embryo (EMB) GSH concentrations ranged lower (3.2 mM) and were only slightly more oxidized than the VYS. Total GSH concentrations in yolk sac fluid (YSF) and amniotic fluid (AF) are >500-fold lower than in tissues and are highly oxidized (YSF E(h)=-121 mV and AF E(h)=-49 mV). Steady-state total Cys concentrations (Cys+2CySS) were significantly lower than GSH in tissues but were otherwise equal in VYS and EMB near 0.5 mM. On gestational day 11, total GSH and Cys concentrations in EMB and VYS increase significantly over the 6h time course while E(h) remains relatively constant. The Eh (GSH/GSSG) in YSF and AF become more reduced over time while E(h) (Cys/CySS) become more oxidized. Addition of L-buthionine-S,R-sulfoximine (BS0) to selectively inhibit GSH synthesis and mimic the effects of some GSH-depleting environmental chemicals significantly decreased VYS and EMB GSH and Cys concentrations and increased Eh over the 6h exposure period, showing a greater overall oxidation. In the YSF, BSO caused a significant increase in total Cys concentrations to 1.7 mM but did not significantly change the E(h) for Cys/CySS. A significant net oxidation was seen in the BSO-treated AF compartment after 6 h. Biotinylated iodoacetamide (BIAM) labeling of proteins revealed the significant thiol oxidation of many EMB proteins following BSO treatment. Quantitative changes in the thiol proteome, associated with developmentally relevant pathways, were detected using isotope coded affinity tag (ICAT) labeling and mass spectroscopy. Adaptive pathways were selectively enriched with increased concentrations of proteins involved in mRNA processing (splicesome) and mRNA stabilization (glycolysis, GAPDH), as well as protein synthesis (aminoacyl-tRNA) and protein folding (antigen processing, Hsp70, protein disulfide isomerase). These results show the ability of chemical and environmental modulators to selectively alter compartmental intracellular and extracellular GSH and Cys concentrations and change their corresponding E(h) within the intact viable conceptus. The altered E(h) were also of sufficient magnitude to alter the redox proteome and change relative protein concentrations, suggesting that the mechanistic links through which environmental factors inform and regulate developmental signaling pathways may be discovered using systems developmental biology techniques.
发育信号控制着生长和分化，这些信号受到环境因素的调控，这些环境因素会产生活性氧（ROS）并改变组织和液体中的稳态氧化还原环境。蛋白质巯基在与谷胱甘肽/谷胱甘肽二硫化物（GSH/GSSG）和半胱氨酸/胱氨酸（Cys/CySS）氧化还原电势（Eh）变化相关的特定时空模式中被选择性地氧化和还原，以调节发育信号。本研究的目的是测量培养的器官发生阶段大鼠胚胎中特定隔室的巯基氧化还原状态，并评估巯基氧化对氧化还原蛋白质组的影响。内脏卵黄囊（VYS）具有最高的初始（0 小时）总细胞内 GSH（GSH+2GSSG）浓度（5.5 mM）和最低的 Eh（-223 mV），这是通过 HPLC 分析确定的。总胚胎（EMB）GSH 浓度较低（3.2 mM），仅比 VYS 略氧化。卵黄囊液（YSF）和羊水（AF）中的总 GSH 浓度比组织低>500 倍，且高度氧化（YSF Eh=-121 mV，AF Eh=-49 mV）。稳态总半胱氨酸浓度（Cys+2CySS）在组织中明显低于 GSH，但在 VYS 和 EMB 中几乎相等，接近 0.5 mM。在妊娠第 11 天，EMB 和 VYS 中的总 GSH 和 Cys 浓度在 6 小时的时间过程中显著增加，而 Eh 保持相对稳定。YSF 和 AF 中的 Eh（GSH/GSSG）随时间变得更还原，而 Eh（Cys/CySS）变得更氧化。L-丁硫氨酸-S，R-亚砜亚胺（BS0）的添加可选择性抑制 GSH 合成并模拟某些 GSH 耗竭环境化学物质的作用，在 6 小时暴露期间显著降低 VYS 和 EMB 的 GSH 和 Cys 浓度并增加 Eh，表现出更大的整体氧化。在 YSF 中，BS0 导致总半胱氨酸浓度显著增加到 1.7 mM，但对半胱氨酸/胱氨酸的 Eh 没有显著影响。BSO 处理后的 AF 隔室在 6 小时后出现明显的净氧化。生物素化碘乙酰胺（BIAM）标记蛋白质显示，BSO 处理后 EMB 中的许多蛋白质发生明显的巯基氧化。使用同位素编码亲和标签（ICAT）标记和质谱检测到与发育相关途径相关的巯基蛋白质组的定量变化。适应性途径被选择性富集，参与 mRNA 加工（剪接体）和 mRNA 稳定（糖酵解、GAPDH）以及蛋白质合成（氨酰-tRNA）和蛋白质折叠（抗原加工、Hsp70、蛋白质二硫键异构酶）的蛋白质浓度增加。这些结果表明，化学和环境调节剂能够选择性地改变完整活胚胎中隔室内细胞内和细胞外 GSH 和 Cys 浓度，并改变其相应的 Eh。Eh 的变化幅度也足以改变氧化还原蛋白质组并改变相对蛋白质浓度，这表明可以使用系统发育生物学技术来发现环境因素告知和调节发育信号通路的机制联系。
