Identification and functional analysis of two unknown PHGPx in embryogenesis

胚胎发生中两种未知PHGPx的鉴定及功能分析

• 批准号: 17590067
• 负责人: IMAI Hirotaka
• 金额: $ 2.3万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590067/
• 关键词: Embryogenesis; Oxidative stress; Transgenic rescue; Proteome Analysis; Lipid; Glutathione Peroxidase; ストレス; プロモーター; プロテオーム; 過酸化脂質

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide. Disruption of PHGPx gene in mice resulted in the embryonic lethality at 8.5 dpc. However, the analysis of role of PHGPx in embryogenesis has remained.In this study, to clarify the role of PHGPx during embryogenesis, we examined about the expression of PHGPx protein, mRNA in embryogenesis and the functional analysis using in vitro culture system and transgenic rescue method.We found that new 18kDa PHGPx was expressed in 7.5dpc-9.5dpc embryos.This PHGPx was associated with other protein via disulfide formation.From 5'RACE analysis, new transcriptional star site in lb exons was found and new translational start site. In vitro embryo culture system, PHGPx KO embryo died and could not formation of ICM, however transfection of non-mitochondrial PHGPx rescued lethality of KO embryo. We developed new strategy for analysis of role of PHGPx during embryogenesis using transgenic rescue. Trans gene of normal PHGPx gene rescued lethality of KO mice, but all mutant of PHGPx gene did not rescued lethality of KO mice. In placenta we found that 20kDa PHGPx was associated with 10kDa other protein and dimmer formation.

磷脂氢过氧化物谷胱甘肽过氧化物酶（PHGPx）是一种独特的抗氧化酶，可以直接还原磷脂氢过氧化物。PHGPx基因的破坏导致小鼠胚胎在8.5 dpc时死亡。然而，PHGPx在胚胎发生中的作用的分析仍然存在。在本研究中，为了阐明PHGPx在胚胎发生中的作用，我们检测了PHGPx蛋白的表达，利用体外培养系统和转基因拯救方法对PHGPx基因在胚胎发生中的作用进行了研究，发现新的18 kDa PHGPx基因在7.5dpc-1中表达，通过5 'RACE分析，在lb外显子上发现了新的转录星星位点和新的翻译起始位点。在体外胚胎培养系统中，PHGPx KO胚胎死亡且不能形成ICM，而转染非线粒体PHGPx可挽救KO胚胎的死亡。我们开发了一种新的策略来分析PHGPx在胚胎发生过程中的作用，使用转基因拯救。正常PHGPx基因的反式基因挽救了KO小鼠的致死性，而PHGPx基因的所有突变体都不能挽救KO小鼠的致死性。在胎盘中，我们发现20 kDa的PHGPx与10 kDa的其他蛋白质和二聚体的形成有关。