Role of Polyamine Oxidase in Apoptosis Studied with RNAi

RNAi 研究多胺氧化酶在细胞凋亡中的作用

• 批准号: 6766143
• 负责人: GLENN D KUEHN
• 金额: $ 7.49万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766143
• 关键词: RNA interference; amine oxidoreductase; apoptosis; cell growth regulation; chemical structure function; corn; electron microscopy; genetic regulation; genetic regulatory element; genetically modified plants; hydrogen peroxide; immunocytochemistry; intermolecular interaction; metabolism; oxidation reduction reaction; plant genetics; plant growth /development; plant proteins; polyamines; protein localization; spermidine; spermine; transcription factor

Polyamine oxidase (PAO) catalyzes oxidative cleavage of polyamines spermidine (spd) or spermine (spm) to yield diaminopropane, H202, and an aminoaldehyde derivative. A voluminous literature correlates the catabolism of spd and spm by PAO in plant and animal cells with the process of programmed cell death (apoptosis). Numerous studies have suggested that the causative agent for eliciting apoptosis is oxidative stress mediated by H202. The gene for PAO was first cloned, sequenced, and characterized in this laboratory from oat seedlings. The availability of this isolated gene offers unique opportunities to gain genetic evidence for potential roles of PAO and polyamine-catabolism in apoptosis in a plant model test system. The hypothesis of this application is: PAO has a causative role in programmed cell death (apoptosis) through H202 produced by oxidation of spd or spm. The specific aims of this proposal are: (1) To define the potential role of PAO as a major generator of cellular H202, and hence as a determinant of apoptosis. The PAO cDNA gene sequence has been used to generate antisense PAO gene segments. Plasmid constructs of these antisense and corresponding sense sequences, ligated to chemically controllable promoters, will be used to produce dsRNA in transformed maize, in order to analyze the consequences on apoptosis of post-translational gene silencing of PAO by RNAi; (2) To exploit the biochemical properties of PAO for depleting cellular levels of polyamines in recombinant cells by controlled expression of a trans-gene for PAO using chemically-activated promoters. This aim seeks to construct gene vectors that can be applied to a spectrum of cells, in order to delineate functions for the polyamines; (3) To confirm that a putative promoter sequence -AGGGACTTTCCG-, which we have discovered in the 5C-promoter region of the PAO gene, is a recognition signal for a pro-apoptotic, H202-activated NF-kappaB-like transcription factor. We also seek to identify an NF-kappaB-like protein in plants which binds to this region; (4) To localize the cellular compartment where PAO resides in natural and recombinant cell systems. In situ labeling of PAO by immunogold antibody reagents, followed by electron microscopy will corroborate localization studies by the GFP fusion protein technique. This information will localize the origin of events that initiate programmed cell death with participation of PAO.

多胺氧化酶(PAO)催化多胺亚精胺(SPD)或精胺(SPM)氧化裂解生成二氨基丙烷、H202和氨基醛衍生物。大量文献将PAO对植物和动物细胞中Spd和Spm的分解代谢与细胞程序性死亡(细胞凋亡)的过程相关联。大量研究表明，H202介导的氧化应激是诱导细胞凋亡的原因。PAO基因是本实验室首次从燕麦幼苗中克隆、测序和鉴定的。这一分离基因的可获得性为在植物模型试验系统中获得PAO和多胺分解代谢在细胞凋亡中的潜在作用的遗传学证据提供了独特的机会。这一应用的假设是：PAO通过以下途径在程序性细胞死亡(细胞凋亡)中起致病作用 SPD或SPM氧化产生的H202。这项建议的具体目的是：(1)明确PAO作为细胞H202的主要生成者的潜在作用，从而作为细胞凋亡的决定因素。PAO基因序列已被用来产生反义PAO基因片段。将这些反义和相应的正义序列连接到化学可控制的启动子上，构建的质粒将用于在转化玉米中产生dsRNA，以分析RNAi抑制PAO翻译后基因沉默对细胞凋亡的影响；(2)利用化学激活的启动子控制PAO的反式基因表达，挖掘PAO在重组细胞中消耗细胞水平多胺的生化特性。这一目标旨在构建可应用于一系列细胞的基因载体，以描述多胺的功能；(3)证实我们在PAO基因5C-启动子区域发现的一个可能的启动子序列-AGGGACTTTCCG-是促凋亡的、H202激活的NF-kappaB样转录因子的识别信号。我们还试图在植物中鉴定与该区域结合的核因子-kappaB样蛋白；(4)在天然和重组细胞系统中定位PAO所在的细胞室。用免疫金抗体试剂原位标记PAO，然后用电子显微镜证实GFP融合蛋白技术的定位研究。这一信息将在PAO的参与下定位启动细胞程序性死亡的事件的起源。