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) 的氧化裂解，产生二氨基丙烷、H 2 O 2 和氨基醛衍生物。大量文献将植物和动物细胞中 PAO 对 spd 和 spm 的分解代谢与程序性细胞死亡（细胞凋亡）过程联系起来。大量研究表明引起细胞凋亡的致病因素是H2O2介导的氧化应激。该实验室首先从燕麦幼苗中克隆、测序并鉴定了 PAO 基因。这种分离基因的可用性提供了独特的机会，可以在植物模型测试系统中获得有关 PAO 和多胺分解代谢在细胞凋亡中潜在作用的遗传证据。本申请的假设是：PAO 通过以下方式在程序性细胞死亡（细胞凋亡）中发挥致病作用： spd或spm氧化产生H202。该提案的具体目标是： (1) 确定 PAO 作为细胞 H2O2 主要生成物的潜在作用，从而作为细胞凋亡的决定因素。 PAO cDNA 基因序列已用于生成反义 PAO 基因片段。这些反义序列和相应有义序列的质粒构建体，连接到化学可控启动子上，将用于在转化玉米中产生dsRNA，以分析RNAi引起的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 转基因的表达，利用 PAO 的生化特性来消耗重组细胞中多胺的细胞水平。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)确认我们在PAO基因的5C启动子区域中发现的推定启动子序列-AGGGACTTTCCG-是促凋亡、H202激活的NF-kappaB样转录因子的识别信号。我们还试图鉴定植物中与该区域结合的 NF-kappaB 样蛋白； (4) 定位天然和重组细胞系统中 PAO 所在的细胞区室。通过免疫金抗体试剂原位标记 PAO，然后通过电子显微镜观察，将证实 GFP 融合蛋白技术的定位研究。该信息将定位在 PAO 参与下引发程序性细胞死亡的事件的起源。