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）的氧化裂解以产生二氨基丙烷、H2 O2和氨基醛衍生物。大量的文献将植物和动物细胞中PAO对spd和spm的催化作用与程序性细胞死亡（凋亡）过程相关联。许多研究表明，引发细胞凋亡的病原体是由H2 O2介导的氧化应激。本实验室首先从燕麦幼苗中克隆了PAO基因，并对其进行了测序和鉴定。这个分离的基因的可用性提供了独特的机会，以获得遗传证据的潜在作用的PAO和多胺-catalysts在植物模型试验系统中的细胞凋亡。本申请的假设是：PAO通过以下途径在程序性细胞死亡（凋亡）中起致病作用： 由spd或spm氧化产生的H202。该建议的具体目的是：（1）确定PAO作为细胞H2 O2的主要产生者的潜在作用，并因此作为细胞凋亡的决定因素。PAO cDNA基因序列已用于产生反义PAO基因片段。将这些反义和相应的有义序列的质粒构建体连接到化学可控启动子上，用于在转化的玉米中产生dsRNA，以分析通过RNAi使PAO的翻译后基因沉默对细胞凋亡的后果;（2）利用PAO的生物化学性质，通过使用化学方法控制PAO的转基因表达，在重组细胞中消耗细胞水平的多胺。启动子激活该目的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的启动子序列-AGGGACTTTCCG-是促凋亡的H2 O2激活的NF-κ B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NF-κ B样蛋白;（4）定位PAO在天然和重组细胞系统中的细胞区室。原位标记PAO的免疫金抗体试剂，然后通过电子显微镜将证实定位研究的GFP融合蛋白技术。该信息将定位在PAO参与下启动程序性细胞死亡的事件的起源。