Role of Polyamine Oxidase in Apoptosis Studies with RNA Interference

多胺氧化酶在 RNA 干扰细胞凋亡研究中的作用

• 批准号: 7631379
• 负责人: GLENN D KUEHN
• 金额: $ 10.61万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-05 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7631379
• 关键词: Anabolism; Animals; Apoptosis; Apoptotic; Applications Grants; Avena sativa; Base Sequence; Binding Sites; Biochemical; Biochemical Reaction; Bioinformatics; Biological; Biological Models; Catabolism; Catalysis; Cells; Copper; DNA; Development Plans; Disease; Disease Resistance; Double-Stranded RNA; Enzymes; Eukaryotic Cell; Event; Funding; Gene Expression; Genes; Genetic Transcription; Grant; Hydrogen Peroxide; Ions; Knowledge; Laboratories; Link; Literature; Location; Mediating; Messenger RNA; Methodology; Methods; NADPH Oxidase; NF-kappa B; Necrotic Lesion; Oats; Oryza sativa; Oxidative Stress; Oxygen; Pathway interactions; Peroxidases; Plant Model; Plants; Plasmid Cloning Vector; Play; Polyamine Catabolism; Polyamines; Prevention; Process; Production; Promoter Regions; Proteins; RNA Interference; Reaction; Reactive Oxygen Species; Research; Research Design; Research Personnel; Respiratory Burst; Rice; Role; Seedling; Seeds; Sequence Analysis; Signal Transduction Pathway; Site; Source; Spermidine; Spermine; System; Techniques; Time; Wound Healing; Yeasts; amine oxidase; crosslink; direct application; knock-down; oxalate oxidase; oxidation; pathogen; polyamine oxidase; promoter; rapid technique; research study; response; transcription factor

DESCRIPTION (provided by applicant): A voluminous literature documents studies that correlate the catabolism of the polyamines, spermidine and spermine, in plant and animal cells with the process of programmed cell death (apoptosis). Polyamine oxidase (PAO) catalyzes oxygen-dependent cleavage of the polyamines to form hydrogen peroxide (H2O2), among other products. Many studies indicate that the causative agent for eliciting apoptosis is oxidative stress mediated by H2O2. Our project hypothesis is: The reaction catalyzed by the enzyme PAO is the major source of cellular H2O2 that causes H2O2-mediated apoptosis in eukaryotic cells. We have gained strong evidence that PAO catalysis is indeed the major source of H2O2 in a plant model system undergoing the hypersensitive response (HR). HR is a pathogen-elicited process mediated by H2O2, which culminates in apoptosis. The origin of the H2O2 is not known. In preliminary studies, we have applied RNA interference (RNAi) methods, using a newly discovered, rapid technique for introducing double-stranded (dsRNA) into plants during HR, and have succeeded in knocking down PAO enzymatic expression with concurrent abolishment of the H2O2 burst that normally accompanies HR. The specific aims of this project are: (1) RNA interference (RNAi) methods that exploit our new technique for introducing dsRNA into plant cells will be used to silence (knockdown) PAO gene expression in Avena sativa L. (oat) seedlings. This knockdown will be shown to result in suppression of five cellular parameters: PAO mRNA levels, PAO enzymatic activity, the H2O2 burst in the HR, catabolic loss of cellular polyamines during the HR, and entry into apoptosis. (2) The existence of an NF-kB-like transcription factor in plants will be established, which may have a role in regulating the expression of PAO by pro-apoptotic, H2O2-activated pathways. The DNA nucleotide sequence (gtgaatttcc) was discovered by us in two locations of the 5'-untranslated promoter region of the PAO gene from Oryza sativa (rice). This sequence contains recognition sites for binding transcription factors that link PAO gene transcription to NF-kB-like factors. We will complete the isolation of this protein to confirm its existence in the plant kingdom. (3) Methodologies, plant seed stocks, and plasmid vectors will be developed in order to exploit a synthetic copper(ll) ion-dependent genetic promoter system, produced by our laboratory, for controlling the synthesis of trans genes in plant and yeast hosts.

描述（由申请人提供）：大量文献记录了与多胺，精子和精子，在植物和动物细胞中与程序性细胞死亡过程（凋亡）相关的研究。多胺氧化酶（PAO）催化了多胺的氧依赖性裂解，形成过氧化氢（H2O2），以及其他产物。许多研究表明，引起凋亡的原因是H2O2介导的氧化应激。我们的项目假设是：由酶PAO催化的反应是导致真核细胞中H2O2介导的凋亡的细胞H2O2的主要来源。我们已经获得了有力的证据，表明PAO催化确实是经历过度反应（HR）的植物模型系统中H2O2的主要来源。 HR是由H2O2介导的病原体引诱过程，该过程最终导致凋亡。 H2O2的起源尚不清楚。在初步研究中，我们使用了新发现的快速技术应用RNA干扰方法（RNAI）方法，将双链（DSRNA）引入HR期间，并成功地将PAO酶促表达与正常伴随HR的H2O2爆发一起击倒PAO酶促表达。 该项目的具体目的是：（1）利用我们将dsRNA引入植物细胞中的新技术的RNA干扰（RNAi）方法将用于沉默（敲低）PAO基因在Avena Sativa L.（OAT）幼苗中的表达。该敲低将显示出五个细胞参数的抑制：PAO mRNA水平，PAO酶活性，H2O2在HR中爆发，HR期间细胞多胺的分解代谢丧失以及进入细胞凋亡。 （2）将建立植物中NF-KB样转录因子的存在，这可能在调节PAO的表达中通过促凋亡，H2O2激活的途径发挥作用。我们在来自Oryza sativa（大米）的PAO基因的5'-非翻译启动子区域的两个位置发现了DNA核苷酸序列（GTGAATTTCC）。该序列包含将PAO基因转录与NF-KB样因子联系起来的结合转录因子的识别位点。我们将完成该蛋白质的隔离，以确认其在植物王国中的存在。 （3）将开发方法，植物种子储备和质粒载体，以利用我们实验室生产的合成铜（LL）离子依赖性遗传启动子系统，用于控制植物和酵母宿主中跨性基因的合成。