POLYAMINE OXIDASE ROLE IN APOPTOSIS

多胺氧化酶(PAO)催化多胺精胺(Spd)或精胺(Spm)氧化裂解生成二氨基丙烷(DAP)、过氧化氢和一种氨基醛衍生物。在植物中，DAP是通过席夫碱还原酶脱羧酶(SBRD)生物合成去甲精胺(NSPD)和去甲精胺(NSPM)的前体。最近，PAO对Spd和Spm的分解代谢被提出。几位研究人员认为这是一种致病因素，通过产生过氧化氢和随后的氧化应激，迫使动物细胞进入程序性细胞死亡(细胞凋亡)。燕麦幼苗PAO基因是最近在本实验室克隆、测序和鉴定的。这一新分离基因的可获得性为在植物模型试验系统中PAO和多胺分解代谢在细胞凋亡中的潜在作用(S)提供了独特的遗传学证据。这一应用的假设是：(1)PAO通过其氧化SPD或SPM产生的H_2O_2引起细胞凋亡；(2)DAP产生的NSPD和NSPM通过反馈抑制PAO和减少H_2O_2的合成而起到抑制细胞凋亡的作用。本研究的具体目的是：(1)利用PAO基因序列，用钛质粒法转化紫花苜蓿植株，分析PAO缺乏症对植物组织细胞凋亡的影响。(2)将催化DAP生物合成NSPD和NSPM的酶SBRD的基因序列用于转化紫花苜蓿植株，以分析SBRD从DAP生物合成NSPD和NSPM过程中PAO解偶联产生H202的后果。目标(1)和(2)是对假设的(I)和(Ii)部分的直接检验。(3)。利用燕麦中的PAO基因序列作为基因探针，尝试从肾脏和肝脏的cDNA文库中分离人的PAO基因。一个具有特征性的人类PAO基因将使开发一种分子生物学方法来研究PAO在产生H202中的作用以及它所谓的诱导动物凋亡的作用。(4)将PAO基因信号肽的cDNA基因序列连接到编码荧光蛋白(GFP)的基因。利用荧光成像技术对构建的融合蛋白在紫花苜蓿组织和转化植株中的表达进行分析，以确定PAO的亚细胞定位。用免疫胶体金抗体试剂原位标记PAO和电子显微镜分析，将证实GFP蛋白技术的定位研究。这些结果将有助于定位启动PAO依赖的细胞凋亡的事件的起源。

Polyamine oxidase (PAO) catalyzes oxidative cleavage of polyamines spermine (spd) or spermine (spm) to produce diaminopropane (dap), H2O2, and an aminoaldehyde derivative. In plants, dap is the precursor for biosynthesis of norspermidine (nspd) and norspermine (nspm) via the enzyme, Schiff base reductase decarboxylase (SBRD). Recently, the catabolism of spd and spm by PAO has been proposed. By several investigators to be a causative agent, through product H2O2 and ensuing oxidative stress, which forces animal cells into programmed cell death (apoptosis). The gene for PAO from oat seedlings was recently cloned, sequenced, and characterized in this laboratory. The availability of this newly isolated gene offers unique opportunities to gain genetic evidence for a potential role(s) of PAO and polyamine-catabolism in apoptosis in a plant model test system. The hypothesis of this application is: (i) PAO has a causative role in apoptosis in cells through H2O2 produced by its oxidation of spd or spm, and (ii) the nspd and nspm produced from dap serve as suppressors of apoptosis through feedback inhibition of PAO and reduction in H2O2 synthesis. The specific aims of this proposal are: (1) The PAO cDNA gene sequence will be used to transform alfalfa plants with Ti-plasmid methods to analyze the consequences of controlled PAO- deficiency on apoptosis in plant tissues. (2) The cDNA gene sequence for the enzyme, SBRD, which catalyzes nspd and nspm biosynthesis from dap, will be used to transform alfalfa plants in order to analyze the consequences of uncoupling generation of H202 by PAO from the biosynthesis of nspd and nspm derived from dap by SBRD. Aims (1) and (2) are direct tests of parts (i) and (ii) of the hypothesis. (3). The PAO cDNA gene sequence from oat will be used as a gene probe to attempt the isolation of the human PAO gene from kidney and liver cDNA libraries. A characterized human PAO gene will make possible the development of a molecular biological approach to investigate the role of PAO in generating H202 and its alleged role to elicit animal apoptosis. (4) The cDNA gene sequence of a signal peptide for the PAO gene will be ligated to a gene coding for a fluorescent protein (GFP). The fusion protein produced from this construction will be analyzed by fluorescent imaging techniques in tissue of alfalfa and out plants transformed with this construction in order to identify the subcellular localization of PAO. In situ labeling of PAO by immunogold antibody reagents and electron microscopic analysis, will corroborate the localization studies by the GFP protein technique. These results will aid in localizing the origin of events that initiate PAO-dependent apoptosis.