POLYAMINE OXIDASE ROLE IN APOPTOSIS

多胺氧化酶（PAO）催化多胺精胺（spd）或精胺（spm）的氧化裂解生成二氨基丙烷（dap）、H2O2和氨基醛衍生物。在植物中，dap是通过希夫碱还原酶脱羧酶（SBRD）生物合成去精胺（nspd）和去精胺（nspm）的前体。近年来，有人提出PAO对spd和spm的分解代谢作用。被一些研究者认为是一种病原体，通过产物H2O2和随后的氧化应激，迫使动物细胞进入程序性细胞死亡（凋亡）。从燕麦幼苗中提取的PAO基因最近在这个实验室被克隆、测序和鉴定。这个新分离基因的可用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证据提供了独特的机会。本应用的假设是：(i) PAO通过其氧化spd或spm产生H2O2导致细胞凋亡；（ii） dap产生的nspd和nspm通过反馈抑制PAO和减少H2O2合成而抑制细胞凋亡。本课题的具体目的是：(1)利用PAO cDNA基因序列，利用ti质粒法转化苜蓿植株，分析PAO控制缺失对植物组织凋亡的影响。(2)利用催化dap合成nspd和nspm的酶SBRD的cDNA基因序列转化苜蓿植物，分析SBRD生物合成dap合成nspd和nspm过程中PAO解偶联生成H202的影响。目标(1)和(2)是对假设(i)和（ii）部分的直接检验。(3)。利用燕麦PAO cDNA基因序列作为基因探针，尝试从肾脏和肝脏cDNA文库中分离人PAO基因。一个特征化的人类PAO基因将使分子生物学方法的发展成为可能，以研究PAO在产生H202中的作用及其引发动物凋亡的作用。(4) PAO基因信号肽的cDNA基因序列将与编码荧光蛋白（GFP）的基因连接。将利用荧光成像技术对该结构所产生的融合蛋白在紫花苜蓿和用该结构转化的植物的组织中进行分析，以确定PAO的亚细胞定位。用免疫金抗体试剂对PAO进行原位标记和电镜分析，将证实GFP蛋白技术的定位研究。这些结果将有助于定位引发pao依赖性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.