ORIGINS AND COMPARTMENTALIZATION OF ANTIZYME PROTEINS

该项目的长期目标是确定抗酶如何维持细胞多胺的动态平衡和防止肿瘤的形成。多胺、亚精胺和精胺，以及它们的二胺前体腐胺，是哺乳动物细胞生长和存活所必需的脂肪族阳离子。多胺的异常积累与阿尔茨海默病和其他神经疾病、细胞凋亡诱导和肿瘤形成有关。作为多胺在癌症中致病作用的证据，正在进行的临床试验正在证明多胺合成的抑制剂是有效的化学预防药物，并且几个多胺类似物在抗癌化疗中有用。抗酶是一种调节蛋白，在组织多胺水平的正常维持中起关键作用，它的下调允许肿瘤形成。它既控制多胺生物合成的第一种酶，也控制负责细胞吸收的转运蛋白。不幸的是，人们对抗酶本身知之甚少。哺乳动物细胞中至少存在四种抗酶形式，其中两种主要形式在分子稳定性和对渗透胁迫的响应上存在差异。一些抗酶蛋白专门与线粒体相关，在线粒体中它没有已知的功能。在理解抗酶在维持多胺水平中的关键作用方面的进展目前由于对多种抗酶形式及其不同细胞位置的混淆而停滞不前。这项研究的目的是解决在哺乳动物细胞中观察到的抗酶蛋白的形成和细胞定位的基本问题。具体地说，这些研究将(A)确定两种主要的抗酶形式是如何从一个基因产生的，以及(B)阐明抗酶蛋白在线粒体中的转位和加工。这些研究将集中在大鼠肝脏(HTC)和仓鼠(CHO)细胞系中发现的抗酶，并将依靠活性分析和免疫反应来检测和定量抗酶蛋白。一些实验将涉及在高度调控的哺乳动物表达系统中，将修饰形式的az-1 cdna导入细胞。将使用HTC细胞制备的线粒体进行体外抗酶蛋白转位实验。这些基础研究将为进一步探索抗酶在多胺动态平衡中的关键作用提供必要的基础。

The long-term goal of this project is to determine how antizyme maintains cellular polyamine homeostasis and prevents tumor formation. The polyamines, spermidine and spermine, and their diamine precursor, putrescine, are aliphatic cations essential for mammalian cell growth and viability. Abnormal accumulations of the polyamines are associated with Alzheimer's disease and other neurological disorders, apoptosis induction and tumor formation. As evidence for the polyamine's causative role in cancer, ongoing clinical trials are proving that inhibitors of polyamine synthesis are effective chemopreventive agents, and several polyamine analogs are useful in anti-cancer chemotherapy. Antizyme is a regulatory protein that serves a critical role in the normal maintenance of tissue polyamine levels, and its down- regulation allows tumor formation. It controls both the first enzyme in polyamine biosynthesis and the transporter responsible for their cellular uptake. Unfortunately, very little is known about antizyme itself. There are at least four antizyme forms in mammalian cells, and the two major ones are distinct in molecular stability and response to osmotic stress. Some antizyme protein is specifically associated with mitochondria, where it has no known function. Progress in understanding the critical role of antizyme in maintenance of polyamine levels is currently stalled by confusion over the multiple antizyme forms and their varied cell locations. The aim of this study is to resolve basic questions regarding formation and cellular localization of the antizyme proteins observed in mammalian cells. Specifically, these studies will (a) determine how the two major antizyme forms are generated from one gene, and (b) elucidate the translocation and processing of antizyme protein in mitochondria. The studies will concentrate on antizyme found in rat liver (HTC) and hamster (CHO) cell lines, and will rely on activity assays and immunoreactivity to detect and quantify antizyme protein. Some experiments will involve cells that have been transfected with modified forms of the az-1 cDNA in a highly regulated mammalian expression system. Antizyme protein translocation experiments will be conducted in vitro using mitochondria prepared from HTC cells. These basic studies will provide an essential foundation for further exploration of the critical role of antizyme in polyamine homeostasis.