ORIGINS AND COMPARTMENTALIZATION OF ANTIZYME PROTEINS

本项目的长期目标是确定抗酶如何维持细胞内多胺稳态并防止肿瘤形成。 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活力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系统疾病、细胞凋亡诱导和肿瘤形成有关。 作为多胺在癌症中的致病作用的证据，正在进行的临床试验证明多胺合成的抑制剂是有效的化学预防剂，并且几种多胺类似物可用于抗癌化疗。抗酶是在组织多胺水平的正常维持中起关键作用的调节蛋白，并且其下调允许肿瘤形成。 它控制多胺生物合成中的第一个酶和负责其细胞摄取的转运蛋白。 不幸的是，对antizyme本身知之甚少。 在哺乳动物细胞中至少有四种抗酶形式，其中两种主要的抗酶在分子稳定性和对渗透胁迫的反应上是不同的。 一些抗酶蛋白特异性地与线粒体相关，在那里它没有已知的功能。 抗酶在维持多胺水平中的关键作用的研究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。 本研究的目的是解决有关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。 具体而言，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，（B）阐明线粒体中抗酶蛋白的移位和加工。 这些研究将集中在大鼠肝（HTC）和仓鼠（CHO）细胞系中发现的抗酶，并将依赖于活性测定和免疫反应性来检测和定量抗酶蛋白。 一些实验将涉及在高度调节的哺乳动物表达系统中用az-1 cDNA的修饰形式转染的细胞。 抗酶蛋白易位实验将使用从HTC细胞制备的线粒体在体外进行。 这些基础研究将为进一步探索抗酶在多胺稳态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.