ORIGINS AND COMPARTMENTALIZATION OF ANTIZYME PROTEINS

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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.