Regulation of polyamine biosynthesis in Saccharomyces cerevisiae

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme. The synthesis of mammalian antizyme was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. In this work, a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian antizyme was identified. Synthesis of yeast antizyme (Oaz1) involves polyamine-regulated frameshifting. New elements, termed OFRE (OAZ1 frameshifting repressor element) and OPRE (OAZ1 polyamine responsive element) that are necessary for the polyamines to regulate frameshifting were mapped in the OAZ1 mRNA. Degradation of yeast ODC by the proteasome depends on Oaz1. Oaz1 mediates the degradation by binding to ODC thereby exposing a degradation signal at the N-terminus of ODC. Using the novel transplantable yeast ODC degradation signal (ODS) identified in this work a new possible role of the shuttle factor Rad23 in ODC degradation was identified. In addition, ODS is shown to interact with multiple 19S lid subunits in the proteasome. Using this novel model system for polyamine regulation another level of its control was discovered. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is efficiently inhibited by polyamines. I propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation. In a second part of the work, peptide aptamers were isolated that inhibit the ubiquitin-dependent turnover of test substrates of the proteasome. These aptamers appear to either inhibit ubiquitylation or the proteasome and thereby lead to a stabilization of test substrates. I Propose that ODS due to its ubiquitin-independent mode of degradation can be used as a tool in aptamer screens that are aimed at identifying additional peptide inhibitors of the proteasome with potential clinical relevance