A Unified Pathway for the Degradation of Ornithine Decarboxylase in Reticulocyte Lysate Requires Interaction with the Polyamine‐Induced Protein, Ornithine Decarboxylase Antizyme

Abstract

Recent studies have provided convincing evidence to add to a number of earlier observations suggesting that the rapid intracellular degradation of mammalian ornithine decarboxylase (ODC) is further accelerated by the action of ornithine decarboxylase antizyme (ODC-Az), a polyamineinduced protein. However, the mechanism whereby ODC-Az exerts its effect in this proteolytic process is mostly unknown. Here, by using reticulocyte-lysate-based synthesis and degradation systems, we demonstrate that interaction of ODC-Az with ODC results in two related outcomes: (a) ODC is inactivated as a result of its monomerization, and (b) ODC degradation is dramatically accelerated. While ODC inactivation requires the integrity of the ODC-Az binding site of ODC and the ODC binding site of ODC-Az, acceleration in ODC degradation also requires the previously characterized carboxyl-terminal destabilizing segment of ODC and a specific segment of ODC-Az that may be functionally distinct from that required for ODC binding. Interestingly, an active ODC variant with a mutant ODC-Az binding site is stable under basal degradation conditions. This, together with the ability of anti-(ODC-Az) antibody to specifically inhibit the basal degradation of ODC in the lysate, suggests that ODC-Az is an essential general mediator of ODC degradation. Based on these observations, we propose a model for the degradation of ODC which always require interaction with antizyme.Short intracellular half-life is a characteristic feature shared by highly regulated proteins, which enables their efficient modulation by upstream cellular regulatory mechanisms [l]. Ornithine decarboxylase (ODC) is a highly regulated enzyme in the biosynthesis of polyamines, which is one of the most labile cellular proteins [l]. Studies performed, both in intact cells and in vitro in a reticulocyte-lysate-based degradation system, revealed that ODC is degraded via a non-lysosomal, ATP-dependent, but ubiquitin-independent, proteolytic pathway [2, 31. Furthermore, genetically engineered ODC variants revealed that the integrity of a segment, encompassing the most carboxyl-terminal 37 amino acids of mouse ODC is required for maintaining its rapid degradation both in cells [4, 51 and in vitro [5].Recent studies have suggested that ODC degradation may be mediated by two distinct proteolytic machinery's [6, 71. According to these studies, the first proteolytic machinery is engaged in degrading ODC under basal metabolic conditions [6, 71 ; the second, that is stimulated by high concentration of polyamines, requires interaction of ODC with a small protein, termed ornithine decarboxylase antizyme (ODC-Az) [6, 71. ODC-Az is a 26-kDa protein that is induced in cells by polyamines [S-lo]. It binds ODC with high affinity and Correspondence to C. Kahana, Department of Molecular Genetics and Virology, The Weizmann Institute of Science, IL-Rehovot IL-76100, IsraelAbbreviations. ODC, ornithine decarboxylase; ODC-Az, ornithine decarboxylase antizyme ; GST, glutathione S-transferase. inhibits its activity ...

Antizyme Affects Cell Proliferation and Viability Solely through Regulating Cellular Polyamines

“…However, these studies did not compare the efficiency of their degradation with the efficiency with which Az stimulates ODC degradation. This is particularly important, as Az acts catalytically in stimulating ODC degradation, being effective even when present in inferior molar amounts (38). We have shown here that the two major forms of Az, Az1 and Az2, efficiently stimulated ODC degradation but failed to stimulate A, HEK-293T cells were cotransfected with constructs encoding FLAG-tagged (f) Az2, ODC, Aurora-A, cyclin D1, and ⌬Np73.…”

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions

“…In agreement with a recent report (Choi et al, 2005), we demonstrate that downregulation of AzI by compatible siRNA results in decreased cell proliferation. These effects of AzI are a result of its binding to Az and neutralizing its ability to target ODC for degradation (Li and Coffino, 1992;Murakami et al, 1992a, b;Mamroud-Kidron et al, 1994) and to inhibit polyamine uptake Mitchell et al, 1994;Suzuki et al, 1994;Sakata et al, 1997;Sakata et al, 2000), as forced expression of an AzI mutant with reduced Az binding displays reduced effect on ODC activity, polyamine uptake or growth properties. As AzI affects cellular polyamine homeostasis by increasing both ODC activity and polyamine uptake, the regulation of polyamine levels in AzI overproducing cells is different than in ODC overproducing cells, in which the polyamines content is dependant solely on augmented ODC activity, as polyamine uptake is actually compromised (Figure 7).…”

Structural Motifs Involved in Ubiquitin-Mediated Processing of the NF-κB Precursor p105: Roles of the Glycine-Rich Region and a Downstream Ubiquitination Domain

“…In addition, we generated the chimeric protein p53-GRR-ODC, which contains the GRR inserted between p53 and ODC. This chimeric protein was constructed in three steps: (i) removal of the C-terminal portion of p105 (residues 435 to 969) and in-frame insertion of the NcoI-NotI fragment containing the entire ODC sequence subcloned in these sites in a Bluescript vector (29), (ii) generation of the SpeI site that follows the last amino acid residue of p53, and (iii) replacement of the SpeI-SpeI fragment of the p50-GRR-ODC that contains p50 amino acid residues 1 to 342 with the SpeISpeI fragment that codes for full-length p53. The sequences of all constructs were confirmed by either the manual (Amersham) or the automatic (Applied Biosystems) dideoxy method using the ABI 310 autosequencer.…”