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 inhibitor (AzI) regulates cellular polyamine homeostasis by binding to the polyamine-induced protein, Antizyme (Az), with greater affinity than ornithine decarboxylase (ODC). AzI is highly homologous to ODC but is not enzymatically active. In order to understand these specific characteristics of AzI and its differences from ODC, we determined the 3D structure of mouse AzI to 2.05 Å resolution. Both AzI and ODC crystallize as a dimer. However, fewer interactions at the dimer interface, a smaller buried surface area, and lack of symmetry of the interactions between residues from the two monomers in the AzI structure suggest that this dimeric structure is nonphysiological. In addition, the absence of residues and interactions required for pyridoxal 59-phosphate (PLP) binding suggests that AzI does not bind PLP. Biochemical studies confirmed the lack of PLP binding and revealed that AzI exists as a monomer in solution while ODC is dimeric. Our findings that AzI exists as a monomer and is unable to bind PLP provide two independent explanations for its lack of enzymatic activity and suggest the basis for its enhanced affinity toward Az.Keywords: structure/function studies; protein crystallization; protein structures-new; antizyme inhibitor; antizyme; ornithine decarboxylase Polyamines are small organic polycations that are essential for cell proliferation and play an important role in regulating other fundamental cellular processes. Elevated polyamine levels are observed in rapidly growing cells including transformed cells; thus, polyamine metabolism has been suggested as a potential target for cancer therapy (Pegg 1988;Marton and Pegg 1995;Wallace and Fraser 2004). The range of intracellular polyamines is determined at the lower limit by their absolute requirement for cellular proliferation and at the upper limit by their cytotoxicity (Poulin et al. 1993;Tobias and Kahana 1995), indicating a need for strict regulation of their intracellular concentration. Multiple pathways such as synthesis, uptake, degradation, and efflux regulate cellular polyamine levels. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the polyamine biosynthesis pathway (Pegg 2006). It is a pyridoxal 59-phosphate (PLP)-dependent enzyme that provides the only route for converting ornithine to putrescine. ODC, which is characterized by a short intracellular half-life, is part of an autoregulatory circuit mediated by a polyamine-induced protein, termed Antizyme (Az). An increased intracellular polyamine concentration increases the synthesis of Az by stimulating ribosomal frameshifting (Rom and Kahana 1994;Matsufuji et al. 1995). Az, in turn, binds to transient ODC monomer subunits with high affinity, preventing their re-association into active homodimers and targeting them for rapid degradation by the 26S proteasome (Murakami et al. 1992). Az also regulates polyamine transport across the plasma membrane via an unknown mechanism Mitchell et al. 1994;Suzuki et al. 1994;Sakata et al. 2000). 3 These authors contri...

Eukaryotic cells have been shown to contain two high-molecular-mass proteases of 700 kDa and 1400kDa (20s and 26s proteases, respectively). It has been suggested that the 20s protease, also known as proteasome, may constitute the catalytic core of the 26s protease. While the role of the free 20s protease in intracellular protein degradation is unclear, the 26s protease is implicated in the degradation of ubiquinated proteins. We have recently demonstrated, that ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells, is degraded via an ATP-dependent but ubiquitin-independent proteolytic pathway. Here we extend these observations by demonstrating that in reticulocyte lysate ODC degradation is inhibited by antibodies raised against the C9 subunit of rat proteasome. Partial fractionation of the lysate demonstrated preferential degradation of ODC in the fraction of the lysate proteins that are precipitated by 38% ammonium sulfate. Since it was demonstrated that the 26s protease precipitates at this concentration of ammonium sulfate while the 20s proteasome remains soluble, our results suggest that the 26s protease is the one degrading ODC.Two high-molecular-mass proteases have been described in a variety of eukaryotic cells including rabbit reticulocytes [l, 21. The first is a 700-kDa proteolytic complex termed proteasome or 20s protease (for review see . It is a cylinder-shaped particle [8, 91 composed of a large number of typical low-molecular-mass subunits [lo, 111, exhibiting various proteolytic activities specific to proteins and peptides [4,12, 131. The physiological role of this protease is still unknown, mainly because of contradictory reports concerning its ATP-dependence [l, 2, 141 which does not reconcile with the absolute requirement for energy in the process of intracellular protein breakdown [15]. The second, is an even larger 1400-kDa proteolytic complex known as the 26s protease. Recent studies have demonstrated that this ATP-dependent protease has clear preference for ubiquinated proteins [l, 21. It has been recently suggested that the 20s proteasome may constitute the catalytic core of the larger 26s protease [ 16 -181.Ornithine decarboxylase (ODC) is a highly regulated enzyme in the biosynthesis pathway of polyamines [19], whose activity is crucial for the process of cellular proliferation [20-231. ODC is one of the most rapidly turned over proteins in mammalian cells [24, 251. We have recently demonstrated that in cells, and in an in v i m reticulocyte-lysatebased degradation mix, ODC degradation occurs via a nonlysosomal, ATP-dependent but ubiquitin-independent pathway [26, 271. In the present study we have further characCorrespondence to C . Kahana, Department of Molecular Genetics and Virology, The Weizmann Institute of Science, Rehovot 76100, Israel Abbreviation. ODC, omithine decarboxylase.Enzyme. Ornithine decarboxylase (EC 4.1.1.17).terized ODC degradation in reticulocyte lysate. We show here that highly specific rabbit polyclonal antibodies raised against the C9...