Zippi Bercovich

The role of polyamines in supporting growth of mammalian cells is mediated through their requirement for translation initiation and elongation

Polyamines are essential cell constituents whose depletion results in growth cessation. Here we have investigated potential mechanisms of action of polyamines in supporting mammalian cell proliferation. We demonstrate that polyamines regulate translation both at the initiation and at the elongation steps. l-α-Difluoromethylornithine treatment resulting in polyamine depletion reduces protein synthesis via inhibition of translation initiation. N1-Guanyl-diaminoheptane (GC7), a spermidine analogue that inhibits eukaryotic initiation factor 5A (eIF5A) hypusination, also caused inhibition of translation initiation. In contrast, depletion of eIF5A by short hairpin RNA inhibits translation elongation as was recently demonstrated in yeast and Drosophila. These results suggest that in addition to competing with spermidine in the hypusination reaction, GC7 also competes with spermidine at yet undefined sites required for translation initiation. Finally, we show that either polyamine depletion or GC7 treatment induced eIF2α phosphorylation and reduced phosphorylation of 4E-BP, thus setting the molecular basis for the observed inhibition of translation initiation.

Mitochondrial localization of antizyme is determined by context-dependent alternative utilization of two AUG initiation codons

Ornithine decarboxylase-antizyme (Az), a polyamine-induced protein that targets ornithine decarboxylase (ODC) to rapid degradation, is synthesized as two isoforms. Studies performed in vitro indicated that the 29 and 24.5 kDa isoforms originate from translation initiation at two alternative initiation codons. Using transient transfections we demonstrate here that also in cells the two isoforms are synthesized from two AUG codons with the second being utilized more efficiently. The more efficient utilization of the second AUG is due to its location within a better sequence context for translation initiation. By using immunostaining we demonstrate that only the less expressed long form of Az is localized in the mitochondria. Moreover, this long isoform of Az and not the more efficiently expressed short isoform is imported into mitochondria in an in vitro uptake assay. Our data therefore demonstrate that a single Az transcript gives rise to two Az proteins with different N-terminal sequence and that the longer Az form containing a potential N-terminal mitochondrial localization signal is transported to mitochondria.

ODCp, a brain- and testis-specific ornithine decarboxylase paralogue, functions as an antizyme inhibitor, although less efficiently than AzI1

ODC (ornithine decarboxylase), the first enzyme in the polyamine biosynthesis pathway in mammalian cells, is a labile protein. ODC degradation is stimulated by Az (antizyme), a polyamine-induced protein, which in turn is regulated by an ODC-related protein termed AzI (Az inhibitor). Recently, another ODCp (ODC paralogue) was suggested to function as AzI, on the basis of its ability to increase ODC activity and inhibit Az-stimulated ODC degradation in vitro. We show in the present study that ODCp is indeed capable of negating Az functions, as reflected by its ability to increase ODC activity and polyamine uptake and by its ability to provide growth advantage in stably transfected cells. However, ODCp is less potent than AzI1 in stimulating ODC activity, polyamine uptake and growth rate. The superiority of AzI1 to ODCp in inhibiting the Az-stimulated ODC degradation is also demonstrated using an in vitro degradation assay. We show that the basis for the inferiority of ODCp as an AzI is its lower affinity towards Az (Az1 and Az3). Further, we show here that ODCp, like AzI, is degraded in a ubiquitin-dependent manner, in a reaction that does not require either interaction with Az or the integrity of its C-terminus. Interaction with Az actually stabilizes ODCp by interfering with its ubiquitination. This results in sequestration of Az into a stable complex with ODCp, which is the central feature contributing to the ability of ODCp to function as AzI.

Antizyme 3 inhibits polyamine uptake and ornithine decarboxylase (ODC) activity, but does not stimulate ODC degradation

Azs (antizymes) are small polyamine-induced proteins that function as feedback regulators of cellular polyamine homoeostasis. They bind to transient ODC (ornithine decarboxylase) monomeric subunits, resulting in inhibition of ODC activity and targeting ODC to ubiquitin-independent proteasomal degradation. Az3 is a mammalian Az isoform expressed exclusively in testicular germ cells and therefore considered as a potential regulator of polyamines during spermatogenesis. We show here that, unlike Az1 and Az2, which efficiently inhibit ODC activity and stimulate its proteasomal degradation, Az3 poorly inhibits ODC activity and fails to promote ODC degradation. Furthermore, Az3 actually stabilizes ODC, probably by protecting it from the effect of Az1. Its inhibitory effect is revealed only when it is present in excess compared with ODC. All three Azs efficiently inhibit the ubiquitin-dependent degradation of AzI (Az inhibitor) 1 and 2. Az3, similar to Az1 and Az2, efficiently inhibits polyamine uptake. The potential significance of the differential behaviour of Az3 is discussed.

Expression Profiling and Biochemical Analysis Suggest Stress Response as a Potential Mechanism Inhibiting Proliferation of Polyamine-depleted Cells

Background: Depletion of cellular polyamines results in cessation of cellular proliferation. Results: Genomic and biochemical analysis demonstrates stress establishment in the polyamine-depleted cells. Conclusion: Establishment of stress response accounts at least in part for growth arrest establishment in polyamine-depleted cells. Significance: Our results suggest a novel view on the mechanistic basis accounting for growth arrest of polyamine-depleted cells. Polyamines are small organic polycations that are absolutely required for cell growth and proliferation; yet the basis for this requirement is mostly unknown. Here, we combined a genome-wide expression profiling with biochemical analysis to reveal the molecular basis for inhibited proliferation of polyamine-depleted cells. Transcriptional responses accompanying growth arrest establishment in polyamine-depleted cells or growth resumption following polyamine replenishment were monitored and compared. Changes in the expression of genes related to various fundamental cellular processes were established. Analysis of mirror-symmetric expression patterns around the G1-arrest point identified a set of genes representing a stress-response signature. Indeed, complementary biochemical analysis demonstrated activation of the PKR-like endoplasmic reticulum kinase arm of the unfolded protein response and of the stress-induced p38 MAPK. These changes were accompanied by induction of key growth-inhibitory factors such as p21 and Gadd45a and reduced expression of various cyclins, most profoundly cyclin D1, setting the basis for the halted proliferation. However, although the induced stress response could arrest growth, polyamine depletion also inhibited proliferation of PKR-like endoplasmic reticulum kinase and p38α-deficient cells and of cells harboring a nonphosphorylatable mutant eIF2α (S51A), suggesting that additional yet unidentified mechanisms might inhibit proliferation of polyamine-depleted cells. Despite lengthy persistence of the stress and activation of apoptotic signaling, polyamine-depleted cells remained viable, apparently due to induced expression of protective genes and development of autophagy.

Antizyme Affects Cell Proliferation and Viability Solely through Regulating Cellular Polyamines

Background: Antizyme is a regulator of cell proliferation, inhibiting this process when overexpressed. Results: Antizyme overexpression does not attenuate cell proliferation and viability in cells whose polyamine supply is secured. Conclusion: Antizyme affects cell proliferation and viability only by modulating polyamine metabolism. Significance: This result emphasizes the functional relationship of antizyme to cellular polyamine metabolism. Antizymes are key regulators of cellular polyamine metabolism that negatively regulate cell proliferation and are therefore regarded as tumor suppressors. Although the regulation of antizyme (Az) synthesis by polyamines and the ability of Az to regulate cellular polyamine levels suggest the centrality of polyamine metabolism to its antiproliferative function, recent studies have suggested that antizymes might also regulate cell proliferation by targeting to degradation proteins that do not belong to the cellular polyamine metabolic pathway. Using a co-degradation assay, we show here that, although they efficiently stimulated the degradation of ornithine decarboxylase (ODC), Az1 and Az2 did not affect or had a negligible effect on the degradation of cyclin D1, Aurora-A, and a p73 variant lacking the N-terminal transactivation domain whose degradation was reported recently to be stimulated by Az1. Furthermore, we demonstrate that, although Az1 and Az2 could not be constitutively expressed in transfected cells, they could be stably expressed in cells that express trypanosome ODC, a form of ODC that does not bind Az and therefore maintains a constant level of cellular polyamines. Taken together, our results clearly demonstrate that Az1 and Az2 affect cell proliferation and viability solely by modulating cellular polyamine metabolism.

Yeast Antizyme Mediates Degradation of Yeast Ornithine Decarboxylase by Yeast but Not by Mammalian Proteasome NEW INSIGHTS ON YEAST ANTIZYME

Mammalian antizyme (mAz) is a central element of a feedback circuit regulating cellular polyamines by accelerating ornithine decarboxylase (ODC) degradation and inhibiting polyamine uptake. Although yeast antizyme (yAz) stimulates the degradation of yeast ODC (yODC), we show here that it has only a minor effect on polyamine uptake by yeast cells. A segment of yODC that parallels the Az binding segment of mammalian ODC (mODC) is required for its binding to yAz. Although demonstrating minimal homology to mAz, our results suggest that yAz stimulates yODC degradation via a similar mechanism of action. We demonstrate that interaction with yAz provokes degradation of yODC by yeast but not by mammalian proteasomes. This differential recognition may serve as a tool for investigating proteasome functions.

Dual Regulatory Role of Polyamines in Adipogenesis.

Background: Polyamines are required for the process of adipogenesis. Results: Polyamine depletion causes inactivation of C/EBPβ that is correlated with elevation of CHOP, and independently causes inhibition of mitotic clonal expansion. Conclusion: Polyamines are required for both main hubs of adipogenesis; completion of MCE and activation of C/EBPβ. Significance: Our results assist clarifying the mechanism by which polyamines support adipogenesis. Adipogenesis is a complex process, accompanied by a chain of interdependent events. Disruption of key events in this cascade may interfere with the correct formation of adipose tissue. Polyamines were demonstrated necessary for adipogenesis; however, the underlying mechanism by which they act has not been established. Here, we examined the effect of polyamine depletion on the differentiation of 3T3-L1 preadipocytes. Our results demonstrate that polyamines are required early in the adipogenic process. Polyamine depletion inhibited the second division of the mitotic clonal expansion (MCE), and inhibited the expression of PPARγ and C/EBPα, the master regulators of adipogenesis. However, it did not affect the expression of their transcriptional activator, C/EBPβ. Additionally, polyamine depletion resulted in elevation of mRNA and protein levels of the stress-induced C/EBP homologous protein (CHOP), whose dominant negative function is known to inhibit C/EBPβ DNA binding activity. Conditional knockdown of CHOP in polyamine-depleted preadipocytes restored PPARγ and C/EBPα expression, but failed to recover MCE and differentiation. Thus, our results suggest that the need for MCE in the adipogenic process is independent from the requirement for PPARγ and C/EBPα expression. We conclude that de novo synthesis of polyamines during adipogenesis is required for down-regulation of CHOP to allow C/EBPβ activation, and for promoting MCE.