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Overproduction of stable ornithine decarboxylase and antizyme in the difluoromethylornithine-resistant cell line DH23b.

ABSTRACT: DH23b cells, a variant of the HTC line selected for their resistance to difluoromethylornithine, exhibit defective feedback regulation of ornithine decarboxylase (ODC) stability and polyamine transport, and accumulate ODC protein to > 1000 times normal concentrations. The components of the polyamine feedback regulation system have been examined in an attempt to understand these unusual responses. Southern-blot analysis revealed an amplification (approx. 10-fold) in ODC DNA sequence without any concomitant increase in antizyme. Moreover, the amplified ODC sequence contains a single base substitution that results in the conversion of Cys-441 into Trp. This modification has previously been shown to cause ODC stability in HMOA cells. Although antizyme activity has not been noted in DH23b cells, Western-blot analysis revealed the accumulation of antizyme protein to > 50 times that induced in parental HTC cells. This increase is consistent with a 6-9-fold increase in the half-life of antizyme in these cells, a consequence of the inability of the mutant ODC-antizyme complex to be degraded by 26 S proteasome. Associated with the stabilization of antizyme in both DH23b and HMOA cells is the appearance of two additional forms of antizyme protein with apparent molecular masses of 22 and 18.5 kDa. It is suggested that these result from proteolytic removal of discrete fragments from the N-terminal end of antizyme, perhaps an indication of an initial step in rapid antizyme turnover.

SUBMITTER: Mitchell JL

PROVIDER: S-EPMC1217557 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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Discovery of a spermatogenesis stage-specific ornithine decarboxylase antizyme: antizyme 3.

Project description:Previous studies with mice overproducing ornithine decarboxylase have demonstrated the importance of polyamine homeostasis for normal mammalian spermatogenesis. The present study introduces a likely key player in the maintenance of proper polyamine homeostasis during spermatogenesis. Antizyme 3 is a paralog of mammalian ornithine decarboxylase antizymes. Like its previously described counterparts, antizymes 1 and 2, it inhibits ornithine decarboxylase, which catalyzes the synthesis of putrescine. Earlier work has shown that the coding sequences for antizymes 1 and 2 are in two different, partially overlapping reading frames. Ribosomes translate the first reading frame, and just before the stop codon for that frame, they shift to the second reading frame to synthesize a trans-frame product. The efficiency of this frameshifting depends on polyamine concentration, creating an autoregulatory circuit. Antizyme 3 cDNA has the same arrangement of reading frames and a potential shift site with definite, although limited, homology to its evolutionarily distant antizyme 1 and 2 counterparts. In contrast to antizymes 1 and 2, which are widely expressed throughout the body, antizyme 3 transcription is restricted to testis germ cells. Expression starts early in spermiogenesis and finishes in the late spermatid phase. The potential significance of antizyme 3 expression during spermatogenesis is discussed in this paper.

| S-EPMC18314 | biostudies-literature

Zinc is required for the expression of ornithine decarboxylase in a difluoromethylornithine-resistant cell line.

Project description:Dilution of quiescent L1210-DFMOr (difluoromethylornithine-resistant) cells in fresh medium containing serum led to the induction of ornithine decarboxylase (ODC) and to the expression of its mRNA, as determined by a sensitive solution-hybridization-RNase-protection assay. Addition of the chelating agent diethylenetriaminepentaacetic acid (DTPA) at seeding time caused an inhibition of the induction of ODC activity by up to 90%, and only Zn2+ of the bivalent metal ions tested was effective in reversing this effect. The inhibition of the induction of ODC activity was accompanied by a marked decrease, prevented by Zn2+ supplementation, of the accumulation of immunoreactive ODC protein and ODC mRNA. DTPA treatment also caused a slight acceleration of ODC turnover. These results indicate that a restricted Zn2+ availability in L1210-DFMOr cells impairs ODC induction remarkably, mainly by affecting the expression of the messenger.

| S-EPMC1138301 | biostudies-other

Ornithine decarboxylase antizyme Oaz3 modulates protein phosphatase activity.

Project description:Ornithine decarboxylase antizyme 3 (Oaz3) is expressed in spermatids, makes up the antizyme family of Oaz genes with Oaz1 and Oaz2, and was proposed to encode a 22 kDa antizyme protein involved in polyamine regulation similar to the 22 kDa OAZ1 and OAZ2 proteins. Here we demonstrate however that the major product encoded by Oaz3 is a 12 kDa protein, p12, which lacks the antizyme domain that interacts with ornithine decarboxylase. We show that p12 does not affect ornithine decarboxylase levels, providing an explanation for the surprising observation made in Oaz3 knock-out male mice, which do not display altered testis polyamine metabolism. This suggested a novel activity for Oaz3 p12. Using immuno-electron microscopy we localized p12 to two structures in the mammalian sperm tail, viz. the outer dense fibers and fibrous sheath, as well as to the connecting piece linking head and tail. We identified myosin phosphatase targeting subunit 3 (MYPT3), a regulator of protein phosphatase PP1β, as a major p12-interacting protein, and show that MYPT3 is present in sperm tails and that its ankyrin repeat binds p12. We show that MYPT3 can also bind protein phosphatase PP1γ2, the only protein phosphatase present in sperm tails, and that p12- MYPT3 interaction modulates the activity of both PP1β and PP1γ2. This is, to our knowledge, the first demonstration of a novel activity for an Oaz-encoded protein.

| S-EPMC3156769 | biostudies-literature

Knocking out Ornithine Decarboxylase Antizyme 1 (<i>OAZ1</i>) Improves Recombinant Protein Expression in the HEK293 Cell Line.

Project description:Creating efficient cell lines is a priority for the biopharmaceutical industry, which produces biologicals for various uses. A recent approach to achieving this goal is the use of non-coding RNAs, microRNA (miRNA) and small interfering RNA (siRNA), to identify key genes that can potentially improve production or growth. The ornithine decarboxylase antizyme 1 (OAZ1) gene, a negative regulator of polyamine biosynthesis, was identified in a genome-wide siRNA screen as a potential engineering target, because its knock down by siRNA increased recombinant protein expression from human embryonic kidney 293 (HEK293) cells by two-fold. To investigate this further, the OAZ1 gene in HEK293 cells was knocked out using CRISPR genome editing. The OAZ1 knockout cell lines displayed up to four-fold higher expression of both stably and transiently expressed proteins, with comparable growth and metabolic activity to the parental cell line; and an approximately three-fold increase in intracellular polyamine content. The results indicate that genetic inactivation of OAZ1 in HEK293 cells is an effective strategy to improve recombinant protein expression in HEK293 cells.

| S-EPMC6024716 | biostudies-literature

Determinants of the differential antizyme-binding affinity of ornithine decarboxylase.

Project description:Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The K(d) value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 ?M, while the K(d) value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 ?M. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the K(d) was 1.4 and 1.2 ?M, respectively. These affinities are 6-fold higher than the WT_ODC K(d), which suggests that residues 119 and 137 play a role in AZ binding.

| S-EPMC3207831 | biostudies-literature

Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2.

Project description:Ornithine decarboxylase (ODC) is the key enzyme in the polyamine biosynthetic pathway. ODC levels are controlled by polyamines through the induction of antizymes (AZs), small proteins that inhibit ODC and target it to proteasomal degradation without ubiquitination. Antizyme inhibitors (AZIN1 and AZIN2) are proteins homologous to ODC that bind to AZs and counteract their negative effect on ODC. Whereas ODC and AZIN1 are well-characterized proteins, little is known on the structure and stability of AZIN2, the lastly discovered member of this regulatory circuit. In this work we first analyzed structural aspects of AZIN2 by combining biochemical and computational approaches. We demonstrated that AZIN2, in contrast to ODC, does not form homodimers, although the predicted tertiary structure of the AZIN2 monomer was similar to that of ODC. Furthermore, we identified conserved residues in the antizyme-binding element, whose substitution drastically affected the capacity of AZIN2 to bind AZ1. On the other hand, we also found that AZIN2 is much more labile than ODC, but it is highly stabilized by its binding to AZs. Interestingly, the administration of the proteasome inhibitor MG132 caused differential effects on the three AZ-binding proteins, having no effect on ODC, preventing the degradation of AZIN1, but unexpectedly increasing the degradation of AZIN2. Inhibitors of the lysosomal function partially prevented the effect of MG132 on AZIN2. These results suggest that the degradation of AZIN2 could be also mediated by an alternative route to that of proteasome. These findings provide new relevant information on this unique regulatory mechanism of polyamine metabolism.

| S-EPMC4066113 | biostudies-literature

Critical factors governing the difference in antizyme-binding affinities between human ornithine decarboxylase and antizyme inhibitor.

Project description:Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC(50) value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K(d)) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K(d) value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K(d) value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the K(d) value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities.

| S-EPMC3084279 | biostudies-literature

Minimal antizyme peptide fully functioning in the binding and inhibition of ornithine decarboxylase and antizyme inhibitor.

Project description:Antizyme (AZ) is a protein with 228 amino acid residues that regulates ornithine decarboxylase (ODC) by binding to ODC and dissociating its homodimer, thus inhibiting its enzyme activity. Antizyme inhibitor (AZI) is homologous to ODC, but has a higher affinity than ODC for AZ. In this study, we quantified the biomolecular interactions between AZ and ODC as well as AZ and AZI to identify functional AZ peptides that could bind to ODC and AZI and inhibit their function as efficiently as the full-length AZ protein. For these AZ peptides, the inhibitory ability of AZ_95-228 was similar to that of AZ_WT. Furthermore, AZ_95-176 displayed an inhibition (IC(50): 0.20 µM) similar to that of AZ-95-228 (IC(50): 0.16 µM), even though a large segment spanning residues 177-228 was deleted. However, further deletion of AZ_95-176 from either the N-terminus or the C-terminus decreased its ability to inhibit ODC. The AZ_100-176 and AZ_95-169 peptides displayed a noteworthy decrease in ability to inhibit ODC, with IC(50) values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC(50) values comparable to that of AZ_WT and formed AZ-ODC complexes with K(d,AZ-ODC) values of 1.5, 5.3 and 5.6 µM, respectively. Importantly, our data also indicate that AZI can rescue AZ peptide-inhibited ODC enzyme activity and that it can bind to AZ peptides with a higher affinity than ODC. Together, these data suggest that these truncated AZ proteins retain their AZI-binding ability. Thus, we suggest that AZ_95-176 is the minimal AZ peptide that is fully functioning in the binding of ODC and AZI and inhibition of their function.

| S-EPMC3170320 | biostudies-literature

Polyamines directly promote antizyme-mediated degradation of ornithine decarboxylase by the proteasome.

Project description:Ornithine decarboxylase (ODC), a ubiquitin-independent substrate of the proteasome, is a homodimeric protein with a rate-limiting function in polyamine biosynthesis. Polyamines regulate ODC levels by a feedback mechanism mediated by ODC antizyme (OAZ). Higher cellular polyamine levels trigger the synthesis of OAZ and also inhibit its ubiquitin-dependent proteasomal degradation. OAZ binds ODC monomers and targets them to the proteasome. Here, we report that polyamines, aside from their role in the control of OAZ synthesis and stability, directly enhance OAZ-mediated ODC degradation by the proteasome. Using a stable mutant of OAZ, we show that polyamines promote ODC degradation in Saccharomyces cerevisiae cells even when OAZ levels are not changed. Furthermore, polyamines stimulated the in vitro degradation of ODC by the proteasome in a reconstituted system using purified components. In these assays, spermine shows a greater effect than spermidine. By contrast, polyamines do not have any stimulatory effect on the degradation of ubiquitin-dependent substrates.

| S-EPMC5349141 | biostudies-literature

Brain neurons express ornithine decarboxylase-activating antizyme inhibitor 2 with accumulation in Alzheimer's disease.

Project description:Polyamines are small cationic molecules that in adult brain are connected to neuronal signaling by regulating inward-rectifier K(+)-channels and different glutamate receptors. Antizyme inhibitors (AZINs) regulate the cellular uptake of polyamines and activate ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis. Elevated levels of ODC activity and polyamines are detected in various brain disorders including stroke and Alzheimer's disease (AD). We originally reported a novel brain- and testis-specific AZIN, called AZIN2, the distribution of which we have now studied in normal and diseased human brain by in situ hybridization and immunohistochemistry. We found the highest accumulation of AZIN2 in a pearl-on-the-string-like distribution along the axons in both the white and gray matter. AZIN2 was also detected in a vesicle-like distribution in the somas of selected cortical pyramidal neurons. Double-immunofluorescence staining revealed co-localization of AZIN2 and N-methyl D-aspartate-type glutamate receptors (NMDARs) in pyramidal neurons of the cortex. Moreover, we found accumulation of AZIN2 in brains affected by AD, but not by other neurodegenerative disorders (CADASIL or Lewy body disease). ODC activity is mostly linked to cell proliferation, whereas its regulation by AZIN2 in post-mitotically differentiated neurons of the brain apparently serves different purposes. The subcellular distribution of AZIN2 suggests a role in vesicular trafficking.

| S-EPMC8094758 | biostudies-literature

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