Role of antizyme in degradation of ornithine decarboxylase in HTC cells.
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ABSTRACT:
SUBMITTER: Murakami Y
PROVIDER: S-EPMC1144794 | biostudies-other | 1985 Mar
REPOSITORIES: biostudies-other
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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
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
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
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
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
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
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
Project description:A new method was developed for the assay of ornithine decarboxylase (ODC)-antizyme complex, in which alpha-difluoromethylornithine (DFMO)-inactivated ODC was used to release active ODC competitively from the complex. ODC-antizyme complex was present in the extracts of hepatoma tissue-culture (HTC) cells and of ODC-stabilized variant HMOA cells, in much larger amounts in the latter. Cellular amounts of the complex fluctuated after a change of medium in a similar manner in HTC and HMOA cells, increasing during the period of ODC decay. After treatment with cycloheximide, the decay of ODC-antizyme complex in HMOA cells was more rapid than the decay of free ODC, but it was much slower than the decay of free ODC or complexed ODC in HTC cells. Administration of putrescine caused a rapid increase in the amount of ODC-antizyme complex in both HTC and HMOA cells, but nevertheless the decay of total ODC (free ODC plus ODC-antizyme complex) was more rapid with putrescine than with cycloheximide. These results suggested the possibility that ODC is degraded through complex-formation with antizyme. In contrast with complexed antizyme, free antizyme was not stabilized in HMOA cells.
| S-EPMC1144645 | biostudies-other
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
Project description:Ornithine decarboxylase isolated from HTC cells was separated into two distinct charged states by salt-gradient elution from DEAE-Sepharose columns. This charge difference between the enzyme forms was maintained in partially purified preparations, but enzyme form II was observed to change to form I in a time-dependent polyamine-stimulated fashion in crude cell homogenates. The enzyme modification that produces this charge diversity between the alternative enzyme states was further investigated for its role in enzyme activity induction, protein stability and rapid turnover. Inhibition of new protein synthesis by cycloheximide resulted in a much more rapid loss of form I enzyme than of form II, suggesting that during normal enzyme turnover the latter enzyme state may be derived from the former. Culture conditions that favour the stabilization of this usually labile enzyme generally induced an increased proportion of the enzyme in the form II charge state. In particular, inhibitors of synthesis of spermidine and spermine induced the stabilization of cellular ornithine decarboxylase and promoted a marked accumulation in form II. Conversely, polyamines added to the cells in culture induced a very rapid loss in both forms of the enzyme, an effect that could not be attributed merely to an inhibition of new enzyme synthesis. It appears that the polyamines, but not putrescine, may be an essential part of the rapid ornithine decarboxylase inactivation process and that they may function in part by stimulating the conversion of the more stable enzyme form II into the less stable enzyme state, form I.
| S-EPMC1144987 | biostudies-other