Multiple active conformers of mouse ornithine decarboxylase.
Ontology highlight
ABSTRACT:
SUBMITTER: Tsirka SE
PROVIDER: S-EPMC1134353 | biostudies-other | 1993 Jul
REPOSITORIES: biostudies-other
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Project description:We have investigated and characterized a novel ornithine decarboxylase (ODC) related protein (ODCrp) also annotated as gm853. ODCrp shows 41% amino acid sequence identity with ODC and 38% with ODC antizyme inhibitor 1 (AZIN1). The Odcrp gene is selectively expressed in the epithelium of proximal tubuli of mouse kidney with higher expression in males than in females. Like Odc in mouse kidney, Odcrp is also androgen responsive with androgen receptor (AR)-binding loci within its regulatory region. ODCrp forms homodimers but does not heterodimerize with ODC. Although ODCrp contains 20 amino acid residues known to be necessary for the catalytic activity of ODC, no decarboxylase activity could be found with ornithine, lysine or arginine as substrates. ODCrp does not function as an AZIN, as it neither binds ODC antizyme 1 (OAZ1) nor prevents OAZ-mediated inactivation and degradation of ODC. ODCrp itself is degraded via ubiquination and mutation of Cys363 (corresponding to Cys360 of ODC) appears to destabilize the protein. Evidence for a function of ODCrp was found in ODC assays on lysates from transfected Cos-7 cells where ODCrp repressed the activity of endogenous ODC while Cys363Ala mutated ODCrp increased the enzymatic activity of endogenous ODC.
| S-EPMC5518511 | biostudies-literature
Project description:The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating prostatic bud induction, and are required for the expression of a subset of prostatic developmental regulatory genes including Notch1 and Nkx3.1.
| S-EPMC4591331 | 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:We have isolated from mouse FM3A cells a variant cell line, termed EXOD-1, that overproduces ornithine decarboxylase (ODC). The cells were resistant to alpha-difluoromethylornithine, an irreversible inhibitor of the enzyme, and produced the enzyme protein to the extent of approx. 3-6% of total cytosolic protein. The rate of ODC synthesis in this cell line accounted for 25-50% of the rate of total protein synthesis. The amounts of the ODC gene and its mRNA in the variant cells were both about 60 times as much as those in wild-type FM3A cells. Upon removal of the inhibitor, the growth of the ODC-overproducing cells was stimulated approx. 2-fold. Under these conditions, the rate of ODC synthesis increased about 4-fold on day 1 and then decreased to near the original level by day 3. The amount of ODC mRNA increased about 1.7-fold on day 1 and 2.5-fold on day 3. No correlation was observed between changes in ODC synthesis rate and in ODC mRNA content, suggesting a translational repression of ODC mRNA due to accumulation of polyamines. In fact, the cellular contents of putrescine and spermidine markedly increased and that of spermine inversely decreased during the same period. Pulse-chase experiments showed that the accumulation of putrescine and spermidine also elicited a rapid degradation of ODC. Excess amounts of newly synthesized putrescine and cadaverine were excreted into the medium, whereas spermidine, spermine and acetylated polyamines were undetectable there. We conclude that ODC regulation upon removal of the inhibitor is dependent on at least three steps, namely the level of mRNA, the translational efficiency of mRNA and the stability of the enzyme, the last two of which are involved in cellular polyamines.
| S-EPMC1132208 | biostudies-other
Project description:Microgram quantities of ornithine decarboxylase (ODC, EC 4.1.1.17)-specific mRNA were synthesized by transcription techniques in vitro, by using a plasmid containing mouse cDNA coding for this enzyme. The homogeneous RNA preparation was then used for cell-free synthesis of ODC protein, in rabbit reticulocyte lysates. Analysis of products translated in vitro by polyacrylamide-gel electrophoresis revealed predominantly one protein produced, with Mr approx. 54,000, which was immunoprecipitable by anti-ODC serum. Two-dimensional gel-electrophoretic analysis showed that the protein ODC synthesized in vitro had a pI of approx. 5.4, similar to the native enzyme isolated from mouse tissues. In addition, quantification of activity and protein amount showed that the enzyme synthesized in vitro had a specific activity of approx. 63,000 units (nmol/min)/mg, consistent with the purified mouse kidney enzyme's specific activity of approx. 47,000 units/mg. An average of nearly 200 pg of ODC protein was produced in vitro from various RNA preparations. These data demonstrate that ODC-specific mRNA and active ODC protein can be produced by 'in vitro' technology, which should prove useful in studying functional and structural characteristics of these molecules.
| S-EPMC1148090 | biostudies-other
Project description:BACKGROUND: The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme. METHODOLOGY AND RESULTS: To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC)-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF) of approximately 1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa) from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size approximately 46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids-aspartate-332, aspartate-361, and tyrosine-323-by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity. CONCLUSION: To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from E. histolytica. Computer modeling revealed that three of the critical residues required for binding of DFMO to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO.
| S-EPMC2217671 | biostudies-literature
Project description:Ornithine Decarboxylase (ODC) a key enzyme in polyamine biosynthesis is often overexpressed in cancers and contributes to polyamine-induced cell proliferation. We noted ubiquitous expression of ODC1 in our published endometrial cancer gene array data and confirmed this in the cancer genome atlas (TCGA) with highest expression in non-endometrioid, high grade, and copy number high cancers, which have the worst clinical outcomes. ODC1 expression was associated with worse overall survival and increased recurrence in three endometrial cancer gene expression datasets. Importantly, we confirmed these findings using quantitative real-time polymerase chain reaction (qRT-PCR) in a validation cohort of 60 endometrial cancers and found that endometrial cancers with elevated ODC1 had significantly shorter recurrence-free intervals (KM log-rank p = 0.0312, Wald test p = 5.59e-05). Difluoromethylornithine (DFMO) a specific inhibitor of ODC significantly reduced cell proliferation, cell viability, and colony formation in cell line models derived from undifferentiated, endometrioid, serous, carcinosarcoma (mixed mesodermal tumor; MMT) and clear cell endometrial cancers. DFMO also significantly reduced human endometrial cancer ACI-98 tumor burden in mice compared to controls (p = 0.0023). ODC-regulated polyamines (putrescine [Put] and/or spermidine [Spd]) known activators of cell proliferation were strongly decreased in response to DFMO, in both tumor tissue ([Put] (p = 0.0006), [Spd] (p<0.0001)) and blood plasma ([Put] (p<0.0001), [Spd] (p = 0.0049)) of treated mice. Our study indicates that some endometrial cancers appear particularly sensitive to DFMO and that the polyamine pathway in endometrial cancers in general and specifically those most likely to suffer adverse clinical outcomes could be targeted for effective treatment, chemoprevention or chemoprevention of recurrence.
| S-EPMC5730160 | biostudies-literature
Project description:Ornithine decarboxylase (ODC) of the fungus Neurospora crassa, encoded by the spe-1 gene, catalyzes an initial and rate-limiting step in polyamine biosynthesis and is highly regulated by polyamines. In N. crassa, polyamines repress the synthesis and increase the degradation of ODC protein. Changes in the rate of ODC synthesis correlate with similar changes in the abundance of spe-1 mRNA. We identify two sequence elements, one in each of the 5' and 3' regions of the spe-1 gene of N. crassa, required for this polyamine-mediated regulation. A 5' polyamine-responsive region (5' PRR) comprises DNA sequences both in the upstream untranscribed region and in the long 5' untranslated region (5'-UTR) of the gene. The 5' PRR is sufficient to confer polyamine regulation to a downstream, heterologous coding region. Use of the beta-tubulin promoter to drive the expression of various portions of the spe-1 transcribed region revealed a 3' polyamine-responsive region (3' PRR) downstream of the coding region. Neither changes in cellular polyamine status nor deletion of sequences in the 5'-UTR alters the half-life of spe-1 mRNA. Sequences in the spe-1 5'-UTR also impede the translation of a heterologous coding region, and polyamine starvation partially relieves this impediment. The results show that N. crassa uses a unique combination of polyamine-mediated transcriptional and translational control mechanisms to regulate ODC synthesis.
| S-EPMC85492 | biostudies-literature
Project description:Ornithine decarboxylase (ODC) catalyzes ornithine decarboxylation to yield putrescine, a key precursor of polyamines, and tropane alkaloids (TAs). Here, to investigate in depth the role of ODC in polyamine/TA biosynthesis and to provide a candidate gene for engineering polyamine/TA production, the ODC gene (HnODC) was characterized from Hyoscyamus niger, a TA-producing plant. Our phylogenetic analysis revealed that HnODC was clustered with ODC enzymes of plants. Experimental work showed HnODC highly expressed in H. niger roots and induced by methyl jasmonate (MeJA). In the MeJA treatment, the production of both putrescine and N-methylputrescine were markedly promoted in roots, while contents of putrescine, spermidine, and spermine were all significantly increased in leaves. By contrast, MeJA did not significantly change the production of either hyoscyamine or scopolamine in H. niger plants. Building on these results, the 50-kDa His-tagged HnODC proteins were purified for enzymatic assays. When ornithine was fed to HnODC, the putrescine product was detected by HPLC, indicating HnODC catalyzed ornithine to form putrescine. Finally, we also investigated the enzymatic kinetics of HnODC. Its K m, V max, and K cat values for ornithine were respectively 2.62 ± 0.11 mM, 1.87 ± 0.023 nmol min-1 μg-1 and 1.57 ± 0.015 s-1, at pH 8.0 and at 30°C. The HnODC enzyme displays a much higher catalytic efficiency than most reported plant ODCs, suggesting it may be an ideal candidate gene for engineering polyamine/TA biosynthesis.
| S-EPMC6400997 | biostudies-literature
Project description:Neuroblastoma is a pediatric malignancy that arises from the neural crest, and patients with high-risk neuroblastoma, which typically harbor amplifications of MYCN, have an extremely poor prognosis. The tyrosine hydroxylase (TH) promoter-driven TH-MYCN transgenic mouse model faithfully recapitulates many hallmarks of human MYCN-amplified neuroblastoma. A key downstream target of Myc oncoproteins in tumorigenesis is ornithine decarboxylase (Odc), the rate-limiting enzyme of polyamine biosynthesis. Indeed, sustained treatment with the Odc suicide inhibitor alpha-difluoromethylornithine (DFMO) or Odc heterozygosity markedly impairs lymphoma development in Emicro-Myc transgenic mice, and these effects are linked to the induction of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1), which is normally repressed by Myc. Here, we report that DFMO treatment, but not Odc heterozygosity, impairs MYCN-induced neuroblastoma and that, in this malignancy, transient DFMO treatment is sufficient to confer protection. The selective anticancer effects of DFMO on mouse and human MYCN-amplified neuroblastoma also rely on its ability to disable the proliferative response of Myc, yet in this tumor context, DFMO targets the expression of the p21(Cip1) Cdk inhibitor, which is also suppressed by Myc oncoproteins. These findings suggest that agents, such as DFMO, that target the polyamine pathway may show efficacy in high-risk, MYCN-amplified neuroblastoma.
| S-EPMC2749594 | biostudies-literature