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P Coffino

Publications and source records attributed to P Coffino.

At least 19 recordsLinked to original sources
PubMed1992

Dominant negative mutants of ornithine decarboxylase.

Conserved lysines of mouse ornithine decarboxylase were individually mutated to arginines. The mutations at amino acid residues 69, 115, and 169 greatly reduced or abolished enzymatic activity. Lysine 69 is the site of Schiff base formation with the cofactor pyridoxal phosphate; the functional role of the other two lysines essential for activity is not known. Coexpression of wild type ornithine decarboxylase along with the lysine 115 to arginine mutant reduced the activity of the former without diminishing the amount of wild type protein. This form of negative complementation was seen when wild type and mutant protein were coexpressed either by in vitro translation or in bacteria. The data are consistent with the conclusion that a wild type and mutant subunit form a heterodimer that is enzymatically inactive.

Amino Acid Sequence↗
PubMed1992

Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation.

Mammalian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is rapidly degraded in cells, an attribute important to the regulation of its activity. Mutant and chimeric ODCs were created to determine the structural requirements for two modes of proteolysis. Constitutive degradation requires the carboxy terminus and is independent of intracellular polyamines. Truncation of five or more carboxy-terminal amino acids prevents this mode of degradation, as do several internal deletions within the 37 carboxy-most amino acids that spare the last five residues. Polyamine-dependent degradation of ODC requires a distinct region outside the carboxy terminus. The ODC of a parasite, Trypanosoma brucei, is structurally very similar to mouse ODC but lacks the carboxy-terminal domain; it is not a substrate for either pathway. The regulatory properties of enzymatically active chimeric proteins incorporating regions of the two ODCs support the conclusion that distinct domains of mouse ODC confer constitutive degradation and polyamine-mediated regulation. Mouse ODC contains two PEST regions. The first was not required for either form of degradation; major deletions within the second ablated constitutive degradation. When mouse and T. brucei ODC RNAs were translated in vitro in a reticulocyte lysate system, the effects of polyamine concentration on ODC protein production and activity were similar for the two mRNAs, which contradicts claims that this system accurately reflects the in vivo effects of polyamines on responsive ODCs.

Animals↗
PubMed1992

Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme.

Intracellular degradation of vertebrate ornithine decarboxylase (ODC) is accelerated by polyamines, the products of the pathway controlled by ODC. Antizyme, a reversible, tightly binding protein inhibitor of ODC activity, is believed to be involved in this process. Mouse and Trypanosoma brucei ODCs are structurally similar, but the trypanosome enzyme, unlike that of the mouse, is not regulated by intracellular polyamines when expressed in hamster cells (L. Ghoda, D. Sidney, M. Macrae, and P. Coffino, Mol. Cell. Biol. 12:2178-2185, 1992). We found that mouse ODC interacts with antizyme in vitro but trypanosome ODC does not. To localize the region necessary for binding, we made a series of enzymatically active chimeric mouse-trypanosome ODCs and tested them for antizyme interaction. Replacing residues 117 to 140 within the 461-amino-acid mouse ODC sequence with the equivalent region of trypanosome ODC disrupted both antizyme binding and in vivo regulation. Formation of an antizyme-ODC complex is therefore required for regulated degradation.

Amino Acid Sequence↗
PubMed1992

Sequestered end products and enzyme regulation: the case of ornithine decarboxylase.

The polyamines (putrescine, spermidine, and spermine) are synthesized by almost all organisms and are universally required for normal growth. Ornithine decarboxylase (ODC), an initial enzyme of polyamine synthesis, is one of the most highly regulated enzymes of eucaryotic organisms. Unusual mechanisms have evolved to control ODC, including rapid, polyamine-mediated turnover of the enzyme and control of the synthetic rate of the protein without change of its mRNA level. The high amplitude of regulation and the rapid variation in the level of the protein led biochemists to infer that polyamines had special cellular roles and that cells maintained polyamine concentrations within narrow limits. This view was sustained in part because of our continuing uncertainty about the actual biochemical roles of polyamines. In this article, we challenge the view that ODC regulation is related to precise adjustment of polyamine levels. In no organism does ODC display allosteric feedback inhibition, and in three types of organism, bacteria, fungi, and mammals, the size of polyamine pools may vary radically without having a profound effect on growth. We suggest that the apparent stability of polyamine pools in unstressed cells is due to their being largely bound to cellular polyanions. We further speculate that allosteric feedback inhibition, if it existed, would be inappropriately responsive to changes in the small, freely diffusible polyamine pool. Instead, mechanisms that control the amount of the ODC protein have appeared in most organisms, and even these are triggered inappropriately by variation of the binding of polyamines to ionic binding sites. In fact, feedback inhibition of ODC might be maladaptive during hypoosmotic stress or at the onset of growth, when organisms appear to require rapid increases in the size of their cellular polyamine pools.

Animals↗
PubMed1992

Role of ornithine decarboxylase suppression and polyamine depletion in the antiproliferative activity of polyamine analogs.

Two transfected cell lines, one carrying a mammalian ornithine decarboxylase (ODC) that is suppressed by polyamines and one carrying a trypanosomal ODC that is not, were used to ask whether ODC suppression is necessary for the antiproliferative activities of two polyamine analogs, N1,N8-bis(ethyl)spermidine (BES) and N1,N14-bis(ethyl)homospermine (BE444). Both analogs accumulated within cells and suppressed S-adenosylmethionine decarboxylase, as well as polyamine-sensitive mouse ODC activity. Neither drug was able to suppress the activity of the polyamine-refractory trypanosome ODC. But, whereas BE444 was able to inhibit growth of both cell lines, BES could inhibit only growth of cells carrying the polyamine-sensitive ODC, under conditions that cause prolonged depletion of endogenous polyamines. We conclude from these studies that the antiproliferative activity of BES, a less potent drug, requires the suppression of ODC. The efficacy of BE444 is enhanced by its ability to suppress ODC. However, it can function without ODC suppression, whereas BES cannot.

Adenosylmethionine Decarboxylase↗
PubMed1991

Expression and post-transcriptional regulation of ornithine decarboxylase during early Xenopus development.

In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid-blastula transition (MBT). ODC activity was stable in cycloheximide-treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post-MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC-deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post-translationally by polyamines, indicating that the post-MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine-mediated regulation.

Animals↗
PubMed1991

Killer polyamines?

Mammalian cells can rapidly make large changes in their rate of polyamine biosynthesis in response to mitogenic and trophic signals. However, cultured cells seem to grow adequately as long as they are supplied a steady but unregulated supply of polyamines. This implies that complex and rapid changes in polyamine synthesis serve a function in a special rather than a general biological context. We suggest that the appropriate context in which regulation of polyamines mediates crucial functions is the mammalian embryo and that one function of polyamines is to act as substrate in an oxidative pathway that arbitrates programmed cell death.

Animals↗
PubMed1990

Trypanosome ornithine decarboxylase is stable because it lacks sequences found in the carboxyl terminus of the mouse enzyme which target the latter for intracellular degradation.

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Mouse ODC is rapidly degraded in mouse cells, whereas ODC within Trypanosoma brucei, a protozoan parasite infesting cattle, is stable. We have expressed cloned ODC genes of both T. brucei and mouse in ODC-deficient Chinese hamster ovary (CHO) cells. The T. brucei enzyme is stable, whereas the mouse ODC similarly expressed in CHO cells is unstable. This shows that the observed difference in intracellular stability is a property of the ODC protein itself, rather than the cellular environment in which it is expressed. A chimeric ODC composed of the amino terminus of trypanosome and the carboxyl terminus of mouse ODC is rapidly degraded in CHO cells, suggesting that peptide sequences in the mouse ODC carboxyl terminus determine its stability.

Amino Acid Sequence↗
PubMed1989

Regulation of mouse ornithine decarboxylase activity by cell growth, serum and tetradecanoyl phorbol acetate is governed primarily by sequences within the coding region of the gene.

To determine the genetic elements required for modulation of ornithine decarboxylase (ODC) activity in response to cell growth or treatment with serum or with tetradecanoyl phorbol acetate, ODC-deficient cells were transfected with a series of recombinant DNAs encoding mouse ODC. All of the transfected cells expressing an intact mouse ODC protein displayed regulation of ODC activity, including those expressing a construct deprived of all ODC-specific sequence information except the protein-coding region. ODC mRNA changed much less than enzymatic activity. A mutation of the protein-coding region that converted ODC from an unstable to a stable intracellular protein attenuated the regulatory response. We conclude that post-transcriptional events associated with ODC degradation dominate the response to these stimuli.

Animals↗
PubMed1989

Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation.

Ornithine decarboxylase (ODC) was converted from a protein with a short intracellular half-life in mammalian cells to a stable protein by truncating 37 residues at its carboxyl terminus. Cells expressing wild-type protein lost ODC activity with a half-life of approximately 1 hour. Cells expressing the truncated protein, however, retained full activity for at least 4 hours. Pulse-chase experiments in which immunoprecipitation and gel electrophoresis were used confirmed the stabilizing effect of the truncation. Thus, a carboxyl-terminal domain is responsible for the rapid intracellular degradation of murine ODC.

Animals↗
PubMed1989

Linkage genetics of mouse ornithine decarboxylase (Odc).

Ornithine decarboxylase (ODC) is synthesized as a single polypeptide in the mouse, but DNA sequences reactive with ODC cDNA probes have been mapped to multiple mouse chromosomes. A DNA fragment from the untranscribed 5' flank of a transcriptionally active ODC gene has been used as a probe to define a restriction fragment length polymorphism associated with the gene. The pattern of inheritance of this polymorphism in recombinant inbred strains of mice and in progeny of a conventional backcross localized it to proximal chromosome 12. This result confirms previous experiments with somatic cell hybrids that mapped the gene to chromosome 12 and further defines the linkage group conserved between proximal chromosome 12 and human chromosome 2p. The other ODC homologs are tentatively identified as cDNA-like pseudogenes.

Alleles↗
PubMed1989

Polyamine-mediated regulation of mouse ornithine decarboxylase is posttranslational.

The activity of ornithine decarboxylase (ODC) is negatively regulated by intracellular polyamines, which thereby mediate a form of feedback inhibition of the initial enzyme in the pathway of their synthesis. This phenomenon has been believed to result, at least in part, from translational regulation. To investigate this further, we performed four series of experiments. First, we found that a chimeric protein encoded by an mRNA containing the ODC 5' leader sequence did not exhibit polyamine-dependent regulation. Second, we showed that transcripts containing the protein-coding sequence of ODC, but no other ODC-derived sequence information, exhibited regulation. Third, we found that the association of ODC mRNA with ribosomes was not altered when intracellular polyamine levels were modulated under conditions previously deemed to cause translational regulation. Last, we carried out experiments to measure the incorporation of [35S]methionine into ODC in polyamine-starved and polyamine-replete cells. Differential incorporation diminished progressively as pulse-label times were shortened; at the shortest labeling time used (4 min), the difference in favor of ODC in polyamine-starved cells was less than twofold. These findings suggest that it is necessary to reevaluate the question of whether polyamines cause alterations of translation of ODC mRNA.

Actins↗
PubMed1988

Trypanosoma brucei ornithine decarboxylase: enzyme purification, characterization, and expression in Escherichia coli.

Ornithine decarboxylase from the African trypanosome is an important target for antitrypanosomal chemotherapy. Despite this, the enzyme had not been previously purified or extensively characterized as it is a very low level protein. In this paper we describe the purification of Trypanosoma brucei brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 10(6) nmol CO2/h/mg of protein in the parasite. T. brucei ornithine decarboxylase had a native molecular weight of 90,000 and a subunit molecular weight of 45,000. The isoelectric point of the protein was 5.0. The Km for ornithine was 280 microM and the Ki for the irreversible inhibitor alpha-difluoromethylornithine (DFMO) was 220 microM with a half-time of inactivation at saturating DFMO concentration of 2.7 min. T. brucei ornithine decarboxylase appears similar to mouse ornithine decarboxylase, further supporting our previous suggestion that the selective toxicity of DFMO to the parasite is not due to catalytic differences between the two proteins. Although a small quantity of T. brucei ornithine decarboxylase was purified from T. brucei, extensive structural and kinetic studies will require a more ample source of the enzyme. We therefore expressed our previously cloned T. brucei ornithine decarboxylase gene in Escherichia coli using a vector that contains an inducible lambda promoter. T. brucei ornithine decarboxylase activity was induced in E. coli to levels that were 50 to 200 fold of that present in the long-slender bloodstream form of T. brucei. Ornithine decarboxylase activity in the crude E. coli lysate was 1500-6000 nmol of CO2/h/mg of protein and represented 0.05-0.2% of the total cell protein. The recombinant T. brucei ornithine decarboxylase was purified to apparent homogeneity from the transformed E. coli. The purified recombinant enzyme had kinetic and physical properties essentially identical to those of the native enzyme.

Animals↗
PubMed1988

Probable cloning artefacts previously interpreted as unusual leader sequences of rodent ornithine decarboxylase mRNAs--a cautionary tale.

Messenger RNAs that have structurally unusual 5' leaders attract interest and provoke conjecture. Cloning and sequencing of two rodent ornithine decarboxylase (ODC) cDNAs, those for mouse [Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673-1677] and, recently as published in this journal, for rat [Van Kranen et al., Gene 60 (1987) 145-155], have indicated the presence of such features. In both cases, the leader is unusually long and contains multiple AUG start codons preceding that which encodes the N terminus of the protein. In addition, the leader of the rat clone contains a 54-nt perfect inverted repeat. Because ODC expression appears to be regulated translationally, functional implications immediately suggest themselves. Certain unusual features of the mouse cDNA have proven artefactual [Brabant et al., Proc. Natl. Acad. Sci. USA 85 (1988) 2200-2204; Katz and Kahana, J. Biol. Chem. 263 (1988) 7604-7609]. It is likely that the putative leader sequence of rat ODC cDNA also resulted from a cloning artefact.

Amino Acid Sequence↗
PubMed1988

Mouse ornithine decarboxylase gene: cloning, structure, and expression.

We used molecular cloning to isolate a functional gene for mouse ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) from a cell line in which that gene had been selectively amplified. The position of the 5' terminus of the mRNA was identified, and the coding sequence was shown to be preceded by a 312- or 313-nucleotide (nt) untranslated leader. The latter is highly G + C rich, particularly in its 5'-most portion. The leader can be anticipated to have extensive and stable secondary structure. The transcription unit of the gene is of relatively small size, approximately equal to 6.2 kilobases (kb) from the start site to the proximal site of polyadenylylation. Sequence analysis of DNA near the transcription start position demonstrated the presence of a "TATA" box, but no "CAAT" box. Functional properties of the cloned gene were tested by transfecting it into cultured cells. Expression of the putative full-length gene efficiently conferred ornithine decarboxylase activity on recipient mutant cells deficient in that activity. To assess the function and strength of the OrnDCase promoter region and to delimit its boundaries, we used a transient expression assay. Upstream of a bacterial chloramphenicol acetyltransferase gene was placed a portion of the OrnDCase gene, including the presumed promoter region, spanning a region from approximately equal to 3.0 kb 5' of the site of transcription initiation to the first 250 nt of the transcript. When expressed in mouse NIH 3T3 cells, this OrnDCase genomic element was comparable in strength to the Rous sarcoma virus long terminal repeat promoter. A similar construct, truncated so as to retain only 264 base pairs of the OrnDCase gene 5' to the site of transcription start, yielded undiminished levels of expression.

Animals↗
PubMed1988

A functional mouse ornithine decarboxylase gene (Odc) maps to chromosome 12: further evidence of homoeology between mouse chromosome 12 and the short arm of human chromosome 2.

We have used a DNA probe specific for a functional mouse ornithine decarboxylase gene (Odc) in conjunction with a panel of Chinese hamster x mouse somatic cell hybrids to assign Odc to mouse chromosome 12. This assignment provides further evidence of genetic homoeology between a region of mouse chromosome 12 and the distal short arm of human chromosome 2.

Animals↗