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S Matsufuji

Publications and source records attributed to S Matsufuji.

At least 19 recordsLinked to original sources
PubMed1998

Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels.

Polyamines are required for entry and progression of the cell cycle. As such, augmentation of polyamine levels is essential for cellular transformation. Polyamines are autoregulated through induction of antizyme, which represses both the rate-limiting polyamine biosynthetic enzyme ornithine decarboxylase and cellular polyamine transport. In the present study we demonstrate that agmatine, a metabolite of arginine via arginine decarboxylase (an arginine pathway distinct from that of the classical polyamines), also serves the dual regulatory functions of suppressing polyamine biosynthesis and cellular polyamine uptake through induction of antizyme. The capacity of agmatine to induce antizyme is demonstrated by: (a) an agmatine-dependent translational frameshift of antizyme mRNA to produce a full-length protein and (b) suppression of agmatine-dependent inhibitory activity by either anti-antizyme IgG or antizyme inhibitor. Furthermore, agmatine administration depletes intracellular polyamine levels to suppress cellular proliferation in a transformed cell line. This suppression is reversible with polyamine supplementation. We propose a novel regulatory pathway in which agmatine acts as an antiproliferative molecule and potential tumor suppressor by restricting the cellular polyamine supply required to support growth.

3T3 Cells↗
PubMed1998

Programmed frameshifting in the synthesis of mammalian antizyme is +1 in mammals, predominantly +1 in fission yeast, but -2 in budding yeast.

The coding sequence for mammalian ornithine decarboxylase antizyme is in two different partially overlapping reading frames with no independent ribosome entry to the second ORF. Immediately before the stop codon of the first ORF, a proportion of ribosomes undergo a quadruplet translocation event to shift to the +1 reading frame of the second and main ORF. The proportion that frameshifts is dependent on the polyamine level and, because the product antizyme is a negative regulator of intracellular polyamine levels, the frameshifting acts to complete an autoregulatory circuit by sensing polyamine levels. An mRNA element just 5' of the shift site and a 3' pseudoknot are important for efficient frameshifting. Previous work has shown that a cassette with the mammalian shift site and associated signals directs efficient shifting in the budding yeast Saccharomyces cerevisiae at the same codon to the correct frame, but that the shift is -2 instead of +1. The product contains an extra amino acid corresponding to the shift site. The present work shows efficient frameshifting also occurs in the fission yeast, Schizosaccharomyces pombe. This frameshifting is 80% +1 and 20% -2. The response of S. pombe translation apparatus to the mammalian antizyme recoding signals is more similar to that of the mammalian system than to that of S. cerevisiae. S. pombe provides a good model system for genetic studies on the mechanism of at least this type of programmed mammalian frameshifting.

Amino Acid Sequence↗
PubMed1997

Characterization of the human antizyme gene.

Antizyme is a polyamine-inducible protein involved in feedback regulation of cellular polyamine levels. Recently, we isolated genomic clones for the human antizyme gene and determined its chromosomal location (Matsufuji et al., Genomics 38 (1996) 112-114). In the present study, we report complete nucleotide sequence and organization of the human antizyme gene. The organizations of human and rat genes are very similar, but their introns show divergency in terms of the length and nucleotide sequence. Luciferase reporter assay revealed that the 5'-flanking region of the human gene had a strong transcriptional activity in NIH-3T3 with and without addition of spermidine. The promoter was also effective in transfected COS7 and HeLa cells. A 223-bp region at the proximity of the transcriptional start points carries several regulatory sequence motifs including a TATA box, CAAT boxes and GC boxes, and was shown to be important for the strong transcriptional activity.

Animals↗
PubMed1996

Reading two bases twice: mammalian antizyme frameshifting in yeast.

Programmed translational frameshifting is essential for the expression of mammalian ornithine decarboxylase antizyme, a protein involved in the regulation of intracellular polyamines. A cassette containing antizyme frameshift signals is found to direct high-level (16%) frameshifting in yeast, Saccharomyces cerevisiae. In contrast to +1 frameshifting in the mammalian system, in yeast the same frame is reached by -2 frameshifting. Two bases are read twice. The -2 frameshifting is likely to be mediated by slippage of mRNA and re-pairing with the tRNA in the P-site. The downstream pseudoknot stimulates frameshifting by 30-fold compared with 2.5-fold in reticulocyte lysates. When the length of the spacer between the shift site and the pseudoknot is extended by three nucleotides, +1 and -2 frameshifting become equal.

Amino Acid Sequence↗
PubMed1996

Superinduction of mouse epidermal ornithine decarboxylase activity by repeated 12-o-tetradecanoylphorbol-13-acetate treatments.

A correlation of the levels of epidermal protein kinase C (PKC) isozymes, steady state levels of ornithine decarboxylase (ODC) mRNA, and ODC antizyme with the induction of ornithine decarboxylase (ODC) activity by a second repeat 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment to mouse skin was determined. A single application of TPA to female CD-1 mouse skin leads to a dramatic induction of ODC activity (approximately 3 nmol CO2/60 min/mg protein) which peaks at about 5 h after treatment. However, a superinduction of ODC activity (approximately 13 CO2/60 min/mg protein) is observed upon the second TPA application at 48 or 72 h after the first TPA treatment. Prior application of a tumor initiating dose of 7,12-dimethylbenz[a]anthracine to mouse skin did not influence the degree of induction of ODC by a repeat TPA treatment. Western Blot analyses using antibodies specific to PKC alpha, beta, gamma, delta and epsilon indicate detectable levels of PKC alpha, beta, delta and epsilon in mouse epidermal extracts. A time course of the effects of a single topical application of 20 nmol of TPA to the mouse skin indicate that none of PKC isozymes (alpha, beta, gamma, delta and epsilon) were completely downregulated at times (72 h) when ODC was overinduced by TPA. TPA-induced steady state levels of ODC mRNA did not correlate with the degree of superinduction of ODC activity by TPA. The second TPA treatment, 72 h after the first TPA treatment, which leads to superinduction of ODC activity did not decrease the levels of the ODC-antizyme. The results indicate that superinduction of mouse epidermal ODC activity is regulated in part post-transcriptionally and may not be the result of either a loss of PKC isoform(s) or a decrease in the levels of ODC antizyme.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase.

The degradation of ornithine decarboxylase (ODC) catalyzed by the 26 S proteasome is accelerated by antizyme, an ODC inhibitory protein induced by polyamines. Previously, we have found another possible regulatory protein of ODC degradation, antizyme inhibitor. Antizyme inhibitor binds to the antizyme with a higher affinity than that of ODC, releasing ODC from ODC-antizyme complex. We report here the cDNA sequence of rat heart antizyme inhibitor. The deduced sequence of the protein is highly similar to, but distinct from, sequences of ODCs from various species. Antizyme inhibitor contains amino acid residues required for formation of active sites of ODC, but it completely lacks ODC activity. Antizyme inhibitor has no homology with peptide sequence in the mammalian ODC carboxyl terminus, which is needed for rapid turnover of ODC. It inhibits antizyme-dependent ODC degradation, but, unlike ODC, its degradation is not accelerated by antizyme.

Amino Acid Sequence↗
PubMed1996

Ornithine decarboxylase antizyme: a novel type of regulatory protein.

Antizyme plays an important regulatory role in the synthesis of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis in higher animals. As well as inactivating polyamine uptake, antizyme is induced by polyamine-enhanced translational frameshifting, and binds to ODC, accelerating its ATP-dependent degradation, a process catalysed by the 26S proteasome.

Animals↗
PubMed1995

Nucleotide sequence of ornithine decarboxylase antizyme cDNA from Xenopus laevis.

An ornithine decarboxylase antizyme cDNA was obtained from Xenopus laevis liver and its sequence was determined. The cDNA consists of two major open reading frames as found in mammalian antizymes, which require +1 ribosomal frameshifting for its translation. Sequences important for frameshifting, namely the frameshift site and downstream stimulatory pseudoknot determined in the rat mRNA, are conserved.

Amino Acid Sequence↗
PubMed1995

Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme.

Rat antizyme gene expression requires programmed, ribosomal frameshifting. A novel autoregulatory mechanism enables modulation of frameshifting according to the cellular concentration of polyamines. Antizyme binds to, and destabilizes, ornithine decarboxylase, a key enzyme in polyamine synthesis. Rapid degradation ensues, thus completing a regulatory circuit. In vitro experiments with a fusion construct using reticulocyte lysates demonstrate polyamine-dependent expression with a frameshift efficiency of 19% at the optimal concentration of spermidine. The frameshift is +1 and occurs at the codon just preceding the terminator of the initiating frame. Both the termination codon of the initiating frame and a pseudoknot downstream in the mRNA have a stimulatory effect. The shift site sequence, UCC-UGA-U, is not similar to other known frameshift sites. The mechanism does not seem to involve re-pairing of peptidyl-tRNA in the new frame but rather reading or occlusion of a fourth base.

Amino Acid Sequence↗
PubMed1994

Forced expression of antizyme abolishes ornithine decarboxylase activity, suppresses cellular levels of polyamines and inhibits cell growth.

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. It is a short-lived protein and negatively regulated by its products, polyamines. Its degradation is accelerated by the binding of antizyme, an ODC-inhibitory protein induced by polyamines. To evaluate the physiological importance of antizyme we examined the effect of forced expression of antizyme on cellular ODC and polyamine levels and cell growth. Antizyme almost completely abolished the induction of ODC by growth stimuli. This may have been caused by antizyme-induced rapid degradation of newly synthesized ODC, since the half-life of ODC complexes with antizyme was less than 5 min. Forced expression of antizyme caused reductions of cellular putrescine and spermidine levels, and inhibited cell growth, which was partially restored by the addition of putrescine. These observations suggested a critically important role of antizyme in polyamine metabolism.

Animals↗
PubMed1994

Functional regions of ornithine decarboxylase antizyme.

Degradation of ornithine decarboxylase, a key enzyme in polyamine biosynthesis, is accelerated by the binding of antizyme, an ornithine decarboxylase inhibitory protein induced by polyamines. In the present study, we examined the effects of a series of deletion mutants of rat antizyme. The results indicated that two regions of antizyme, one internal (amino acids 122-144) and the other near the C-terminus (amino acids 211-218) are necessary for its binding to ornithine decarboxylase and inhibition of its activity, and an additional internal region (amino acids 88-118, especially 113-118) is necessary for its destabilization.

Animals↗
PubMed1993

Involvement of the proteasome and antizyme in ornithine decarboxylase degradation by a reticulocyte lysate.

Ornithine decarboxylase (ODC) degradation in a freshly prepared reticulocyte lysate was examined. Immunodepletion of proteasomes from the reticulocyte lysate resulted in almost complete loss of ODC degradation. In contrast with the previously reported degradation in extracts of hepatoma tissue-culture (HTC) and Chinese-hamster ovary (CHO) cells or that by the purified 26 S proteasome, efficient degradation of ODC was observed in the lysate without exogenous antizyme, an ODC protein inhibitor induced by polyamines, owing to the presence of a significant amount of antizyme in the lysate. The degradation of ODC in the lysate was strongly suppressed on inactivation of antizyme in the lysate with antizyme inhibitor, a protein which binds to the antizyme and releases ODC from the ODC-antizyme complex. Thus the main pathway for ODC degradation in a reticulocyte lysate was essentially the same as that characterized previously in extracts of HTC and CHO cells, namely an ATP- and antizyme-dependent 26 S proteasome-catalysed pathway that is presumed to be responsible for ODC degradation in whole cells.

Animals↗
PubMed1993

Single amino-acid replacement is responsible for the stabilization of ornithine decarboxylase in HMOA cells.

The half-life of ornithine decarboxylase (ODC) in HMOA cells, a variant cell line derived from hepatoma tissue culture (HTC) cells, is markedly increased compared with that in the parental cell line. In the present study, we examined which of the three relevant factors is responsible for the ODC stabilization in HMOA cells, namely ODC itself, a regulatory protein antizyme and an ODC-degrading activity. SDS/PAGE analysis of radiolabeled ODC revealed that ODC from HMOA cells migrated somewhat faster than that from HTC cells, suggesting that HMOA ODC was structurally altered. Direct sequencing of reverse-transcription/polymerase-chain-reaction (RT-PCR) products of ODC mRNA from HMOA cells revealed a T to G replacement, causing a Cys441-->Trp replacement near the C-terminus. No alteration was found in the whole coding region of antizyme mRNA. An authentic mutant ODC cDNA with the same replacement was transfected and expressed in C55.7 ODC-deficient Chinese hamster ovary cells. Upon cycloheximide treatment, the mutant ODC activity did not decrease appreciably for at least 3 h, whereas wild-type ODC activity decreased with a half-life of 1 h. In-vitro-synthesized mutant ODC with the Cys441-->Trp (or Ala) replacement was also stable in a reticulocyte-lysate ODC-degradation system. Metabolically labeled and purified mouse ODC was degraded in HMOA cell extracts in the presence of ATP and antizyme as rapidly as in HTC cell extracts, indicating that HMOA cells have a normal ODC degrading activity. These results indicated that the single amino acid replacement, Cys441-->Trp, is responsible for the stabilization of ODC in HMOA cells and that Cys441 is important for rapid ODC turnover.

Animals↗
PubMed1993

Failure of treatment with alpha-difluoromethylornithine against secondary multilocular echinococcosis in mice.

In AKR/J mice inoculated intraperitoneally with protoscoleces of Echinococcus multilocularis, the oral administration of alpha-difluoromethylornithine (DFMO) in drinking water had no obvious effect on the development of hydatid cysts. The DFMO treatment also caused no apparent depletion of three major polyamines (putrescine, spermidine, and spermine) in the cysts, although a slight reduction in the putrescine level was detected. The failure of DFMO treatment to reduce the growth of cysts in mice may result from the absence of ornithine decarboxylase in the parasite.

Animals↗
PubMed1992

Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is the most rapidly turned over mammalian enzyme. We have shown that its degradation is accelerated by ODC antizyme, an inhibitory protein induced by polyamines. This is a new type of enzyme regulation and may be a model for selective protein degradation. Here we report the identification of the protease responsible for ODC degradation. Using a cell-free degradation system, we demonstrate that immunodepletion of proteasomes from cell extracts causes almost complete loss of ATP- and antizyme-dependent degradation of ODC. In addition, purified 26S proteasome complex, but not the 20S proteasome, catalyses ODC degradation in the absence of ubiquitin. These results strongly suggest that the 26S proteasome, widely viewed as specific for ubiquitin-conjugated proteins, is the main enzyme responsible for ODC degradation. The 26S proteasome may therefore have a second role in ubiquitin-independent proteolysis.

Adenosine Triphosphate↗