Shin-ichi Hayashi

Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme.

Abstract 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.

ATP-Dependent Inactivation and Sequestration of Ornithine Decarboxylase by the 26S Proteasome Are Prerequisites for Degradation

ABSTRACT The 26S proteasome is a eukaryotic ATP-dependent protease, but the molecular basis of its energy requirement is largely unknown. Ornithine decarboxylase (ODC) is the only known enzyme to be degraded by the 26S proteasome without ubiquitinylation. We report here that the 26S proteasome is responsible for the irreversible inactivation coupled to sequestration of ODC, a process requiring ATP and antizyme (AZ) but not proteolytic activity. Neither the 20S proteasome (catalytic core) nor PA700 (the regulatory complex) by itself contributed to this ODC inactivation. Analysis with a C-terminal mutant ODC revealed that the 26S proteasome recognizes the C-terminal degradation signal of ODC exposed by attachment of AZ, and subsequent ATP-dependent sequestration of ODC in the 26S proteasome causes irreversible inactivation, possibly unfolding, of ODC and dissociation of AZ. These processes may be linked to the translocation of ODC into the 20S proteasomal inner cavity, centralized within the 26S proteasome, for degradation.

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeneity from the livers of thioacetamide- and DL-alpha-hydrazino-delta aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5.10(5)-fold with an 8% yield; the specific activity of the final enzyme preparation was 1.1.10(6) nmol CO2/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50000 on SDS-polyacrylamide gels. The isoelectric point of the enzyme was pH 4.1.

Translocation of ornithine decarboxylase to the surface membrane during cell activation and transformation

Ornithine decarboxylase (ODC) is highly up‐regulated in proliferating and transforming cells. Here we show that upon induction, an initial cytosolic increase of ODC is followed by translocation of a fraction of the enzyme to the surface membrane. ODC membrane translocation is mediated by a p47phox membrane‐targeting motif‐related sequence, as indicated by reduced ODC activity in the membrane fraction of cells treated with a competing, ODC‐derived (amino acids 165–172) peptide, RLSVKFGA, which is homologous to the p47phox membrane‐targeting sequence. p47phox membrane translocation is known to be dependent on the phosphorylation of the targeting motif. Analogously, overexpressed ODC.S167A, a mutant ODC lacking the putative phosphorylation site Ser67, is unable to move to the surface membrane. Cells blocked with the RLSVKFGA peptide showed defective transformation, indicating that the motif‐mediated translocation of ODC is prerequisite to its biological function. Constitutive targeting of ODC to the membrane using a plasmid encoding the chimeric protein, wild‐type ODC with C‐terminal linkage to the farnesylation motif of K‐ras, caused impaired cytokinesis with an accumulation of polykaryotic cells. Impaired cytokinesis confirms that ODC is involved in mitotic cytoskeletal rearrangement events and pinpoints the importance of relevant membrane targeting to its physiological function.

CLONING AND SEQUENCING OF A HUMAN CDNA ENCODING ORNITHINE DECARBOXYLASE ANTIZYME INHIBITOR

We report here cloning and sequencing human antizyme inhibitor from a human kidney cDNA library. Amino acid sequence deduced from the nucleotide sequence shows 92.9% identity to that of rat antizyme inhibitor. Northern blot analysis reveals that antizyme inhibitor is expressed in human liver.

Molecular mechanism for the regulation of hepatic ornithine decarboxylase

A single injection of thioacetamide into starved rats induced a 40- to 100-fold increase in hepatic ODC activity. However, immunotitratable ODC protein increased by only 5-fold because of the presence of significant amounts of inactive ODC protein in the liver of untreated starved rats. Polysomal ODC-mRNA activity also increased only 5-fold, a significant amount being present in control liver. Furthermore, the peak of polysomal ODC-mRNA activity preceded that of ODC activity or ODC protein by several hours. These results indicate that the enzyme induction is due not only to increase in polysomal ODC-mRNA activity, but also to some translational and/or post-translational regulation. Exogenously administered diamines or polyamines cause rapid decay of ODC activity and induce antizyme that binds to ODC and inactivates it. Another protein factor, antizyme inhibitor, was found in the liver of thioacetamide-treated or protein-fed rats. Antizyme inhibitor binds to antizyme and reactivates ODC in the ODC-antizyme complex. A small, but significant, amount of antizyme was found in the liver of starved rats. Only small amounts of ODC-antizyme complex were detected in rat liver and cultured hepatocytes, even during the period of rapid ODC decay caused by exogenously added diamines. On the other hand, the complex was present in HTC cells and more especially in ODC-stabilized HMOA cells, even under physiological conditions. On addition of 10(-2) M putrescine, the amount of complex first increased and then decreased in both types of cells. Decay of total ODC activity (free plus complexed ODC) was more rapid with putrescine than with cycloheximide. These results suggest that antizyme plays an essential role in the degradation of ODC by a catalytic effect both in the presence and absence of exogenous putrescine and that antizyme inhibitor stabilizes ODC by removing antizyme.

Two-Step Purification of Mouse Kidney Ornithine Decarboxylase

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4.1.1.17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1.0 x 10(6)-1.4 x 10(6) nmol/h.mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54,000 and a minor band of Mr 51,000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [14C]difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.

Affinity labeling of purified ornithine decarboxylase by α-difluoromethylornithine

Abstract Ornithine decarboxylase ( l -ornithine carboxy-lyase, EC 4.1.1.17) purified from rat liver was affinity-labeled by α-[5- 14 C]difluoromethylornithine. On analysis by SDS-polyacrylamide gel electrophoresis, the radioactivity migrated as a single major peak that coincided with a single protein band of M r 50 000. Calculation from bound radioactivity indicated that ornithine decarboxylase has two active sites, one for each subunit, and that pure enzyme should have a specific activity of about 1.4·10 6 nmol CO 2 /h per mg protein.

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.

Expression of insulin receptor substrate-1 in hepatocytes : an investigation using monoclonal antibodies

To investigate the expression and subcellular distribution of insulin receptor substrate-1 in hepatocytes, which are major targets of insulin along with muscle and adipose tissue, we obtained monoclonal antibodies by immunizing mice with a fusion protein consisting of the C-terminal portion of the human insulin receptor substrate-1 and glutathione-S-transferase. Two of the monoclonal antibodies (designated as 7B3 and 6G5) were found to be useful for immunohistochemical studies. Using 6G5 we demonstrate a high level of expression of insulin receptor substrate-1 in liver cirrhosis hepatocytes and variable expression in hepatocellular carcinoma cells. These results suggest that insulin receptor substrate-1 may play a role in liver regeneration during cirrhosis and that an insulin signaling cascade may be involved in hepatocarcinogenesis.