Comparison of the basic Escherichia coli antizyme 1 and antizyme 2 with the ribosomal proteins S20/L26 and L34

The two basic Escherichia coli proteins that inhibit ornithine and arginine decarboxylase and were named provisionally antizyme 1 and antizyme 2 (Heller, J.S., Rostomily, R., Kyriakidis, D.A., and Canellakis, E.S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5181-5184) are shown to have long identical sequences with the ribosomal proteins S20/L26 and L34, respectively. We have also isolated ribosomal proteins from purified E. coli ribosomes by established methodology and further purified them by our purification procedure for antizymes 1 and 2. Of the various basic ribosomal proteins, two were found to have the same properties as antizyme 1 and 2. These results indicate that these two basic E. coli antizymes are ribosomal proteins. The nature of the acidic antizyme remains to be elucidated.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

The antizyme family: polyamines and beyond

The family of antizymes functions as regulators of polyamine homeostasis. They are a class of small, inhibitory proteins, whose expression is regulated by a unique ribosomal frameshift mechanism. They have been shown to inhibit cell proliferation and possess anti-tumor activity. Antizymes bind ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. They inhibit its enzymatic activity and promote the ubiquitin-independent degradation of ODC by the 26S proteasome. In addition, they also negatively regulate polyamine transport. Antizyme-mediated, ubiquitin-independent degradation of ODC is conserved from yeast to man. But recent data suggest that this degradation pathway might not be restricted to ODC alone and could involve newly discovered antizyme binding partners. Interestingly, antizyme proteins have been strictly preserved over a vast evolutionary timeframe. Antizymes consequently represent an important class of proteins that regulate cell growth and metabolism by a diverse set of mechanisms that include protein degradation, inhibition of enzyme activity, small molecule transport and other, potentially not yet discovered properties.     

Purification and properties of the antizymes of Escherichia coli to ornithine decarboxylase

The purification of the antizymes to ornithine decarboxylase of Escherichia coli to homogeneity is detailed. An acidic component, pI 3.8, and two basic histone-like proteins, pI above 9.5, are described. The two latter proteins constitute approximately 90% of the total antizyme activity.     

Study of the surface of Escherichia coli ribosomes and ribosomal particles by the tritium bombardment method

A new technique of atomic tritium bombardment has been used to study the surface topography of Escherichia coli ribosomes and ribosomal subunits. The technique provides for the labeling of proteins exposed on the surface of ribosomal particles, the extent of protein labeling being proportional to the degree of exposure. The following proteins were considerably tritiated in the 70S ribosomes: S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L1, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27. A conclusion is drawn that these proteins are exposed on the ribosome surface to an essentially greater extent than the others. Dissociation of 70S ribosomes into the ribosomal subunits by decreasing Mg2+ concentration does not lead to the exposure of additional ribosomal proteins. This implies that there are no proteins on the contacting surfaces of the subunits. However, if a mixture of subunits has been subjected to centrifugation in a low Mg2+ concentration at high concentrations of a monovalent cation, proteins S3, S5, S7, S14, S18 and L16 are more exposed on the surface of the isolated 30S and 50S subunits than in the subunit mixture or in the 70S ribosomes. The exposure of additional proteins is explained by distortion of the native quaternary structure of ribosomal subunits as a result of the separation procedure. Reassociation of isolated subunits at high Mg2+ concentration results in shielding of proteins S3, S5, S7 and S18 and can be explained by reconstitution of the intact 30S subunit structure.     

Subcellular distribution of ribosomal proteins in Dictyostelium discoideum

The distribution of ribosomal proteins in monosomes, polysomes, the postribosomal cytosol, and the nucleus was determined during steady-state growth in vegetative amoebae. A partitioning of previously reported cell-specific ribosomal proteins between monosomes and polysomes was observed. L18, one of the two unique proteins in amoeba ribosomes, was distributed equally among monosomes and polysomes. However S5, the other unique protein, was abundant in monosomes but barely visible in polysomes. Of the developmentally regulated proteins, D and S6 were detectable only in polysomes and S14 was more abundant in monosomes. The cytosol revealed no ribosomal proteins. On staining of the nuclear proteins with Coomassie blue, about 18, 7 from 40S subunit and 11 from 60S subunit, were identified as ribosomal proteins. By in vivo labeling of the proteins with [35S]methionine, 24 of the 34 small subunit proteins and 33 of the 42 large subunit proteins were localized in the nucleus. For the majority of the ribosomal proteins, the apparent relative stoichiometry was similar in nuclear preribosomal particles and in cytoplasmic ribosomes. However, in preribosomal particles the relative amount of four proteins (S11, S30, L7, and L10) was two- to four-fold higher and of eight proteins (S14, S15, S20, S34, L12, L27, L34, and L42) was two-to four-fold lower than that of cytoplasmic ribosomes.     

Polyamine stimulation of ribosomal synthesis and activity in a polyamine-dependent mutant of Escherichia coli

A polyamine-dependent mutant of Escherichia coli KK101 was isolated by treatment of E. coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine. In the absence of putrescine, doubling time of the mutant was 496 min. The mutation was accompanied by a change in the nature of the 30 S ribosomal subunits. Addition of putrescine to the mutant stimulated the synthesis of proteins and subsequently, this led to stimulation of RNA and DNA synthesis. Under these conditions, we determined which proteins were preferentially synthesized. Putrescine stimulated the synthesis of ribosomal protein S1 markedly, but stimulated ribosomal proteins S4, L20, and X1, and RNA polymerase slightly. The amounts of initiation factors 2 and 3 synthesized were not influenced significantly by putrescine. The preferential stimulation of the synthesis of ribosomal protein S1 occurred as early as 20 min after the addition of putrescine, while stimulation of the synthesis of the other ribosomal proteins and RNA polymerase appeared at 40 min. The stimulation of the synthesis of ribosomal RNA also occurred at 40 min after addition of putrescine. Our results indicate that putrescine can stimulate both the synthesis and the activity of ribosomes. The increase in the activity of ribosomes was achieved by the association of S1 protein to S1-depleted ribosomes. The early stimulation of ribosomal protein S1 synthesis after addition of putrescine may be important for stimulation of cell growth by polyamines.     

Analyses of ornithine decarboxylase antizyme mRNA with a cDNA cloned from rat liver

Ornithine decarboxylase antizyme is a unique inhibitory protein induced by polyamines and involved in the regulation of ornithine decarboxylase. A cDNA was isolated from a rat liver cDNA library by the screening with monoclonal antibodies to rat liver antizyme as probes. The expression products of the cDNA in bacterial systems inhibited rat ornithine decarboxylase activity in a manner characteristic of antizyme and rabbit antisera raised against its direct expression product reacted to rat liver antizyme, confirming the authenticity of the cDNA. On RNA blot analysis with the cDNA probe, an antizyme mRNA band of 1.3 kb was detected in rat tissues. Antizyme mRNA did not increase upon administration of putrescine, an inducer of antizyme, and its half-life after actinomycin D treatment was as long as 12 h in rat liver, suggesting that antizyme mRNA is constitutively expressed and antizyme synthesis is regulated at the translational level. Similar-sized mRNAs hybridizable to the cDNA were also found in various mammalian and non-mammalian vertebrate tissues under physiological conditions. In addition, chicken and frog antizymes showed immunocrossreactivity with rat antizyme. The ubiquitous presence and the evolutionally conserved structure of antizyme in vertebrate tissues suggest that it has an important function.     

Phosphorylation of ribosomal proteins influences subunit association and translation of poly (U) in Streptomyces coelicolor

The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.     

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.     

Identification of the ribosomal proteins phosphorylated by the ribosome-associated casein kinase type II from cryptobiotic gastrulae of the brine shrimp Artemia sp

Phosphorylation of the ribosomal proteins by the extra-ribosomal protein kinase was investigated "in situ" and with purified 40 S or 60 S ribosomal proteins from cryptobiotic embryos of Artemia sp. Ribosomal proteins that were most readily phosphorylated in 80 S ribosomes included S6 and S8 of the 40 S subunit and proteins L9, L13 and L18 of the 60 S subunit. Several additional polypeptides were phosphorylated when purified 40 S or 60 S ribosomal proteins were separately incubated in the reconstituted system. The possible functions of ribosomal phosphorylation in protein synthesis will be discussed.     

An analysis of alterations in ribosomal conformation using reductive methylation

Optimal conditions for reductive alkylation of ribosomal proteins in their native and denatured states were examined. The relative accessibility of rat liver ribosomal proteins to reductive alkylation was then examined. Intact ribosomes were firs labeled with [14C]formaldehyde and NaBH4. The proteins were then separated from RNA, denatured in 6 M guanidine, and labeled again using formaldehyde and NaB3H4. The relative accessibility of individual proteins to labeling in the intact state could thus be determined from their 3H/14C ratios following separation by two-dimensional electrophoresis. The results suggest that proteins S6, S11, S26, L3, and L35 are less accessible to labeling while proteins S1, S15, L11, L12, L16, and L24 appear relatively more accessible. The accessibility of individual proteins in ribosomes in different conformational states were then compared. The results indicated that S3, L7, and L36 are likely to be involved in a structural difference when normal polysomes and normal monomers are compared. Also, that S26 and L35, and probably S3, S20, L7, L8, L24, L27, L28 and L34 appear to be involved in a ribosomal conformation change induced by ethionine intoxication.     

Structure function relationships in the ribosomal stalk proteins of archaebacteria.

The ribosomal L12 protein gene of Sulfolobus solfataricus (SsoL12) has been subcloned and overexpressed in Escherichia coli. Five protein L12 mutants were designed: two NH2-terminal and two COOH-terminal truncated mutants and one mutant lacking the highly charged part of the COOH-terminal region. The mutant protein genes were overexpressed in E. coli and the products purified. The amino acid composition was verified and the NH2 terminally truncated mutants were subjected to Edman degradation. The SsoL12 protein was selectively removed from entire S. solfataricus ribosomes by an ethanol wash. The remaining ribosomal core particles showed a substantial decrease in the in vitro translational activity. S. solfataricus L12 protein overexpressed in E. coli (SsoL12e) was incorporated into these ribosomal cores and restored their translational activity. Mutants lacking any part of the COOH-terminal region could be incorporated into these cores, as proven by two-dimensional polyacrylamide gels of the reconstituted particles. Mutant SsoL12 MC2 (residue 1-70) was sufficient for dimerization and incorporation into ribosomes. In contrast to the COOH terminally truncated mutants, L12 proteins lacking the 12 highly conserved NH2-terminal residues or the entire NH2-terminal region (44 amino acids) are unable to bind to ribosomes, suggesting that the SsoL12 protein binds with its NH2-terminal portion to the ribosome. None of the mutants could significantly increase the translational activity of the core particles suggesting that every deleted part of the protein was needed directly or indirectly for translational activity. Our results suggest that the COOH terminally truncated mutants were bound to ribosomes but not functional for translation. Cores preincubated with these COOH terminally truncated mutants regained activity when a second incubation with the entire overexpressed SsoL12e protein followed. This finding suggests that archaebacterial L12 proteins are freely exchanged on the ribosome.     

Reversible modification of Escherichia coli ribosomes with 2,3-dimethylmaleic anhydride. A new method to obtain protein-deficient ribosomal particles.

Treatment of Escherichia coli ribosomes with the protein reagent 2,3-dimethylmaleic anhydride is accompanied by inactivation of polypeptide polymerization and by dissociation of ribosomal proteins. Regeneration of the modified amino groups at pH 6.0 is followed by reactivation and reconstitution of the ribosomes. Prior to regeneration of the amino groups, ribosomal particles and split proteins can be separated by centrifugation, which allows the preparation of new protein-deficient particles. The ribosomal particles obtained by three successive treatments with 2,3-dimethyl-maleic anhydride at a molar ratio of reagent to ribosome equal to 16,000 lack proteins S1, S2, S3, S5, S10, S13, S14, L7, L8, L10, L11, L12, and L20 and have lost part of proteins S4, L1, L6, L16, and L25. This new procedure to obtain protein-deficient ribosomal particles is mild and might be useful to dissociate other protein-containing structures in addition to ribosomes.     

Systemic overexpression of antizyme 1 in mouse reduces ornithine decarboxylase activity without major changes in tissue polyamine homeostasis

Polyamines, spermidine, spermine and their precursor putrescine, are ubiquitous cell components essential for normal cell growth. Increased polyamine levels and enhanced biosynthesis have been associated with malignant transformation and tumor formation, and thus, the polyamines have been considered to be a meaningful target to cancer therapies. However, clinical cancer treatment trials using inhibitors of polyamine synthesis have been unsuccessful probably due to compensatory uptake of polyamines from extracellular sources. The antizyme proteins regulate both polyamine biosynthesis and transport, and thus, the antizymes could provide an efficient approach to control cellular proliferation compared to the mere inhibition of biosynthesis. To define the role of antizymes in proliferative processes associated with the whole animal, we have generated transgenic mice overexpressing mouse antizyme 1 gene under its own regulatory sequences. Antizyme 1 protein was abundantly expressed in various organs and the expressed antizyme protein was functional as ornithine decarboxylase activity was significantly reduced in all tissues analyzed. However, antizyme 1 overexpression caused only minor changes in tissue polyamine levels demonstrating the challenges in using the “antizyme approach” to deplete polyamines in a living animal. Neither were there any changes in cellular proliferation in the proliferative tissues of transgenic animals. Interestingly though, there was occurrence of abnormally high level of apoptosis in the non-proliferating part of the colon epithelia. Otherwise, the transgenic founder mice appeared healthy and out of seven founders six were fertile. However, none of the founders could transmit the transgene suggesting that the antizyme 1 overexpression may be deleterious to transgenic gametes.     

Biosynthesis of ribosomal proteins by poly(A)-containing mRNAs from rat liver in a wheat germ cell-free system and sizes of mRNAs coding ribosomal proteins

(1) Poly(A)-containing mRNAs from total polysomal RNA of regenerating rat liver were incubated with [3H]leucine in a wheat germ cell-free system. Ribosomal proteins were purified as described previously [1], and with two-dimensional gel electrophoresis. The proteins on the gel except for less basic protein had appreciable radioactivity, whereas the surrounding areas had very low radioactivity. Acetic acid-soluble proteins labeled in this system were subjected to three-dimensional gel electrophoresis [2]. Except for L1 and L2 proteins, each of the ribosomal proteins, including less basic ones, showed a major radioactive peak coinciding with the protein band on SDS gel. Thus, the wheat germ cell-free system completely translates almost all mRNAs for individual ribosomal proteins. Equimolar amounts of almost all ribosomal proteins were synthesized in the presence of the saturating concentration of mRNAs. (2) Free polysomes from regenerating rat liver were fractionated into three sizes. Each class of polysomes was incubated with [3H]leucine. Ribosomal proteins with molecular weights of 40 000 to 21 000 were mainly synthesized by Fraction B (5-14 monomeric ribosomes), L1 and L2 [2] with 60 000 and 54 000, by Fraction C (greater than 15 monomeric ribosomes) and B, and ribosomal proteins smaller than 20 000 by Fractions A (less than pentamer) and B. (3) mRNAs from rat liver total polysomes were fractionated into seven classes by size and each was translated in the wheat germ extract. Ribosomal proteins with molecular weights of 54 000 to 30 000 were mainly synthesized by mRNAs of 12 to 14.5 S, ribosomal proteins of 35 000 to 22 000 by those of 9.5 to 12 S, ribosomal proteins of 22 000 to 13 000 by those of 7 to 9.5 S, and smaller ribosomal proteins by those smaller than 7 S. These results indicate that individual ribosomal proteins are synthesized by monocistronic mRNAs, the lengths of which are proportional to the molecular weights of the corresponding ribosomal proteins.