Cinnamomin: a multifunctional type II ribosome-inactivating protein

Plant ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can irreversibly inactivate ribosomes by specifically removing the conserved adenine base from the "Sarcin/Ricin domain" of the 28S RNA in ribosome. Cinnamomin is a novel type II RIP isolated in our laboratory from the mature seeds of camphor tree. Besides site-specific deadenylation of the A4324 in the Sarcin/Ricin domain of rat ribosome, this protein could also release the adenine base from DNA molecules at multiple sites and from AMP, ADP, dAMP and adenosine. Furthermore, cinnamomin displays cytotoxicity to carcinoma cells and insect larvae by modifying their ribosomal RNA. These functions possessed by cinnamomin shed a new light on the possible application of cinnamomin in the field of immunotoxin design and transgenic reagents. In this review, we introduce the major recent results on cinnamomin obtained in our laboratory, including purification of this protein, characterization of its enzymatic mechanism, structure and function, gene pattern, physiological role and its biological implications in cytotoxicity.     

Minireview: enzymatic properties of ribosome-inactivating proteins (RIPs) and related toxins.

Ribosome-inactivating proteins (RIPs) are a group of proteins that inhibit protein synthesis in eucaryotic cells. While the biological effects have been well characterized, the underlying enzymatic mechanisms have not been elucidated until recently. Two different mechanisms have been identified. Plant and bacterial RIPs act as N-glycosidases. They cleave a single N-glycosidic bond between adenine and ribose at a specific nucleotide A-4324 of the 28S rRNA of the 60S ribosomal subunit. On the other hand, the fungal RIPs act as ribonucleases and cleave a single phosphodiester bond between G-4325 and A-4326 of the same rRNA, just one nucleotide away from the site of action of plant/bacterial RIPs. Other protein synthesis inhibitory proteins act by their ADP-ribosyltransferase activity which modify and thus inactivate elongation factor-2. Recently, some toxins have been shown to possess deoxyribonuclease activity which may also account for their toxicity.     

Substrate specificity of a maize ribosome-inactivating protein differs across diverse taxa.

The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain-activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes.     

Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes,and correlation with antifungal activity

Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.     

Maize Ribosome-Inactivating Protein Uses Lys158–Lys161 to Interact with Ribosomal Protein P2 and the Strength of Interaction Is Correlated to the Biological Activities

Ribosome-inactivating proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk protein P2 via Lys158–Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the ‘FGLFD’ motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (K_D) of their interaction, which are 1037.50±65.75 µM, 611.70±28.13 µM and 194.84±9.47 µM respectively.Despite MOD and TCS target at the same ribosomal protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal proteins for targeting the sarcin-ricin loop and the importance of protein-protein interaction for ribosome-inactivating activity.     

Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system.     

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.     

Quantitative analysis of the protein composition of yeast ribosomes

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.     

Molecular cloning and in-depth bioinformatics analysis of type II ribosome-inactivating protein isolated from Sambucus ebulus

Plant ribosome–inactivating proteins (RIPs) are N–glycosidases which inhibit protein synthesis through depurination of the ribosomal RNA sequence. Type II RIPs are heterodimer proteins which can bind to cell surfaces. The cytotoxicity of these RIPs is different. Sambucus spp. are a rich source of RIP proteins with different properties. In the present study, a type II RIP was isolated from S. ebulus plant that grows widely in the north of Iran, and different bioinformatics tools were used for the evaluation of physicochemical, functional and 3D protein characteristics. The results showed significant differences among isolated RIP and other Sambucus RIP proteins. The study of these differences can not only expand our insight into the functioning mechanisms of plant RIPs but also provide information about a novel RIP protein with potential biological applications.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.     

The mechanism of action of trichosanthin on eukaryotic ribosomes--RNA N-glycosidase activity of the cytotoxin.

Trichosanthin is a ribosome-inactivating protein from root tubers of Trichosanthes kirilowii Maxim. In this paper, the mechanism of action of trichosanthin on eukaryotic ribosomes was studied. A fragment of about 450 nucleotides was released from 28S ribosomal RNA after treatment of rat liver ribosome with trichosanthin and its isolated ribosomal RNAs were treated with aniline. Analysis of nucleotide sequence of 5' terminus of this fragment revealed that the aniline-sensitive site of the phosphodiester bond was between positions A4324 and G4325 in the 28S rRNA. Adenine was recovered by ion-exchange column chromatography from the 50% ethanol soluble fraction of the reaction mixture in which rat liver ribosomes were treated with trichosanthin. Thin-layer chromatographic analysis indicated that 1 mol of adenine was released from 1 mol of ribosomes. When the ribosomes were incubated with trichosanthin in the presence of inorganic [32P]phosphate, little incorporation of radioactivity into 28S rRNA was observed, indicating that the release of adenine was not mediated by phosphorolysis. These results demonstrate that trichosanthin inactivates the ribosomes by cleaving the N-C glycosidic bond of adenylic acid at 4324 of 28S rRNA in a hydrolytic fashion.     

Type 1 ribosome-inactivating proteins depurinate plant 25S rRNA without species specificity.

Four different type 1 ribosome-inactivating proteins (RIPs) with RNA N-glycosidase activity were tested for their ability to attack the large rRNA of plant ribosomes derived from tobacco plants, as well as from the plant species from which the particular RIP had been isolated. Incubation of tobacco ribosomes with RIPs isolated from either Phytolacca americana L. (pokeweed), Dianthus barbatus L. (carnation), Spinacia oleracea L. (spinach) or Chenopodium amaranthicolor Coste and Reyn. (chenopodium) rendered the 25S rRNA sensitive to aniline-catalyzed hydrolysis, generating a single rRNA-fragment of about 350 nucleotides. The same fragment was generated when rRNAs from pokeweed, carnation, spinach or chenopodium ribosomes were aniline-treated without any deliberate treatment of the ribosomes with the respective RIP. This indicated that ribosomes from all RIP-producing plants were already inactivated by their own RIPs during preparation. These results demonstrate that plant ribosomes are generally susceptible to RIP attack, including modification by their own RIPs. Direct sequencing of the newly generated fragments revealed that a single N-glycosidic bond at an adenosine residue within the highly conserved sequence 5'-AGUACGAGAGGA-3' was cleaved by all of the RIPs investigated, a situation also found in animal, yeast and Escherichia coli ribosomes.     

The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA.     

Analysis of the protein composition of yeast ribosomal subunits by two-dimensional polyacrylamide gel electrophoresis

The number of proteins in yeast ribosomal subunits was determined by two-dimensional polyacrylamide gel electrophoresis. The 40S subunit obtained after dissociation of ribosomes at high ionic strength contains 30 different protein species (including six acidic proteins). The 60S subunit, obtained in the same way contains 39 different species (including 1 acidic protein). While the total number of protein species found in yeast ribosomes, thus, is in close agreement with those reported for other eukaryotic organisms, the distribution between acidic and basic proteins is quite different. When the ribosomes were dissociated at low ionic strength, four extra protein spots appeared in the electropherograms of both 40S and 60S subunits. We consider these proteins to be nonribosomal.     

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.     

On the role of cyclic AMP-independent protein kinases in the modification of yeast ribosomal proteins in vivo

Two proteins of yeast 40S ribosome subunit and four proteins of the 60S ribosome subunit were labelled in vivo with [32P]orthophosphate. Five of these proteins were phosphorylated by protein kinase 3, an enzyme which is cyclic AMP-independent and uses ATP and GTP as phosphoryl donors. Two proteins, belonging to the 60S ribosome subunit were phosphorylated by another, highly specific, cyclic AMP-independent protein kinase 1 B. Both in vivo and in vitro the most extensively phosphorylated protein species were acidic proteins, L44, L45 (according to the nomenclature of Kruiswijk & Planta, Molec. Biol. Rep., 1, 409-415, 1974) possibly corresponding to bacterial L7 and L12 proteins. The 40S ribosomal protein, S9, analogous to mammalian S6 protein, was phosphorylated in vivo but was not phosphorylated in vitro by either of the cyclic AMP-independent protein kinases. The obtained results clearly indicate that cyclic AMP-independent yeast protein kinases might be involved in the modification in vivo of some ribosomal proteins, in particular of the strongly acidic proteins of 60S ribosome subunit.     

A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export

The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.     

Solution Structure of an Active Mutant of Maize Ribosome-Inactivating Protein (MOD) and Its Interaction with the Ribosomal Stalk Protein P2

Ribosome-inactivating proteins (RIPs) are N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of ribosomal RNA. This modification renders the ribosome unable to bind the elongation factors, thereby inhibiting the protein synthesis. Maize RIP, a type III RIP, is unique compared to the other type I and type II RIPs because it is synthesized as a precursor with a 25-residue internal inactivation region, which is removed in order to activate the protein. In this study, we describe the first solution structure of this type of RIP, a  28-kDa active mutant of maize RIP (MOD). The overall protein structure of MOD is comparable to those of the other type I RIPs and the A-chain of type II RIPs but shows significant differences in specific regions, including (1) shorter β6 and αB segments, probably for accommodating easier substrate binding, and (2) an α-helix instead of an antiparallel β-sheet in the C-terminal domain, which has been reported to be involved in binding ribosomal protein P2 in some RIPs. Furthermore, NMR chemical shift perturbation experiments revealed that the P2 binding site on MOD is located at the N-terminal domain near the internal inactivation region. This relocation of the P2 binding site can be rationalized by concerted changes in the electrostatic surface potential and 3D structures on the MOD protein and provides vital clues about the underlying molecular mechanism of this unique type of RIP.     

Functional interaction in establishment of ribosomal integrity between small subunit protein rpS6 and translational regulator rpL10/Grc5p

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.     

Functional interaction in establishment of ribosomal integrity between small subunit protein rpS6 and translational regulator rpL10/Grc5p

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.