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PubMed2005

The 1.4 anstroms structure of dianthin 30 indicates a role of surface potential at the active site of type 1 ribosome inactivating proteins.

Ribosome inactivating proteins (RIPs) are plant proteins with enzymatic activity identified as rRNA N-glycosidase (EC 3.2.2.22), which cleaves the N-glycosidic bond of a specific adenine on the ricin/sarcin region of rRNA, thus causing inhibition of protein synthesis. They also depurinate extensively DNA and other polynucleotides. The three-dimensional structure of dianthin 30, a type 1 (single-chain) RIP of Dianthus caryophyllus (leaves), is now described at 1.4 angstroms, a resolution never achieved before for any RIP. The fold typical of RIPs is conserved, despite some differences in the loop regions. The general structure comparison by superimposed alpha-carbon (249 atoms) and the sequence alignment by structure for dianthin 30 and saporin-S6 give a root mean square deviation of 0.625 angstroms. Despite the differences reported for the biological activities of the two RIPs, their structures fit quite well and both show a protein segment containing strands beta7, beta8, and beta9 shorter than other RIPs. However, the surface electrostatic potential in the active site region neatly distinguishes dianthin 30 from saporin-S6. The possible relationship between the charge distribution and the behavior of the proteins toward different substrates is discussed.

Amino Acid Sequence↗

PubMed2004

The structure of ribosome inactivating proteins.

Ribosome Inactivating Proteins, RIPs, depurinate an invariant adenine from the 28S rRNA of eukaryotic ribosomes; they have evolved to near enzymatic perfection for this task. The N-glycosidase fold is conserved in plant and bacterial enzymes. RIPs can form complexes with cell surface recognition proteins that dramatically increase the cytotoxicity of the molecule.

Drug Design↗

PubMed2000

Current status of ribosome inactivating proteins.

Ribosome inactivating proteins (RIPs) are a group of naturally occurring plant proteins with a RNA-N-glycosidases activity which depurinate rRNA at a specific universally conserved position (i.e. cleavage of N-glycosidic bond of a specific adenine of 28S rRNA). These proteins are found in different parts of plants, in concentrations ranging from a few micrograms to several hundred mg per 100 g of plant tissues. RIPs exist in two forms, type 1 having a single polypeptide chain with a molecular mass of approximately 30 kDa possessing N-glycosidase activity; and type 2 with two or four polypeptide chains having a molecular mass of approximately 60 kDa and approximately 120 kDa respectively showing lectin activity along with N-glycosidase moiety. Such biomolecules causing cytotoxicity are being exploited for designing immunotoxins/hormonotoxins using heterobifunctional conjugates. These carrier conjugates with the RIPs can influence cellular trafficking and inhibition of protein synthesis. We are witnessing a novel protein from plants that can be utilised for various therapeutical treatments ranging from cancers, AIDS and other viral diseases of present times.

Plant Proteins↗

PubMed2004

Cinnamomin--a versatile type II ribosome-inactivating protein.

Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can specifically act on the universally conserved sarcin/ricin domain (S/R domain) of the largest RNA in ribosome and thus irreversibly inactivate ribosome for protein synthesis. Cinnamomin is a multifunctional type II RIP isolated in our laboratory from the mature seeds of the camphor tree. This protein has been extensively studied with regard to its purification, characteristics, structure and function, genetic expression, enzymatic mechanism, physiological role in seed cell and toxicity to cancer cells and insect larvae. The research results of cinnamomin obtained in our laboratory are summarized in this review. Understanding of cinnamomin and the relative new proteins will help expand our knowledge of RIPs and may accelerate theoretical study and the development of their potential applications.

Algal Proteins↗

PubMed1991

Effects of plant ribosome-inactivating proteins on ribosomes from Musca domestica.

1. Ribosomes from M. domestica larvae were isolated and their susceptibility to the action of several ribosome-inactivating proteins (RIPs) from plants was tested. 2. Ribosome-inactivating proteins inhibited, to different extents, phenylalanine polymerization by ribosomes. 3. Analysis of RNA from RIP-treated ribosomes showed the appearance of an aniline-cleavable rRNA fragment resulting from the N-glycosidase activity of the RIPs. 4. The release of adenine from saporin 6-treated M. domestica ribosomes was demonstrated by h.p.l.c. analysis.

Adenine↗

PubMed2004

Enzymatic activity of toxic and non-toxic type 2 ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) display adenine polynucleotide glycosylase activity on different nucleic acid substrates, which at the ribosomal level is responsible for the arrest of protein synthesis. Some type 2 RIPs, namely ricin and related proteins, are extremely toxic to mammalian cells and animals whilst other type 2 RIPs (non-toxic type 2 RIPs) display three to four logs less toxicity. We studied whether a correlation exists between toxicity on cells and enzymatic activity on nucleic acids. All type 2 RIPs differ in their depurinating activity on the different substrates with differences of up to one to two logs. The toxicity of type 2 RIPs is independent of their enzymatic activity on nucleic acids or on ribosomes.

Abrin↗

PubMed1995

Topological requirements for recognition and cleavage of DNA by ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) were demonstrated to exhibit a unique enzymatic activity on cleaving supercoiled double-stranded DNA into the nicked or linear form. Although there is an interaction between supercoiled DNA and RIP, no sequence-specific recognition was involved. Instead, RIPs recognize supercoiled DNA by conformational specificity. Negatively supercoiled DNA is the preferential conformation in the action of RIPs. When double-stranded DNA occurs in the supercoiled form, even if with lower linking number, RIPs can still convert it into nicked or linear form. Terminal-labelling experiments indicated that radioactivity was incorporated into putative 5'-ends of nicked or linear DNA generated by RIPs. We conclude that RIPs act as a novel supercoil-dependent endonuclease when they cleavage supercoiled DNA. The impossibility that contaminating enzymes in the RIP preparations cleaved the supercoiled DNA is briefly discussed.

Algal Proteins↗

PubMed1987

Effect of ribosome-inactivating proteins on ribosomes from Tetrahymena pyriformis and Acanthamoeba castellanii.

The effect of ribosome-inactivating proteins type 1 (single-chain) and type 2 (two-chain, toxins) on polyphenylalanine polymerization by Tetrahymena pyriformis and Acanthamoeba castellanii ribosomes has been studied. The reaction catalysed by tetrahymena ribosomes was inhibited by two ribosome-inactivating proteins type 1 (dianthin 32 and, less effectively, momordin) whereas the reaction catalysed by amoeba ribosomes was inhibited, in a decreasing order of activity, by three ribosome-inactivating proteins type 1 (dianthin 32, saporin 6 and bryodin) and by two toxins (abrin and volkensin).

Acanthamoeba↗

PubMed2002

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.

Catalysis↗

PubMed1999

Identity elements in bovine tRNA(Trp) required for the specific stimulation of gelonin, a plant ribosome-inactivating protein.

Ribosome-inactivating proteins (RIPs) are RNA-N-glycosidases widely present in plants that depurinate RNA in ribosomes at a specific universally conserved position, A4324, in the rat 28S rRNA. A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. Among cofactor-dependent RIPs, gelonin is specifically and uniquely stimulated by tRNA(Trp). The active species are avian (chicken) and mammalian (beef, rat, and rabbit) tRNA(Trp), whereas yeast tRNA(Trp) is completely devoid of stimulating activity. In the present article, bovine and yeast tRNA(Trp) with unmodified bases were prepared by assembly of the corresponding genes from synthetic oligonucleotides followed by PCR and T7 RNA polymerase transcription of the amplified products. The two synthetic tRNAs were fully active (bovine) or inactive (yeast) as the wild-type tRNAs. Construction of chimeric tRNA(Trp) transcripts identified the following bovine nucleotides as recognition elements for gelonin-stimulating activity: G26 and bp G12-C23 in the D arm and G57, A59, and bp G51-C63 and U52-A62 in the T arm. Among single-stranded nucleotides, A59 has a prominent role, but full expression of the gelonin-stimulating activity requires an extensive cooperation between nucleotides in both arms.

Animals↗

PubMed1992

Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites.

Saporin-S6, a ribosome-inactivating protein (RIP) from Saponaria officinalis released more than 1 mol of adenine/mol of ribosomes from house fly (Musca domestica) larvae and from rat liver. The release of adenine from rat liver ribosomes by several RIPs (plant enzymes with RNA N-glycosidase activity) was examined. Saporins, pokeweed antiviral protein from roots of Phytolacca americana (PAP-R), and trichokirin from Trichosanthes kirilowii seeds depurinated rat liver ribosomes at more than one site. Up to 33 mol of adenine were released from 1 mol of ribosomes. This property is not common to all RIPS.

Animals↗

PubMed2006

Isolation and purification of ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in plants, fungi, and bacteria. RIPs inhibit protein synthesis by virtue of their enzymatic activity, selectively cleaving a specific adenine residue from a highly conserved, surface-exposed, stem-loop (S/R loop) structure in the 28S rRNA of ribosomes. Some RIPs also exhibit a number of other enzymatic activities such as RNase, DNase, phospholipase, and superoxide dismutase (SOD). RIPs are considered to be plant defense-related proteins as they are able to inhibit the multiplication and growth of several pathogenic virus, fungi, and bacteria either alone or in conjugation with other defense-related proteins. The mechanism of inhibitory activity of RIPs against fungal pathogens seems to be by directly inhibiting fungal growth rather than depurinating host plant ribosomes and causing cell death as previously envisaged. This chapter describes the protocol used to isolate and purify RIPs from plant tissues.

Glycoside Hydrolases↗

PubMed2004

Genetics of ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) are a heterogeneous group of enzymes found mainly in plants and a few bacteria that possess N-glycosidase activity on ribosomes and a related polynucleotide adenosine glycosidase activity on naked nucleic acids. They encompass single enzymatic chains, heterodimeric toxic lectins and related agglutinins. Plants commonly produce several RIP isoforms encoded by multi-gene families. The toxic lectins possess adaptations related to their cytotoxic role.

Animals↗

PubMed2001

Comparison of the embryotoxic effects of saporin, agrostin (type 1 ribosome-inactivating proteins) and ricin (a type 2 ribosome-inactivating protein).

The effects of two type I ribosome-inactivating proteins, saporin and agrostin, and the type 2 ribosome-inactivating protein ricin and its constituent A and B chains, on the development of cultured mouse embryos, were investigated. Saporin and agrostin had similar embryotoxicity which was approximately 10(5) times weaker than that of ricin and its A and B chains, and the embryotoxicity of saporin and agrostin increased gradually with the dosage. In contrast, there was an abrupt rise in embryotoxictiy of ricin-A chain and B chain as the dose was raised from 10 to 20 ng x ml(-1). Saporin and agrostin did not affect the heart-beat and otic placode, but at 10 ng x ml(-1) ricin and 40 ng x ml(-1) ricin-A chain and B chain, all of the treated embryos exhibited abnormalities in heart-beat, yolk sac circulation, body axis, optic placode, otic placode, forelimb buds, branchial apparatus and cranial neural tube.

Animals↗

PubMed1992

Characterization of Mirabilis antiviral protein--a ribosome inactivating protein from Mirabilis jalapa L.

A protein was purified from root tubers of Mirabilis jalapa to homogeneity by ion-exchange chromatography on CM-Sepharose CL-6B and FPLC on Mono-S column. The purified protein was confirmed to be Mirabilis antiviral protein (MAP). However, in addition to its antiviral property, the MAP was demonstrated to possess abortifacient activity in pregnant mice, inhibitory effect on cell-free protein synthesis and antiproliferative effect on tumor cells. As judged from its biological and physiochemical properties, MAP is a type I ribosome-inactivating protein.

Abortifacient Agents↗

PubMed2003

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.

Algal Proteins↗

PubMed1995

Effects and molecular action of ribosome-inactivating proteins on ribosomes from Streptomyces lividans.

The effects of 29 type 1 and 2 type 2 ribosome-inactivating proteins (RIPs) from plants on polyuridylic acid-directed polyphenylalanine synthesis carried out by purified ribosomes from Streptomyces lividans were studied. Only dianthin 32, saporins R1 and R3, momordin I, trichokirin, Hura crepitans RIP 5 from latex, crotins 2 and 3, and PAPs C, R, and S, inhibited polyphenylalanine synthesis. Both the type 2 RIPs ricin and volkensin were ineffective on translation. The magnesium concentration affected the inhibition of translation to a considerable extent. Upon treatment with inhibitory RIPs, extraction of rRNA and further treatment with acid aniline, S. lividans ribosomes released an RNA fragment of about 130 nucleotides. The 5' terminal sequence of this rRNA fragment was 5'-GAGGACCGGGACGGACGAACCUCUGGUGUGCCAGUUGU-3', similar to the sequence obtained in Escherichia coli. This indicates that the most probable molecular action of these RIPs on S. lividans and E. coli ribosomes is the same: depurination of the rRNA at a site relevant to the translation mechanism and that has been highly conserved throughout evolution.

Base Sequence↗

PubMed2005

Ribosome inactivating proteins and apoptosis.

Ribosome inactivating proteins (RIPs) are protein toxins that are of plant or microbial origin that inhibit protein synthesis by inactivating ribosomes. Recent studies suggest that RIPs are also capable of inducing cell death by apoptosis. Though many reports are available on cell death induced by RIPs, the mechanism involved is not well studied. Comparison of pathways of apoptosis and cellular events induced by various RIPs suggests a central role played by mitochondria, probably acting as an integrator of cellular stress and cell death. The purpose of this review is to compare the various apoptotic pathways that may be involved and propose a general pathway in RIP-induced cell death.

Animals↗