Bacterial expression and enzymatic activity analysis of ME1, a ribosome-inactivating protein from Mirabilis expansa (original) (raw)
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The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50%1 inhibitory concentrations.
Ribosome-inactivating proteins: progress and problems
Cellular and Molecular Life Sciences, 2006
Ribosome-inactivating proteins (RIPs), mostly from plants, are enzymes which depurinate rRNA, thus inhibiting protein synthesis. They also depurinate other polynucleotide substrates. The biological activity of RIPs is not completely clarified, and sometimes independent of the inhibition of protein synthesis. There are differences in the cytotoxicity of RIPs and, consequently, in their toxicity to animals. Some RIPs are potent toxins, the best known being ricin, a potential biological weapon. New toxins have recently been identified. RIPs cause apop-totic and necrotic lesions, and induce production of cytokines causing inflammation. RIPs are potentially useful in agriculture and medicine because (i) they have antiviral activity and (ii) they are used for the preparation of conjugates with antibodies ('immunotoxins') or other carriers, rendering them specifically toxic to the cell target of the carrier, which may be helpful in therapy. The distribution, mechanism of action and role in nature of RIPs are not completely understood, and we can expect several future developments in their practical application.
Site-Specific Inactivation of Bacterial Genes Involved in Plant Rotting
Biotechnology & Biotechnological Equipment, 1995
The soft rot bacterium Erwinia carotovora subspecies carotovora strain ECJ4 contains multiple pectate lyase genes. Genes pe/9.5 and pell0.5, coding for pectate lyases (pi 9.5 and 10.5), were cloned on a plasmid, transformed into Escherichia coli, and expressed cata~yti ca!ly active products. The isoenzymes were subcloned separate~y and sequenced. Along most of their lengths, the genes are homologous to each other and to the pelBC family of Erwinia extracellular pectate lyase genes. However, the amino acid sequence of the Pell0.5 carboxyterminus diverges markedly from the PelBC consensus. We constructed hybrid genes and their products were found to be pectolytic. The pe/9. 5 and pell 0. 5 genes were mutagenized by marker exchange mutagenesis. A double mutant, pel9.5-pell0.5-, was obtained by mobilization of pLAJJ5 into the pell0.5::kan mutant and exchange recombination by the above method. Hybridization of EcoRI digests of the mutant chromosomal DNAs with pe/9.5-, pell0.5-, kan-, and let-specific probes gave profiles consistent with single-site insertions of the kan and tet genes in the corresponding pel genes. In planta accumulation of Pe/9.5 and Pell 0. 5 mRNAs was induced within 3 to 4 hr after inoculation, reaching maxima between 6 and 12 hr with Pell 0. 5 peaking at 6 hr and Pe/9. 5 at 9 hr. The induction of the two genes was regulated differential~y and the level of their mRNAs was higher during compatible than during incompatible conditions. The three mutants, obtained here, especially the double mutant, have diminished virulence compared to the wild type.
FEBS letters, 2015
We investigated the antimicrobial activity of PD-L4, a type 1 RIP from Phytolacca dioica. We found that this protein is active on different bacterial strains both in a native and denatured/alkylated form and that this biological activity is related to a cryptic peptide, named PDL440-65, identified by chemical fragmentation. This peptide showed the same antimicrobial activity of full-length protein and possessed, similarly to several antimicrobial peptides, an immunomodulatory effect on human cells. It assumes an alpha-helical conformation when interact with mimic membrane agents as TFE and likely bacterial membranes are a target of this peptide. To date PDL440-65 is the first antimicrobial peptide identified in a type 1 RIP.
Revising the Taxonomic Distribution, Origin and Evolution of Ribosome Inactivating Protein Genes
PLoS ONE, 2013
Ribosome inactivating proteins are enzymes that depurinate a specific adenine residue in the alpha-sarcin-ricin loop of the large ribosomal RNA, being ricin and Shiga toxins the most renowned examples. They are widely distributed in plants and their presence has also been confirmed in a few bacterial species. According to this taxonomic distribution, the current model about the origin and evolution of RIP genes postulates that an ancestral RIP domain was originated in flowering plants, and later acquired by some bacteria via horizontal gene transfer. Here, we unequivocally detected the presence of RIP genes in fungi and metazoa. These findings, along with sequence and phylogenetic analyses, led us to propose an alternative, more parsimonious, hypothesis about the origin and evolutionary history of the RIP domain, where several paralogous RIP genes were already present before the three domains of life evolved. This model is in agreement with the current idea of the Last Universal Common Ancestor (LUCA) as a complex, genetically redundant organism. Differential loss of paralogous genes in descendants of LUCA, rather than multiple horizontal gene transfer events, could account for the complex pattern of RIP genes across extant species, as it has been observed for other genes.
The maize (Zea mays) b-32 protein shows RIP activity in yeast cells
Maydica, 2018
Ribosome-inactivating proteins (RIPs) are either single-chain (type 1) or two-chain (type 2) toxins. They are toxic to eukaryotic cells by cleaving a N-glycosidic bond in an extremely conserved loop located in the 28S RNA. This releases a specific adenine and inactivates the ribosome, ultimately inhibiting protein synthesis. Plant RIPs have been intensely investigated because of their projected antiviral, antifungal and insecticidal activity. RIPs also have biomedical applications as the toxic mojety of immunotoxins. Given their biotechnological potentials, it is strategic to develop platforms to rapidly evaluate the activity of recombinant RIPs. This investigation fills this need in that it reports that the yeast Saccharomyces cerevisiae is a model system to assess the impact of genetic manipulations on the functionality of a recombinant Zea mays RIP named b-32.
2013
It is reported that some plants contain molecules that inactivate the ribosomes by inhibiting the protein synthesis through their N-glycosidase enzymatic activity. The molecules are identified to be group of proteins namely ribosomal inactivating proteins (RIPs). The RIPs have received a lot of attention in recent biomedical research because of their unique biological and enzymatic activities towards animal and human cells. RIPs can be toxic or non toxic based on their cytotoxicity. The first RIP, Ricin was identified from Ricinus communis seeds (castor beans) and this one was important milestone for plant protein identification. This paper reviews elaborately on recent progress in RIP studies- classification, biological and enzymatic activity and also its potential applications in medical research. Information on structural aspects of RIP compiled from the protein data bank and represented in this review paper will provide insight into researchers who aspire to work on cytotoxic dr...
The Streptomyces coelicolor genome encodes a type I ribosome-inactivating protein
Microbiology, 2010
Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in numerous plants, but also constitute a subunit of the bacterial Shiga toxin. Classification of plant RIPs is based on the absence (type I) or presence (type II) of an additional lectin module. In Shiga toxin, sugar binding is mediated by a distinct RIP-associated homopentamer. In the genome of two actinomycetes, we identified RIP-like proteins that resemble plant type I RIPs rather than the RIP subunit (StxA) of Shiga toxin. Some representatives of β- and γ-proteobacteria also contain genes encoding RIP-like proteins, but these are homologous to StxA. Here, we describe the isolation and initial characterization of the RIP-like gene product SCO7092 (RIPsc) from the Gram-positive soil bacterium Streptomyces coelicolor. The ripsc gene was expressed in Escherichia coli as a recombinant protein of about 30 kDa, and displayed the characteristic N-glycosidase activity causing specific rRNA depurination. In Str...