Ribosome-inactivating Protein Research Articles

Evolution and natural selection of ribosome-inactivating proteins in bacteria, fungi, and plants

Ribosome-inactivating proteins (RIPs) are found in bacteria, fungi, and plants, with a wide range of biological resistances such as anti-fungal, anti-viral, anti-insect, and anti-tumor. They can be roughly divided into proactive defense bacterial or fungal types and passive defense plant types. We identified 1592 RIP genes in bacteria, fungi, and plants. Approximately 88 % of the 764 bacterial RIPs were Shiga or Shiga-like toxins which were exotoxins and could rapidly enter cells to possess strong biotoxicity, and about 98 % of fungal RIPs were predicted as secreted proteins. RIPs were not detected in non-seed plants such as algae, bryophytes, and ferns. However, we found RIPs in some flowering and non-flowering seed plants. The existence of plant RIPs might be related to the structure of seeds or fruits, which might be associated with whether seeds are easy to survive and spread. The evolutionary characteristics of RIPs were different between dicotyledons and monocotyledons. In addition, we also found that RIP2 genes might emerge very early and be plant-specific. Some plant RIP1 genes might evolve from RIP2 genes. This study provides new insights into the evolution of RIPs.

Isolation of Ribosome-Inactivating Proteins from Seeds of Nigella sativa

Isolation of Ribosome-Inactivating Proteins from Seeds of Nigella sativa

The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2.

The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 μM) with little concomitant cytotoxicity (CC50 ~ 2 μM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 μM) but also the viral inhibition (IC50 ~ 1 μM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.

Ribosome inactivating proteins in insects: HGT, gene expression, and functional implications

Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that depurinate an adenine residue in the conserved alpha-sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. Previously, we reported the existence of these toxins in insects, whose presence is restricted to mosquitoes from the Culicinae subfamily (e.g., Aedes aegypti) and whiteflies from the Aleyrodidae family (e.g., Bemisia tabaci). Both groups of genes are derived from two independent horizontal gene transfer (HGT) events and are evolving under purifying selection. Here, we report and characterize the occurrence of a third HGT event in the Sciaroidea superfamily, which supports the recurrent acquisition of RIP genes by insects. Transcriptomic experiments, available in databases, allowed us to describe the temporal and spatial expression profiles for these foreign genes in these organisms. Furthermore, we found that RIP expression is induced after infection with pathogens and provided, for the first time, transcriptomic evidence of parasite SRL depurination. This evidence suggests a possible role of these foreign genes as immune effectors in insects.

Nature's Needle: Harnessing Bacterial Molecular Syringes

Nature's Needle: Harnessing Bacterial Molecular Syringes

Plant Toxic Proteins: Their Biological Activities, Mechanism of Action and Removal Strategies.

Plants evolve to synthesize various natural metabolites to protect themselves against threats, such as insects, predators, microorganisms, and environmental conditions (such as temperature, pH, humidity, salt, and drought). Plant-derived toxic proteins are often secondary metabolites generated by plants. These proteins, including ribosome-inactivating proteins, lectins, protease inhibitors, α-amylase inhibitors, canatoxin-like proteins and ureases, arcelins, antimicrobial peptides, and pore-forming toxins, are found in different plant parts, such as the roots, tubers, stems, fruits, buds, and foliage. Several investigations have been conducted to explore the potential applications of these plant proteins by analyzing their toxic effects and modes of action. In biomedical applications, such as crop protection, drug development, cancer therapy, and genetic engineering, toxic plant proteins have been utilized as potentially useful instruments due to their biological activities. However, these noxious metabolites can be detrimental to human health and cause problems when consumed in high amounts. This review focuses on different plant toxic proteins, their biological activities, and their mechanisms of action. Furthermore, possible usage and removal strategies for these proteins are discussed.

The toxins of vertically transmitted Spiroplasma.

Vertically transmitted (VT) microbial symbionts play a vital role in the evolution of their insect hosts. A longstanding question in symbiont research is what genes help promote long-term stability of vertically transmitted lifestyles. Symbiont success in insect hosts is due in part to expression of beneficial or manipulative phenotypes that favor symbiont persistence in host populations. In Spiroplasma, these phenotypes have been linked to toxin and virulence domains among a few related strains. However, these domains also appear frequently in phylogenetically distant Spiroplasma, and little is known about their distribution across the Spiroplasma genus. In this study, we present the complete genome sequence of the Spiroplasma symbiont of Drosophila atripex, a non-manipulating member of the Ixodetis clade of Spiroplasma, for which genomic data are still limited. We perform a genus-wide comparative analysis of toxin domains implicated in defensive and reproductive phenotypes. From 12 VT and 31 non-VT Spiroplasma genomes, ribosome-inactivating proteins (RIPs), OTU-like cysteine proteases (OTUs), ankyrins, and ETX/MTX2 domains show high propensity for VT Spiroplasma compared to non-VT Spiroplasma. Specifically, OTU and ankyrin domains can be found only in VT-Spiroplasma, and RIP domains are found in all VT Spiroplasma and three non-VT Spiroplasma. These domains are frequently associated with Spiroplasma plasmids, suggesting a possible mechanism for dispersal and maintenance among heritable strains. Searching insect genome assemblies available on public databases uncovered uncharacterized Spiroplasma genomes from which we identified several spaid-like genes encoding RIP, OTU, and ankyrin domains, suggesting functional interactions among those domain types. Our results suggest a conserved core of symbiont domains play an important role in the evolution and persistence of VT Spiroplasma in insects.

Expression, purification, characterization, and cytotoxic evaluation of the ML1-STxB fusion protein.

Targeted delivery of a toxin substance to cancer cells is one of the most recent cancer treatment options. Mistletoe Lectin-1 (ML1) in Viscum album L. is a Ribosome-inactivating proteins with anticancer properties. Therefore, it appears that a recombinant protein with selective permeability can be generated by fusing ML1 protein with Shiga toxin B, which can bind to Gb3 receptor that is abundantly expressed on cancer cells. In this study, we sought to produce and purify a fusion protein containing ML1 fused to STxB and evaluate its cytotoxic activities. The ML1-STxB fusion protein coding sequence was cloned into the pET28a plasmid, then was transformed into E. coli BL21-DE3 cells. Following induction of protein expression, Ni-NTA affinity chromatography was used to purify the protein. Using SDS-PAGE and western blotting, the expression and purification processes were validated. On the SkBr3 cell line, the cytotoxic effects of the recombinant proteins were evaluated. On SDS-PAGE and western blotting membrane, analysis of purified proteins revealed a band of approximately 41kDa for rML1-STxB. Ultimately, statistical analysis demonstrated that rML1-STxB exerted significant cytotoxic effects on SkBr3 cells at 18.09 and 22.52ng/L. The production, purification, and encapsulation of rML1-STxB fusion protein with potential cancer cell-specific toxicity were successful. However, additional research must be conducted on the cytotoxic effects of this fusion protein on other malignant cell lines and in vivo cancer models.

New Insights on Saporin Resistance to Chemical Derivatization with Heterobifunctional Reagents.

Saporin is a type 1 ribosome-inactivating protein widely used as toxic payload in the construction of targeted toxins, chimeric molecules formed by a toxic portion linked to a carrier moiety. Among the most used carriers, there are large molecules (mainly antibodies) and small molecules (such as neurotransmitters, growth factors and peptides). Some saporin-containing targeted toxins have been used for the experimental treatment of several diseases, giving very promising results. In this context, one of the reasons for the successful use of saporin lies in its resistance to proteolytic enzymes and to conjugation procedures. In this paper, we evaluated the influence of derivatization on saporin using three heterobifunctional reagents, namely 2-iminothiolane (2-IT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]toluene (SMPT). In order to obtain the highest number of inserted -SH groups with the lowest reduction of saporin biological activities, we assessed the residual ability of saporin to inhibit protein synthesis, to depurinate DNA and to induce cytotoxicity after derivatization. Our results demonstrate that saporin maintains an excellent resistance to derivatization processes, especially with SPDP, and permit us to define reaction conditions, in which saporin biological properties may not be altered. Therefore, these findings provide useful information for the construction of saporin-based targeted toxins, especially with small carriers.

Structural and functional characterization of the cytotoxic protein ledodin, an atypical ribosome-inactivating protein from shiitake mushroom (Lentinula edodes).

We have purified ledodin, a cytotoxic 22-kDa protein from shiitake mushroom (Lentinula edodes) consisting of a 197 amino acid chain. Ledodin possessed N-glycosylase activity on the sarcin-ricin loop of mammalian 28S rRNA and inhibited protein synthesis. However, it was not active against insect, fungal, and bacterial ribosomes. In vitro and in silico studies suggested that ledodin exhibits a catalytic mechanism like that of DNA glycosylases and plant ribosome-inactivating proteins. Moreover, the sequence and structure of ledodin was not related to any protein of known function, although ledodin-homologous sequences were found in the genome of several species of fungi, some edible, belonging to different orders of the class Agaricomycetes. Therefore, ledodin could be the first of a new family of enzymes widely distributed among this class of basidiomycetes. The interest of these proteins lies both, in the fact that they can be a toxic agent of some edible mushrooms and in their application in medicine and biotechnology.

Anti-human immunodeficiency virus-1 activity of MoMo30 protein isolated from the traditional African medicinal plant Momordica balsamina

BackgroundPlants are used in traditional healing practices of many cultures worldwide. Momordica balsamina is a plant commonly used by traditional African healers as a part of a treatment for HIV/AIDS. It is typically given as a tea to patients with HIV/AIDS. Water-soluble extracts of this plant were found to contain anti-HIV activity.MethodsWe employed cell-based infectivity assays, surface plasmon resonance, and a molecular-cell model of the gp120-CD4 interaction to study the mechanism of action of the MoMo30-plant protein. Using Edman degradation results of the 15 N-terminal amino acids, we determined the gene sequence of the MoMo30-plant protein from an RNAseq library from total RNA extracted from Momordica balsamina.ResultsHere, we identify the active ingredient of water extracts of the leaves of Momordica balsamina as a 30 kDa protein we call MoMo30-plant. We have identified the gene for MoMo30 and found it is homologous to a group of plant lectins known as Hevamine A-like proteins. MoMo30-plant is distinct from other proteins previously reported agents from the Momordica species, such as ribosome-inactivating proteins such as MAP30 and Balsamin. MoMo30-plant binds to gp120 through its glycan groups and functions as a lectin or carbohydrate-binding agent (CBA). It inhibits HIV-1 at nanomolar levels and has minimal cellular toxicity at inhibitory levels.ConclusionsCBAs like MoMo30 can bind to glycans on the surface of the enveloped glycoprotein of HIV (gp120) and block entry. Exposure to CBAs has two effects on the virus. First, it blocks infection of susceptible cells. Secondly, MoMo30 drives the selection of viruses with altered glycosylation patterns, potentially altering their immunogenicity. Such an agent could represent a change in the treatment strategy for HIV/AIDS that allows a rapid reduction in viral loads while selecting for an underglycosylated virus, potentially facilitating the host immune response.

Computational design of fusion proteins against ErbB2-amplified tumors inspired by ricin toxin.

Although the anti-cancer activity of ricin is well-known, its non-specific targeting challenges the development of ricin-derived medicines. In the present study, novel potential ribosome-inactivating fusion proteins (RIPs) were computationally engineered by incorporation of an ErbB2-dependant penetrating peptide (KCCYSL, MARAKE, WYSWLL, MARSGL, MSRTMS, and WYAWML), a linker (either EAAAK or GGGGS) and chain A of ricin which is responsible for the ribosome inactivation. Molecular dynamics simulations assisted in making sure that the least change is made in conformation and dynamic behavior of ricin chain A in selected chimeric protein (CP). Moreover, the potential affinity of the selected CPs against the ligand-uptaking ErbB2 domain was explored by molecular docking. The results showed that two CPs (CP2 and 10) could bind the receptor with the greatest affinity.

Streptavidin-Saporin: Converting Biotinylated Materials into Targeted Toxins.

Streptavidin-Saporin can be considered a type of 'secondary' targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the 'Molecular Surgery' capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.

Genetic modification of crop plants with ribosome-inactivating protein genes for enhanced resistance to pathogens and pests

Genetic modification of crop plants with ribosome-inactivating protein genes for enhanced resistance to pathogens and pests

Recombinant anti-HIV MAP30, a ribosome inactivating protein: against plant virus and bacteriophage

The ribosome inactivating proteins (RIPs) efficiently decrease the microbial infections in plants. Momordicacharantia MAP30 is a type I RIP that has not been investigated against plant viruses or bacteriophages. To evaluate of these activities, the recombinant MAP30 (rMAP30) was produced in the hairy roots of Nicotianatabacum. Inoculation of 3 μg of transgenic total protein or 0.6 μg of rMAP30 against 0.1 μg of TMV reduced the leaf necrotic spots to 78.23% and 82.72%, respectively. The treatment of 0.1 μg of CMV with rMAP30 (0.6 μg) showed the reduction in the leaf necrotic spots to 85.8%. While the infection was increased after rMAP30 dilution. In the time interval assays, the leaves were first inoculated with 1 μg of rMAP30 or 0.1 μg of purified TMV or CMV agent for 6 h, then virus or protein was applied in order. This led the spot reduction to 35.22% and 67% for TMV, and 38.61% and 55.31% for CMV, respectively. In both the pre- and co-treatments of 1:10 or 1:20 diluted bacteriophage with 15 μg of transgenic total protein, the number and diameter of the plaques were reduced. The results showed that the highest inhibitory effect was observed in the pre-treatment assay of bacteriophage with transgenic total protein for 24 h. The decrease in the growth of bacteriophage caused more growth pattern of Escherichiacoli. The results confirm that rMAP30 shows antibacterial activity against Streptococcusaureus and E.coli, antifungal activity against Candidaalbicans, and antiviral activity against CMV and TMV. Moreover, rMAP30 exhibits anti-phage activity for the first time. According to our findings, rMAP30 might be a valuable preservative agent in foods and beverages in the food industry as well as an antiviral and antimicrobial mixture in agriculture.

Evolution of codon and amino acid usage in bacterial protein toxins

Toxin proteins are secreted by most pathogens as an integral part of pathogenic mechanism(s). The toxins act by either damaging the host cell membrane (for example, pore-forming toxins and RTX toxins) or by modulation of important cellular pathways (for example, inhibition of protein translation by ribosome-inactivating proteins). The mechanism of action of these toxins provides the pathogen with strategies for adaptation in the unfavorable host environment. Though, secreted by different pathogenic species, the protein toxins seem to share common features that allow the protein to bind to specific molecules and enter the host cell. Earlier studies have suggested role of several events like horizontal gene transfer and insertion-deletion mutations in evolution of protein toxins. The present study involving 125 bacterial protein toxins secreted by 49 pathogenic bacteria focuses on the role and constraints of the bacterial genome on evolution of codon and amino acid usage in respective bacterial protein toxins. We compare the nucleotide composition, codon and dinucleotide usage trends between different classes of bacterial protein toxins and between individual toxins and the parent bacterial genome expressing the toxin(s)

Plant Toxins as Potential Alternatives to Botulinum Toxin for Eye-Movement Disorder Therapy

The most successful alternative to traditional surgery for ocular muscle spasm treatment is the intramuscular injection of botulinum toxin (BTX), which allows the maintenance of the muscle dynamics and the absence of scars. However, the main BTX disadvantage is its nonpermanent effect. A possible way for overcoming this obstacle could be represented by the enzymatic surgery using plant toxins known as ribosome-inactivating proteins (RIPs). In this paper, two highly toxic RIPs, namely, ricin and stenodactylin, were considered in a preliminary study for their possible use in the treatment of strabismus and oculofacial dystonias, as alternatives to BTX. Both RIPs showed a strong cytotoxic effect against rhabdomyosarcoma cell lines and myotube differentiated cells, with stenodactylin being about 10-fold more toxic than ricin. Moreover, stenodactylin showed a much higher cytotoxicity on myoblasts than on rhabdomyosarcoma cells. In our experimental conditions, stenodactylin did not damage conjunctival cells. Despite the limitations due to in vitro experiments, our data show that the high cytotoxicity of stenodactylin allows the use of a very low dose and, consequently, of very low injection volumes. This can represent a great advantage in the case of in vivo locoregional treatment. Furthermore, it is possible to modulate the chemoablation of myocytes while destroying myoblasts, thus reducing regenerative phenomena. The risk of cytotoxicity to surrounding tissues would be strongly reduced by the low injected volume and the relative resistance of conjunctival cells. In conclusion, our data suggest that stenodactylin and ricin could represent potential candidates to substitute BTX in ocular dystonia therapy.

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On the Cover: Expanding role of ribosome-inactivating proteins: From toxins to therapeutics by Sharma et al.; Article first published: 7 October 2022; https://doi.org/10.1002/iub.2675

Abrin inactivating mitochondrial ribosome induced anther abortion in cotton CMS line LD6A by damaging mitochondrial membrane structure

Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we aimed to elucidate the molecular pathways, regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) lines were analyzed for physiological and proteomic changes during the development process. Transmission electron micrographs of anther tapetum showed a relatively indistinct inner ridge of CMS than of LD6B mitochondria at tetrad stage. Furthermore, at pollen mother cell and tetrad stages, CMS anthers accumulated a significantly higher reactive oxygen species than the maintainer line. At the protein level, CMS anthers induced significantly higher Abrin (XP_016705148.2), lower tricarboxylic acid cycle enzyme (malate dehydrogenase, malic enzyme, isocitrate dehydrogenase), and histone acetyltransferase expression than its maintainer line. Suggesting that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein Abrin in CMS line damaged mitochondrial membrane. This inhibited energy supply from the non-degraded tapetum, and consequently induced pollen sterility in LD6A. These data provide new insights into CMS mechanism in cotton crops that can be used for developing new CMS germplasm resources.

Reducing the Immunogenicity of Pulchellin A-Chain, Ribosome-Inactivating Protein Type 2, by Computational Protein Engineering for Potential New Immunotoxins

Pulchellin is a plant biotoxin categorized as a type 2 ribosome-inactivating protein (RIPs) which potentially kills cells at very low concentrations. Biotoxins serve as targeting immunotoxins (IT), consisting of antibodies conjugated to toxins. ITs have two independent protein components, a human antibody and a toxin with a bacterial or plant source; therefore, they pose unique setbacks in immunogenicity. To overcome this issue, the engineering of epitopes is one of the beneficial methods to elicit an immunological response. Here, we predicted the tertiary structure of the pulchellin A-chain (PAC) using five common powerful servers and adopted the best model after refining. Then, predicted structure using four distinct computational approaches identified conformational B-cell epitopes. This approach identified some amino acids as a potential for lowering immunogenicity by point mutation. All mutations were then applied to generate a model of pulchellin containing all mutations (so-called PAM). Mutants’ immunogenicity was assessed and compared to the wild type as well as other mutant characteristics, including stability and compactness, were computationally examined in addition to immunogenicity. The findings revealed a reduction in immunogenicity in all mutants and significantly in N146V and R149A. Furthermore, all mutants demonstrated remarkable stability and validity in Molecular Dynamic (MD) simulations. During docking and simulations, the most homologous toxin to pulchellin, Abrin-A was applied as a control. In addition, the toxin candidate containing all mutations (PAM) disclosed a high level of stability, making it a potential model for experimental deployment. In conclusion, by eliminating B-cell epitopes, our computational approach provides a potential less immunogenic IT based on PAC.