Activity Of Ribosome-inactivating Proteins Research Articles

Antifungal Activity of Ribosome-Inactivating Proteins

The control of crop diseases caused by fungi remains a major problem and there is a need to find effective fungicides that are environmentally friendly. Plants are an excellent source for this purpose because they have developed defense mechanisms to cope with fungal infections. Among the plant proteins that play a role in defense are ribosome-inactivating proteins (RIPs), enzymes obtained mainly from angiosperms that, in addition to inactivating ribosomes, have been studied as antiviral, fungicidal, and insecticidal proteins. In this review, we summarize and discuss the potential use of RIPs (and other proteins with similar activity) as antifungal agents, with special emphasis on RIP/fungus specificity, possible mechanisms of antifungal action, and the use of RIP genes to obtain fungus-resistant transgenic plants. It also highlights the fact that these proteins also have antiviral and insecticidal activity, which makes them very versatile tools for crop protection.

Synthesis of biologically active Shiga toxins in cell-free systems

Shiga toxins (Stx) produced by pathogenic bacteria can cause mild to severe diseases in humans. Thus, the analysis of such toxins is of utmost importance. As an AB5 toxin, Stx consist of a catalytic A-subunit acting as a ribosome-inactivating protein (RIP) and a B-pentamer binding domain. In this study we synthesized the subunits and holotoxins from Stx and Stx2a using different cell-free systems, namely an E. coli- and CHO-based cell-free protein synthesis (CFPS) system. The functional activity of the protein toxins was analyzed in two ways. First, activity of the A-subunits was assessed using an in vitro protein inhibition assay. StxA produced in an E. coli cell-free system showed significant RIP activity at concentrations of 0.02 nM, whereas toxins synthesized in a CHO cell-free system revealed significant activity at concentrations of 0.2 nM. Cell-free synthesized StxA2a was compared to StxA2a expressed in E. coli cells. Cell-based StxA2a had to be added at concentrations of 20 to 200 nM to yield a significant RIP activity. Furthermore, holotoxin analysis on cultured HeLa cells using an O-propargyl-puromycin assay showed significant protein translation reduction at concentrations of 10 nM and 5 nM for cell-free synthesized toxins derived from E. coli and CHO systems, respectively. Overall, these results show that Stx can be synthesized using different cell-free systems while remaining functionally active. In addition, we were able to use CFPS to assess the activity of different Stx variants which can further be used for RIPs in general.

Biological Activities of Ribosome-Inactivating Proteins

After more than 50 years of research, studies on the structure and biological activities of ribosome-inactivating proteins (RIPs) continue to provide a field of great interest within the scientific community, both for the health risks they pose and their applications in medicine and biotechnology [...].

Integrated Transcriptome and Proteome Analysis Provides Insight into the Ribosome Inactivating Proteins in Plukenetia volubilis Seeds.

Plukenetia volubilis is a highly promising plant with high nutritional and economic values. In our previous studies, the expression levels of ricin encoded transcripts were the highest in the maturation stage of P. volubilis seeds. The present study investigated the transcriptome and proteome profiles of seeds at two developmental stages (Pv-1 and Pv-2) using RNA-Seq and iTRAQ technologies. A total of 53,224 unigenes and 6026 proteins were identified, with functional enrichment analyses, including GO, KEGG, and KOG annotations. At two development stages of P. volubilis seeds, 8815 unique differentially expressed genes (DEGs) and 4983 unique differentially abundant proteins (DAPs) were identified. Omics-based association analysis showed that ribosome-inactivating protein (RIP) transcripts had the highest expression and abundance levels in Pv-2, and those DEGs/DAPs of RIPs in the GO category were involved in hydrolase activity. Furthermore, 21 RIP genes and their corresponding amino acid sequences were obtained from libraries produced with transcriptome analysis. The analysis of physicochemical properties showed that 21 RIPs of P. volubilis contained ricin, the ricin_B_lectin domain, or RIP domains and could be divided into three subfamilies, with the largest number for type II RIPs. The expression patterns of 10 RIP genes indicated that they were mostly highly expressed in Pv-2 and 4 transcripts encoding ricin_B_like lectins had very low expression levels during the seed development of P. volubilis. This finding would represent valuable evidence for the safety of oil production from P. volubilis for human consumption. It is also notable that the expression level of the Unigene0030485 encoding type I RIP was the highest in roots, which would be related to the antiviral activity of RIPs. This study provides a comprehensive analysis of the physicochemical properties and expression patterns of RIPs in different organs of P. volubilis and lays a theoretical foundation for further research and utilization of RIPs in P. volubilis.

Structural Distinctive 26SK, a Ribosome-Inactivating Protein from Jatropha curcas and Its Biological Activities.

Ribosome-inactivating proteins (RIPs) are a group of proteins exhibiting N-glycosidase activity leading to an inactivation of protein synthesis. Thirteen predicted Jatropha curcas RIP sequences could be grouped into RIP types 1 or 2. The expression of the RIP genes was detected in seed kernels, seed coats, and leaves. The full-length cDNA of two RIP genes (26SK and 34.7(A)SK) were cloned and studied. The 34.7(A)SK protein was successfully expressed in the host cells while it was difficult to produce even only a small amount of the 26SK protein. Therefore, the crude proteins were used from E. coli expressing 26SK and 34.7(A)SK constructs and they showed RIP activity. Only the cell lysate from 26SK could inhibit the growth of E. coli. In addition, the crude protein extracted from 26SK expressing cells displayed the effect on the growth of MDA-MB-231, a human breast cancer cell line. Based on in silico analysis, all 13 J. curcas RIPs contained RNA and ribosomal P2 stalk protein binding sites; however, the C-terminal region of the P2 stalk binding site was lacking in the 26SK structure. In addition, an amphipathic distribution between positive and negative potential was observed only in the 26SK protein, similar to that found in the anti-microbial peptide. These findings suggested that this 26SK protein structure might have contributed to its toxicity, suggesting potential uses against pathogenic bacteria in the future.

Antiviral Activity of Ribosome-Inactivating Proteins.

Ribosome-inactivating proteins (RIPs) are rRNA N-glycosylases from plants (EC 3.2.2.22) that inactivate ribosomes thus inhibiting protein synthesis. The antiviral properties of RIPs have been investigated for more than four decades. However, interest in these proteins is rising due to the emergence of infectious diseases caused by new viruses and the difficulty in treating viral infections. On the other hand, there is a growing need to control crop diseases without resorting to the use of phytosanitary products which are very harmful to the environment and in this respect, RIPs have been shown as a promising tool that can be used to obtain transgenic plants resistant to viruses. The way in which RIPs exert their antiviral effect continues to be the subject of intense research and several mechanisms of action have been proposed. The purpose of this review is to examine the research studies that deal with this matter, placing special emphasis on the most recent findings.

Functional Assays for Measuring the Catalytic Activity of Ribosome Inactivating Proteins.

Ribosome-inactivating proteins (RIPs) are potent toxins that inactivate ribosomes by catalytically removing a specific adenine from the α-sarcin/ricin loop (SRL) of the large rRNA. Direct assays for measuring depurination activity and indirect assays for measuring the resulting translation inhibition have been employed to determine the enzyme activity of RIPs. Rapid and sensitive methods to measure the depurination activity of RIPs are critical for assessing their reaction mechanism, enzymatic properties, interaction with ribosomal proteins, ribotoxic stress signaling, in the search for inhibitors and in the detection and diagnosis of enteric infections. Here, we review the major assays developed for measuring the catalytic activity of RIPs, discuss their advantages and disadvantages and explain how they are used in understanding the catalytic mechanism, ribosome specificity, and dynamic enzymatic features of RIPs.

A novel adenine-releasing assay for ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) are toxic enzymes that are mostly biosynthesized by plants. RIPs are N-glycosidases that cleave an essential adenine molecule from the 28S rRNA. This is followed by the irreversible inhibition of protein synthesis leading to cell death. By fusing RIPs to cancer cell specific targeting ligands RIPs have been utilized for targeted anti-tumor therapy. The anti-tumoral efficiency of such conjugates depends significantly on the N-glycosidase activity of the RIP domain. Different methods have been developed in order to determine the N-glycosidase activity of RIPs and RIP domain containing anti-tumor toxins. However the existing methods are elaborate and include radioassays, HPLC and enzymatic conversion assays. Here, a simple and cost effective N-glycosidase assay is presented, which is based on the direct determination of the released adenine by thin-layer chromatography (TLC) and TLC-densitometry. An adenine based single stranded oligonucleotide is used as substrate. Following TLC development the released adenine is quantified on silica glass plates by UV absorbance at 260nm.

Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila.

While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the α-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea’s defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.

Ribosome-Inactivating Proteins from Plants: A Historical Overview.

This review provides a historical overview of the research on plant ribosome-inactivating proteins (RIPs), starting from the first studies at the end of eighteenth century involving the purification of abrin and ricin, as well as the immunological experiments of Paul Erlich. Interest in these plant toxins was revived in 1970 by the observation of their anticancer activity, which has given rise to a large amount of research contributing to the development of various scientific fields. Biochemistry analyses succeeded in identifying the enzymatic activity of RIPs and allowed for a better understanding of the ribosomal machinery. Studies on RIP/cell interactions were able to detail the endocytosis and intracellular routing of ricin, thus increasing our knowledge of how cells handle exogenous proteins. The identification of new RIPs and the finding that most RIPs are single-chain polypeptides, together with their genetic sequencing, has aided in the development of new phylogenetic theories. Overall, the biological properties of these proteins, including their abortifacient, anticancer, antiviral and neurotoxic activities, suggest that RIPs could be utilized in agriculture and in many biomedical fields, including clinical drug development.

Formulation of Medium Viscosity Chitosan-Pectin –MJ Protein Nanoparticles Conjugated with Anti-Ep-CAM and Its Cytotoxicity Against T47D Breast Cancer Cell Lines

Chitosan nanoparticle could become potential formula to protect protein degradation during therapy,since chitosan nanoparticles have “proton sponge hypothesis” mechanism on its protection. Chitosan and pectinis used as basic formula of drug delivery because of its biodegradable and biocompatible properties. Chitosanpectin nanoparticles can be formulated by polyelectrolit complex. EpCAM showed excessive expression inepithelial cancer cells thus can be used as a therapeutic biomarker. MJ protein, a Ribosome-Inactivating Proteins(RIPs) isolated from Mirabilis jalapa L had a higher cytotoxicity on malignant cells than normal cells. MJ proteinneed to be formulated to protect from proteosome degradation in endosome. The aim of this research was todevelop MJ protein-chitosan-pectin nanoparticles and conjugated with anti EpCAM for breast cancer therapy.Mj protein was extracted from M.jalapa leaves. RIPs activity was assayed by supercoiled DNA cleavage. MJprotein were loaded into chitosan nanoparticles using medium viscous chitosan and pectin as cross-linker withpolyelectrolit complex method. Anti EpCAM was conjugated to MJ protein-chitosan-pectin nanoparticles bycarbodiimide reaction and characterized for its entrapment efficiency, morphology by transmission electronmicroscope, particles size, and zeta potential. MJ protein nanoparticles conjugated anti EpCAM and withoutanti EpCAM were cytotoxicity assayed toward T47D and Vero cell lines. MJ protein was able to cleave thesupercoiled DNA into linear and nicked-circular ones. The nanoparticles optimal concentration of mediumviscous chitosan: MJ protein: pectin was 0.01%: 0.01%: 1% (m/v). A high entrapment efficiency of MJ proteinnanoparticles was 98.97 ± 0.07%. Morphology nanoparticles showed an amorphic structure with 200.00 nmparticles size. The nanoparticles conjugated anti EpCAM showed average particles size 367.67nm, polydispersityindex 0.332, and zeta potential +39.97mV. MJ protein-chitosan-pectin nanoparticles conjugated anti EpCAMand unconjugated both had higher cytotoxicity with the IC50 57.64 μg/mL and 46.84 μg/mL respectivelyagainst T47D and 99.38 μg/mL and 111.34 μg/mL against Vero cell lines compared to MJ protein with IC50 of3075.61 μg/mL against T47D and 3286.88 μg/mL against Vero cell lines. Both MJ protein-nanoparticles couldincrease the cytotoxicity effects about 50 times compared to the unformulated MJ protein activity, howeverhad less specificity toward T47D and Vero cell lines.

Mono-PEGylation of Alpha-MMC and MAP30 from Momordica charantia L.: Production, Identification and Anti-Tumor Activity.

PEGylation is a well-established and effective strategy to decrease immunogenicity, which can increase the stability and in vivo half-life time. However, the generation of multi-site modified products is inevitable due to the lysine chemistry, which will bring difficulties in subsequent research, such as purification and quantification. Site-specific modification by mPEG-succinimidyl carbonate (mPEG-SC) is a widely used method for N-terminal conjugation. In this study, we used it for site-directed modification on two ribosome-inactivating proteins (RIPs), alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30), from Momordica charantia L. According to the optimization of previous modification conditions, we compared Macro-Cap SP with SP-Sepharose FF chromatography for separating the final mPEGylated RIPs. Two kinds of methods both can obtain homogenous mPEGylated RIPs which were identified by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing electrophoresis (IEF), and matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) analysis. We also used iodine staining method to detect the amount of unmodified PEG. Furthermore, the inhibition activity of both mPEGylated and non-PEGylated RIPs against human lung adenocarcinoma epithelial A549 cells was detected. All of the results suggested that the mPEGylated α-MMC/MAP30 might be potentially developed as new anti-tumor drugs.

Ribosome-inactivating proteins from apple have strong aphicidal activity in artificial diet and in planta

The apple (Malus domestica) genome contains gene sequences encoding type-1 and type-2 ribosome-inactivating proteins (RIPs). Both types of proteins contain a RIP domain with N-glycosidase activity, but the type-2 RIPs possess an additional domain with lectin activity. Here we investigated the activity of RIPs from apple against two sucking-piercing aphids that are important in agriculture, in particular pea aphids (Acyrthosiphon pisum) and green peach aphids (Myzus persicae).When the recombinant RIPs were dosed in an artificial liquid diet against pea aphids a strong aphicidal activity was observed. Based on LC50s, the type-2 RIP (33 mg/L) was about 10 times more active than the type-1 RIP (341 mg/L), and the LC50 of the recombinant protein corresponding to the type-2 RIP lectin domain alone was 106 mg/L. In in planta experiments with transgenic tobacco plants expressing the type-1 RIP or the type-2 RIP and infected with green peach aphids, mortalities of the nymphal stages amounted to approximately 50% for both types of transgenic lines when compared to wild type plants. In addition, significant sublethal effects were observed in the surviving aphids with a reduction in fecundity, intrinsic rate of increase, net reproductive rate and doubling time of the insect population. The insecticidal activity of the type-1 RIP and type-2 RIP from apple is discussed in relation to the domain structure of the RIPs and potential use in plant protection.

Biological and antipathogenic activities of ribosome-inactivating proteins from Phytolacca dioica L.

BackgroundThe species from the genus Phytolacca constitute one of the best sources of ribosome-inactivating proteins (RIPs) that have been used both in the therapy against virus and tumors and in the construction of transgenic plants resistant to virus, bacteria, fungi and insects. Here we investigate new activities of three representative RIPs from Phytolacca dioica (dioicin 2, PD-S2 and PD-L4). ResultsThe three RIPs displayed, in addition to already reported activities, rRNA N-glycosylase activities against plant, bacterial and fungal ribosomes. Additionally dioicin 2 and PD-L4 displayed endonuclease activity on a supercoiled plasmid DNA, and dioicin 2 and PD-S2 arrested the growth of the fungus Penicillium digitatum. Furthermore, dioicin 2 induced caspase activation and apoptosis in cell cultures. ConclusionsThe different activities of the RIPs from Phytolacca dioica may explain the antipathogenic properties attributed to these RIPs in plants and their antiviral and antitumoral effects. In spite of the similarity in their rRNA N-glycosylase and DNA polynucleotide:adenosine glycosylase activities, they differed in their activities against viral RNA, plasmid DNA, fungi and animal cultured cells. This suggests that the presence of isoforms might optimize the response of the plant against several types of pathogens. General significanceRIPs from Phytolacca can induce plant resistance or tumor cell death not only by means of ribosome inactivation but also by the activities found in this report. Furthermore, the induction of cell death by different mechanisms turns these RIPs into more useful tools for cancer treatment rendering the selection of RIP-resistant mutants impossible.

Biological activities of ribosome-inactivating proteins and their possible applications as antimicrobial, anticancer, and anti-pest agents and in neuroscience research.

Ribosome-inactivating proteins (RIPs) are enzymes which depurinate ribosomal RNA (rRNA), thus impeding the process of translation resulting in inhibition of protein synthesis. They are produced by various organisms including plants, fungi and bacteria. RIPs from plants are linked to plant defense due to their antiviral, antifungal, antibacterial, and insecticidal activities in which they can be applied in agriculture to combat microbial pathogens and pests. Their anticancer, antiviral, embryotoxic, and abortifacient properties may find medicinal applications. Besides, conjugation of RIPs with antibodies or other carriers to form immunotoxins has been found useful to research in neuroscience and anticancer therapy.

Anti-tumor activities and apoptotic mechanism of ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) belong to a family of enzymes that attack eukaryotic ribosomes and potently inhibit cellular protein synthesis. RIPs possess several biomedical properties, including anti-viral and anti-tumor activities. Multiple RIPs are known to inhibit tumor cell proliferation through inducing apoptosis in a variety of cancers, such as breast cancer, leukemia/lymphoma, and hepatoma. This review focuses on the anti-tumor activities of RIPs and their apoptotic effects through three closely related pathways: mitochondrial, death receptor, and endoplasmic reticulum pathways.

An evolutionary arms race between plants and viruses (568.2)

Common pokeweed plant (Phytolacca Americana) produces pokeweed antiviral protein (PAP) as a defense mechanism against foreign pathogenic invaders. PAP is a ribosome inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein, and is a potent antiviral agent against many plant, animal, and human viruses. This study aims to elucidate the mechanism of RNA depurination and to understand how PAP recognizes and targets various RNAs. Here, we investigating interactions between PAP and Turnip mosaic virus genome linked protein (VPg). VPg functions as a cap analog in cap-independent translation, and potentially target PAP to uncapped IRES-containing RNA. Fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nM); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate, and competes with structured RNA derived from tobacco etch virus (TEV) for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms, and also suggests the possible use of this protein against the cytotoxic activity of RIPs.

Protein Synthesis Inhibition Activity by Strawberry Tissue Protein Extracts during Plant Life Cycle and under Biotic and Abiotic Stresses

Ribosome-inactivating proteins (RIPs), enzymes that are widely distributed in the plant kingdom, inhibit protein synthesis by depurinating rRNA and many other polynucleotidic substrates. Although RIPs show antiviral, antifungal, and insecticidal activities, their biological and physiological roles are not completely understood. Additionally, it has been described that RIP expression is augmented under stressful conditions. In this study, we evaluated protein synthesis inhibition activity in partially purified basic proteins (hereafter referred to as RIP activity) from tissue extracts of Fragaria × ananassa (strawberry) cultivars with low (Dora) and high (Record) tolerance to root pathogens and fructification stress. Association between the presence of RIP activity and the crop management (organic or integrated soil), growth stage (quiescence, flowering, and fructification), and exogenous stress (drought) were investigated. RIP activity was found in every tissue tested (roots, rhizomes, leaves, buds, flowers, and fruits) and under each tested condition. However, significant differences in RIP distribution were observed depending on the soil and growth stage, and an increase in RIP activity was found in the leaves of drought-stressed plants. These results suggest that RIP expression and activity could represent a response mechanism against biotic and abiotic stresses and could be a useful tool in selecting stress-resistant strawberry genotypes.

Inhibition of Pokeweed Antiviral Protein (PAP) by Turnip Mosaic Virus Genome‐Linked Protein (VPg).

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and an RNA N‐glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap‐binding protein, and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and Turnip mosaic virus genome linked protein (VPg) were investigated. VPg can function as a cap analog in cap‐independent translation, and potentially target PAP to uncapped IRES‐containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP‐VPg interactions. PAP binds to VPg with high affinity (29.5 nM); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate, and competes with structured RNA derived from tobacco etch virus (TEV) for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms, and also suggests the possible use of this protein against the cytotoxic activity of RIPs.

Targeting ricin to the ribosome

The plant toxin ricin is highly toxic for mammalian cells and is of concern for bioterrorism. Ricin belongs to a family of functionally related toxins, collectively referred to as ribosome inactivating proteins (RIPs), which disable ribosomes and halt protein synthesis. Currently there are no specific antidotes against ricin or related RIPs. The catalytic subunit of ricin is an N-glycosidase that depurinates a universally conserved adenine residue within the sarcin/ricin loop (SRL) of the 28S rRNA. This depurination activity inhibits translation and its biochemistry has been intensively studied. Yet, recent developments paint a more complex picture of toxicity, with ribosomal proteins and cellular signaling pathways contributing to the potency of ricin. In particular, several studies have now established the importance of the ribosomal stalk structure in facilitating the depurination activity and ribosome specificity of ricin and other RIPs. This review highlights recent developments defining toxin–ribosome interactions and examines the significance of these interactions for toxicity and therapeutic intervention.