Search

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., Competing Interests: The authors declare no conflicts of interest.

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that irreversibly inhibit protein synthesis and consequently cause cell death. Recently, an RIP called ledodin has been found in shiitake; it is cytotoxic, strongly inhibits protein synthesis, and shows rRNA N-glycosylase activity. In this work, we isolated and characterized a 50 kDa cytotoxic protein from shiitake that we named edodin. Edodin inhibits protein synthesis in a mammalian cell-free system, but not in insect-, yeast-, and bacteria-derived systems. It exhibits rRNA N-glycosylase and DNA-nicking activities, which relate it to plant RIPs. It was also shown to be toxic to HeLa and COLO 320 cells. Its structure is not related to other RIPs found in plants, bacteria, or fungi, but, instead, it presents the characteristic structure of the fold type I of pyridoxal phosphate-dependent enzymes. Homologous sequences have been found in other fungi of the class Agaricomycetes; thus, edodin could be a new type of toxin present in many fungi, some of them edible, which makes them of great interest in health, both for their involvement in food safety and for their potential biomedical and biotechnological applications.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The ribosome inactivating proteins (RIPs) efficiently decrease the microbial infections in plants. Momordica charantia 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 Nicotiana tabacum. 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 Escherichia coli. The results confirm that rMAP30 shows antibacterial activity against Streptococcus aureus and E. coli, antifungal activity against Candida albicans, 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., (© 2023. The Author(s).)

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry ( Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture.

Ribosome-inactivating proteins (RIPs) are known as RNA N-glycosylases. They depurinate the major rRNA, damaging ribosomes and inhibiting protein synthesis. Here, new single-chain (type-1) RIPs named sodins were isolated from the seeds (five proteins), edible leaves (one protein) and roots (one protein) of Salsola soda L. Sodins are able to release Endo's fragment when incubated with rabbit and yeast ribosomes and inhibit protein synthesis in cell-free systems (IC 50 = 4.83-79.31 pM). In addition, sodin 5, the major form isolated from seeds, as well as sodin eL and sodin R, isolated from edible leaves and roots, respectively, display polynucleotide:adenosine glycosylase activity and are cytotoxic towards the Hela and COLO 320 cell lines (IC 50 = 0.41-1200 nM), inducing apoptosis. The further characterization of sodin 5 reveals that this enzyme shows a secondary structure similar to other type-1 RIPs and a higher melting temperature (Tm = 76.03 ± 0.30 °C) and is non-glycosylated, as other sodins are. Finally, we proved that sodin 5 possesses antifungal activity against Penicillium digitatum .

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients' gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells' growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.

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.

Ribosome-inactivating proteins (RIPs) are N-glycosidases, which depurinate a specific adenine residue in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. This loop is important for anchoring elongation factor (EF-G for prokaryote or eEF2 for eukaryote) in mRNA translocation. Translation is inhibited after the attack. RIPs therefore may have been applied for anti-cancer, and anti-virus and other therapeutic applications. The main obstacles of treatment with RIPs include short plasma half-life, non-selective cytotoxicity and antigenicity. This review focuses on the strategies used to improve the pharmacological properties of RIPs on human immunodeficiency virus (HIV) and cancers. Coupling with polyethylene glycol (PEG) increases plasma time and reduces antigenicity. RIPs conjugated with antibodies to form immunotoxins increase the selective toxicity to target cells. The prospects for future development on the engineering of RIPs for improving their pharmacological properties are also discussed.

Alpha-momorcharin (α-MMC), a member of the ribosome-inactivating protein (RIP) family, has been found in the seeds of Momordica charantia (bitter melon). α-MMC contributes a number of pharmacological activities; however, its inflammatory properties have not been well studied. Here, we aim to determine the inflammatory responses induced by recombinant α-MMC and identify the underlying mechanisms using cell culture and animal models. Recombinant α-MMC was generated in Rosetta™(DE3)pLysS and purified by the way of nitrilotriacetic acid (NTA) chromatography. Treatment of recombinant α-MMC at 40 μg/mL exerted sub-lethal cytotoxic effect on THP-1 monocytic cells. Transcriptional profiling revealed that various genes coding for cytokines and other proinflammatory proteins were upregulated upon recombinant α-MMC treatment in THP-1 cells, including MCP-1, IL-8, IL-1β, and TNF-α. Recombinant α-MMC was shown to activate IKK/NF-κB and JNK pathways and the α-MMC-induced inflammatory gene expression could be blocked by IKKβ and JNK inhibitors. Furthermore, murine inflammatory models further demonstrated that α-MMC induced inflammatory responses in vivo. We conclude that α-MMC stimulates inflammatory responses in human monocytes by activating of IKK/NF-κB and JNK pathways, raising the possibility that consumption of α-MMC-containing food may lead to inflammatory-related diseases., Competing Interests: The authors declare no conflict of interest.

Bougainvillea ( Bougainvillea spectabilis Willd.) is a plant widely used in folk medicine and many extracts from different tissues of this plant have been employed against several pathologies. The observation that leaf extracts of Bougainvillea possess antiviral properties led to the purification and characterization of a protein, named bouganin, which exhibits typical characteristics of type 1 ribosome-inactivating proteins (RIPs). Beyond that, bouganin has some peculiarities, such as a higher activity on DNA with respect to ribosomal RNA, low systemic toxicity, and immunological properties quite different than other RIPs. The sequencing of bouganin and the knowledge of its three-dimensional structure allowed to obtain a not immunogenic mutant of bouganin. These features make bouganin a very attractive tool as a component of immunotoxins (ITs), chimeric proteins obtained by linking a toxin to a carrier molecule. Bouganin-containing ITs showed very promising results in the experimental treatment of both hematological and solid tumors, and one bouganin-containing IT has entered Phase I clinical trial. In this review, we summarize the milestones of the research on bouganin such as bouganin chemico-physical characteristics, the structural properties and de-immunization studies. In addition, the in vitro and in vivo results obtained with bouganin-containing ITs are summarized.

Ribosome Inactivating Proteins (RIPs) isolated from Mirabilis jalapa L. (MJ protein) leaves showed high cytotoxic effect on malignant. Chitosan nanoparticles have frequently been used in protein delivery applications. The aim of this study was to develop targeted drug delivery system of RIP MJ for breast cancer therapy with chitosan nanoparticles conjugated antiEpCAM antibody. RIP MJ nanoparticles were prepared using low viscous chitosan and pectin using polyelectrolit complex method, followed by conjugation process with antiEpCAM antibody. Characterization of this formula was then carried out for its entrapment efficiency, particles size, zeta potential, morphology using transmission electron microscope (TEM) and cytotoxic assay against T47D and Vero cell line. The optimal concentration of MJ protein; low viscous chitosan; pectin for preparing AntiEpCAM conjugated of RIP MJ nanoparticles was 0.1%; 0.01%;1% (m/v) respectively and showed satisfactory formula with the average particle size of 376.8±105.2nm, polydispersity index (PI) 0.401, zeta potential 43,71 mV, high entrapment efficiency 98,97±0,12%. Transmission electron microscope (TEM) imaging showed a spherical and homogenous structure for nanoparticles. The in vitro cytotoxicity analysis showed that RIP MJ nanoparticle had more cytotoxic effect compared to unformulated RIP against T47D cell-lines. AntiEpCAM conjugated RIP MJ nanoparticles however, increased cytotoxic effect of RIPs on Vero cell-lines not for T47D cell-lines. Chitosan-Pectin nanoparticles suitable for delivering protein to target cancer cells.

Vascular endothelial growth factor receptor-2 (VEGFR2) is the main pro-angiogenic receptor expressed by endothelial cells (ECs). Using surface plasmon resonance, immunoprecipitation, enzymatic digestion, immunofluorescence and cross-linking experiments with specific sugar-binding lectins, we demonstrated that VEGFR2 bears both α,1-fucose and α(2,6)-linked sialic acid (NeuAc). However, only the latter is required for VEGF binding to VEGFR2 and consequent VEGF-dependent VEGFR2 activation and motogenic response in ECs. Notably, downregulation of β-galactoside α(2,6)-sialyltransferase expression by short hairpin RNA transduction inhibits VEGFR2 α(2,6) sialylation that is paralleled by an increase of β-galactoside α(2,3)-sialyltransferase expression. This results in an ex-novo α(2,3)-NeuAc sialylation of the receptor that functionally replaces the lacking α(2,6)-NeuAc, thus allowing VEGF/VEGFR2 interaction. In keeping with the role of VEGFR2 sialylation in angiogenesis, the α(2,6)-NeuAc-binding lectin Sambucus nigra (SNA) prevents VEGF-dependent VEGFR2 autophosphorylation and EC motility, proliferation and motogenesis. In addition, SNA exerts a VEGF-antagonist activity in tridimensional angiogenesis models in vitro and in the chick-embryo chorioallantoic membrane neovascularization assay and mouse matrigel plug assay in vivo. In conclusion, VEGFR2-associated NeuAc plays an important role in modulating VEGF/VEGFR2 interaction, EC pro-angiogenic activation and neovessel formation. VEGFR2 sialylation may represent a target for the treatment of angiogenesis-dependent diseases.

Targeted cancer therapy provides the basis for the arrest of tumor growth in aggressive pancreatic carcinoma; however, a number of protein-based targeted toxins lack efficacy due to insufficient endosomal escape after being endocytosed. Therefore, we tested a fusion protein of the ribosome-inactivating protein dianthin and human epidermal growth factor in combination with a glycosylated triterpene (SO1861) that serves as an endosomal escape enhancer. In vitro investigations with the pancreatic carcinoma cell lines BxPC-3 and MIA PaCa-2 revealed no significant differences to off-target cells in the half maximal inhibitory concentration (IC 50 ) for the fusion protein. In contrast, combination with SO1861 decreased the IC 50 for BxPC-3 cells from 100 to 0.17 nm, whereas control cells remained unaffected. Monotherapy of BxPC-3 xenografts in CD-1 nude mice led to a 51.7% average reduction in tumor size (40.8 mm 3 ) when compared to placebo; however, combined treatment with SO1861 resulted in a more than 13-fold better efficacy (3.0 mm 3 average tumor size) with complete regression in 80% of cases. Immunohistochemical analyses showed that tumor cells with lower target receptor expression are, in contrast to the combination therapy, able to escape from the monotherapy, which finally results in tumor growth. At the effective concentration, we did not observe liver toxicity and saw no other side effects with the exception of a reversible skin hardening at the SO1861 injection site, alongside an increase in platelet counts, plateletcrit, and platelet distribution width. In conclusion, combining a targeted toxin with SO1861 is proven to be a very promising approach for pancreatic cancer treatment., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Ovarian carcinoma (OC) patients encounter the severe challenge of clinical management owing to lack of screening measures, chemoresistance and finally dearth of non-toxic therapeutics. Cancer cells deploy various defense strategies to sustain the tumor microenvironment, among which deregulated apoptosis remains a versatile promoter of cancer progression. Although recent research has focused on identifying agents capable of inducing apoptosis in cancer cells, yet molecules efficiently breaching their survival advantage are yet to be classified. Here we identify lectin, Sambucus nigra agglutinin (SNA) to exhibit selectivity towards identifying OC by virtue of its specific recognition of α-2, 6-linked sialic acids. Superficial binding of SNA to the OC cells confirm the hyper-sialylated status of the disease. Further, SNA activates the signaling pathways of AKT and ERK1/2, which eventually promotes de-phosphorylation of dynamin-related protein-1 (Drp-1). Upon its translocation to the mitochondrial fission loci Drp-1 mediates the central role of switch in the mitochondrial phenotype to attain fragmented morphology. We confirmed mitochondrial outer membrane permeabilization resulting in ROS generation and cytochrome-c release into the cytosol. SNA response resulted in an allied shift of the bioenergetics profile from Warburg phenotype to elevated mitochondrial oxidative phosphorylation, altogether highlighting the involvement of mitochondrial dysfunction in restraining cancer progression. Inability to replenish the SNA-induced energy crunch of the proliferating cancer cells on the event of perturbed respiratory outcome resulted in cell cycle arrest before G2/M phase. Our findings position SNA at a crucial juncture where it proves to be a promising candidate for impeding progression of OC. Altogether we unveil the novel aspect of identifying natural molecules harboring the inherent capability of targeting mitochondrial structural dynamics, to hold the future for developing non-toxic therapeutics for treating OC.

The enzymes called ribosome-inactivating proteins (RIPs) that are able to depurinate  nucleic acids and arrest vital cellular functions, including protein synthesis, are still a frontline  research field, mostly because of their promising medical applications. The contributions of Stirpe  to the development of these studies has been one of the most relevant. After a short biographical  introduction, an overview is offered of the main results obtained by his investigations during last  55 years on his main research lines: hyperuricaemia, xanthine oxidoreductase and RIPs.

To improve the pharmacological properties of maize ribosome-inactivating protein (maize RIP) for targeting HIV-infected cells, the previously engineered TAT-fused active form of maize RIP (MOD) was further engineered for cysteine-directed PEGylation. In this work, two potential antigenic sites, namely Lys-78 and Lys-264, were identified. They were mutated to cysteine residue and conjugated with PEG 5k or PEG 20k . The resultant PEG derivatives of MOD variants were examined for ribosome-inactivating activity, circulating half-life and immunogenicity. Our results showed that MOD-PEG conjugates had two- to five-fold lower biological activity compared to the wild-type. Mutation of the two sites respectively did not decrease the anti-MOD IgG and IgE level in mice, but the conjugation of PEG did dramatically reduce the antigenicity. Furthermore, pharmacokinetics studies demonstrated that attachment of PEG 20k prolonged the plasma half-life by five-fold for MOD-K78C and 17-fold for MOD-K264C, respectively. The site-specific mutation together with PEGylation therefore generated MOD derivatives with improved pharmacological properties., Competing Interests: The authors declare no conflict of interest.

Alpha-Momorcharin (α-MMC) is a ribosome inactivating protein from Momordica charantia with anti-tumor activity. Previously, we had observed that modification of α-MMC with polyethylene glycol (PEG) could reduce toxicity, but it also reduces its anti-tumor activity in vitro. This study aims to investigate whether the metabolism-extended properties of α-MMC resulting from PEGylation could preserve its anti-tumor efficacy in vivo through pharmacokinetics and antitumor experiments. The pharmacokinetics experiments were conducted in rats using the TCA (Trichloroacetic Acid) method. Antitumor activity in vivo was investigated in murine mammary carcinoma (EMT-6) and human mammary carcinoma (MDA-MB-231) transplanted tumor mouse models. The results showed that PEGylation increased the plasma half-life of α-MMC in rats from 6.2-7.5 h to 52-87 h. When administered at 1 mg/kg, α-MMC-PEG and α-MMC showed similar anti-tumor activities in vivo, with a T/C% of 38.56% for α-MMC versus 35.43% for α-MMC-PEG in the EMT-6 tumor model and 36.30% for α-MMC versus 39.88% for α-MMC-PEG in the MDA-MB-231 tumor model (p > 0.05). Importantly, at the dose of 3 mg/kg, all the animals treated with α-MMC died while the animals treated with α-MMC-PEG exhibited only moderate toxic reactions, and α-MMC-PEG exhibited improved anti-tumor efficacy with a T/C% (relative tumor growth rate) of 25.18% and 21.07% in the EMT-6 and MDA-MB-231 tumor models, respectively. The present study demonstrates that PEGylation extends the half-life of α-MMC and alleviates non-specific toxicity, thereby preserving its antitumor efficacy in vivo, and a higher lever of dosage can be used to achieve better therapeutic efficacy.

Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs) is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

A fungal strain, R9, was isolated from the South Atlantic sediment sample and identified as Aspergillus fumigatus. An antifungal protein, AfAFPR9, was purified from the culture supernatant of Aspergillus fumigatus R9. AfAFPR9 was identified to be restrictocin, which is a member of the ribosome-inactivating proteins (RIPs), by MALDI-TOF-TOF-MS. AfAFPR9 displayed antifungal activity against plant pathogenic Fusarium oxysporum, Alternaria longipes, Colletotrichum gloeosporioides, Paecilomyces variotii, and Trichoderma viride at minimum inhibitory concentrations of 0.6, 0.6, 1.2, 1.2, and 2.4 μg/disc, respectively. Moreover, AfAFPR9 exhibited a certain extent of thermostability, and metal ion and denaturant tolerance. The iodoacetamide assay showed that the disulfide bridge in AfAFPR9 was indispensable for its antifungal action. The cDNA encoding for AfAFPR9 was cloned from A. fumigatus R9 by RTPCR and heterologously expressed in E. coli. The recombinant AfAFPR9 protein exhibited obvious antifungal activity against C. gloeosporioides, T. viride, and A. longipes. These results reveal the antifungal properties of a RIP member (AfAFPR9) from marine-derived Aspergillus fumigatus and indicated its potential application in controlling plant pathogenic fungi.

Sambucus (Adoxaceae) species have been used for both food and medicine purposes. Among these, Sambucus nigra L. (black elder), Sambucus ebulus L. (dwarf elder), and Sambucus sieboldiana L. are the most relevant species studied. Their use has been somewhat restricted due to the presence of bioactive proteins or/and low molecular weight compounds whose ingestion could trigger deleterious effects. Over the last few years, the chemical and pharmacological characteristics of Sambucus species have been investigated. Among the proteins present in Sambucus species both type 1, and type 2 ribosome-inactivating proteins (RIPs), and hololectins have been reported. The biological role played by these proteins remains unknown, although they are conjectured to be involved in defending plants against insect predators and viruses. These proteins might have an important impact on the nutritional characteristics and food safety of elderberries. Type 2 RIPs are able to interact with gut cells of insects and mammals triggering a number of specific and mostly unknown cell signals in the gut mucosa that could significantly affect animal physiology. In this paper, we describe all known RIPs that have been isolated to date from Sambucus species, and comment on their antiviral and entomotoxic effects, as well as their potential uses.

Ribosome-inactivating proteins (RIPs) are endowed with several medicinal properties, including antiviral activity. We demonstrate here that the recently identified type I RIP from Momordica balsamina also possesses antiviral activity, as determined by viral growth curve assays and single-round infection experiments. Importantly, this activity is at play even as doses where the RIP has no cytotoxic effect. In addition, balsamin inhibits HIV-1 replication not only in T cell lines but also in human primary CD4(+) T cells. This antiviral compound exerts its activity at a viral replicative step occurring later than reverse-transcription, most likely on viral protein translation, prior to viral budding and release. Finally, we demonstrate that balsamin antiviral activity is broad since it also impedes influenza virus replication. Altogether our results demonstrate that type I RIP can exert a potent anti-HIV-1 activity which paves the way for new therapeutic avenues for the treatment of viral infections.

Alpha-momorcharin (α-MMC) is type-1 ribosome inactivating proteins (RIPs) with molecular weight of 29 kDa and has lots of biological activity. Our recent study indicated that the α-MMC purified from seeds of Momordica charantia exhibited distinct antiviral and antifungal activity. Tobacco plants pre-treated with 0.5 mg/mL α-MMC 3 days before inoculation with various viruses showed less-severe symptom and less reactive oxygen species (ROS) accumulation compared to that inoculated with viruses only. Quantitative real-time PCR analysis revealed that the replication levels of viruses were lower in the plants treated with the α-MMC than control plants at 15 days post inoculation. Moreover, the coat protein expression of viruses was almost completely inhibited in plants which were treated with the α-MMC compared with control plants. Furthermore, the SA-responsive defense-related genes including non-expressor of pathogenesis-related genes 1 (NPR1), PR1, PR2 were up-regulated and activities of some antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) were increased after the α-MMC treatment. In addition, the α-MMC (500 μg/mL) revealed remarkable antifungal effect against phytopathogenic fungi, in the growth inhibition range 50.35-67.21 %, along with their MIC values ranging from 100 to 500 μg/mL. The α-MMC had also a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of Sclerotinia sclerotiorum. The α-MMC showed a remarkable antiviral and antifungal effect and hence could possibly be exploited in crop protection for controlling certain important plant diseases.

Researchers have proposed that VAA-I, a specific plant lectin found in Viscum album, has therapeutic effects on cancer and autoimmune diseases. VAA-I has shown some promising treatment results in some types of tumor cell lines, especially SMMC-7721 cells (human hepatocellular carcinoma cells). However, few details are known about the mechanism and process of cell death induced by VAA-I in tumor cells. In this study, the cell morphology results showed that SMMC-7721 cells treated with VAA-I exhibited several features typical of apoptotic cell death, which was confirmed by the Caspase inhibition assay. Fluo-3-acetoxymethyl ester (AM) fluorescence imaging techniques showed that rVAA-I significantly elevated the intracellular calcium level ([Ca2+]i) in SMMC-7721 cells. These findings suggest that apoptosis may play the most important role in SMMC-7721 cell death induced by rVAA-I. Finally, in the SMMC-7721 cells treated with rVAA-I, a series of genes in the p38 mitogen-activated protein kinase (MAPK) signaling pathway were expressed differentially, and further found that PI 3-kinase pathway is involved in rVAA-I signal transduction in SMMC-7721 cells.

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.

Background: Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L. have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity. However, strong immunogenicity and short plasma half-life limit their clinical application. To solve this problem, the two proteins were modified with (mPEG)(2)-Lys-NHS (20 kDa)., Methodology/principal Findings: In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation. Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds. The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot. Two polyethylene glycol (PEG)ylated proteins were synthesized and purified. Homogeneous mono-, di-, or tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1. Furthermore, both PEGylated proteins showed about 60%-70% antitumor and antivirus activities, and at the same time decreased 50%-70% immunogenicity when compared with their unmodified counterparts., Conclusion/significance: α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research. Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities. All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents. According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects. Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body.

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

Maize ribosome-inactivating protein (RIP) is a plant toxin that inactivates eukaryotic ribosomes by depurinating a specific adenine residue at the α-sarcin/ricin loop of 28S rRNA. Maize RIP is first produced as a proenzyme with a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP with full N-glycosidase activity. This naturally occurring switch-on mechanism provides an opportunity for targeting the cytotoxin to pathogen-infected cells. Here, we report the addition of HIV-1 protease recognition sequences to the internal inactivation region and the activation of the maize RIP variants by HIV-1 protease in vitro and in HIV-infected cells. Among the variants generated, two were cleaved efficiently by HIV-1 protease. The HIV-1 protease-activated variants showed enhanced N-glycosidase activity in vivo as compared to their un-activated counterparts. They also possessed potent inhibitory effect on p24 antigen production in human T cells infected by two HIV-1 strains. This switch-on strategy for activating the enzymatic activity of maize RIP in target cells provides a platform for combating pathogens with a specific protease.

Alpha-momorcharin (alpha-MMC) is a ribosome-inactivating protein (RIP) with excellent cytotoxicity to tumor cells. However, its strong immunogenicity and short plasma half-life limit its clinical applications. To overcome this, we have to PEGylated alpha-MMC using a branched 20 kDa (mPEG) (2)-Lys-NHS. Homogeneous mono-, di- and tri-PEGylated alpha-MMCs were synthesized, purified and characterized. In vitro and in vivo analysis indicated that the serial PEG-conjugates preserved moderate anti-tumor activity with 36% acute toxicity and at most 66% immunogenicity decrease. These results suggested the potential application of alpha-MMC-PEG conjugates as an anti-tumor agent.

Synthesis of nitric oxide (NO) is one of the important effector functions of innate immune cells. Although several reports have indicated mistletoe lectins induce immune cells to produce cytokines, studies regarding the activities of the lectins in the production of NO have been very limited. Here, we report on the induction of NO synthesis in a murine macrophage cell line, RAW264.7, by Korean mistletoe lectin (KML-IIU). When the macrophage cells were treated with KML-IIU in the presence of a suboptimal concentration of IFN-gamma, NO production was induced in a concentration-dependent manner. Significantly higher levels of NO were induced by subchains of the KML-IIU (A and B), which have lower toxicities, as compared to the hololectin. Furthermore, expression of the inducible nitric oxide synthase (iNOS) gene was elevated in accordance with the level of NO production. When the synthase was inhibited by iNOS inhibitors (L-NIL and L-NAME), NO production was specifically reduced in a concentration-dependent manner. Our studies demonstrate that the KML-IIU and its subchains induce NO production in murine macrophage cells via activation of the iNOS gene expression, suggesting that the KML-IIU subchains may be used as an immunomodulator to enhance the effector functions of innate immune cells.

The gene encoding a single-chain, ribosome-inactivating protein (SCRIP) was cloned from bitter melon (Momordica charantia L.) leaves infected with the fungus, Sphaerotheca fuliginea, by RT-PCR. The ORF was 861 bp. The ribosome-inactivating protein was expressed in E. coli and, when purified, it inhibited the growth of Fusarium solani [corrected] Northern blot analysis revealed that RIP transcripts rapidly accumulated in leaves 1-day post inoculation with Sphaerotheca fuliginea and reached a peak at 3 d. The expression pattern of RIP induced by methyl jasmonate and salicylic acid were different from that of pathogen-induced expression. Mechanical wounding, silver nitrate and osmotic stress stimulated only a slight accumulation of RIP transcripts. Abscisic acid also induced transcription of RIPs. The signal compounds, ethylene and okadaic acid, induced a moderate accumulation of RIP transcripts.

Maize ribosome-inactivating protein is classified as a class III or an atypical RNA N-glycosidase. It is synthesized as an inactive precursor with a 25-amino acid internal inactivation region, which is removed in the active form. As the first structural example of this class of proteins, crystals of the precursor and the active form were diffracted to 2.4 and 2.5 A, respectively. The two proteins are similar, with main chain root mean square deviation (RMSD) of 0.519. In the precursor, the inactivation region is found on the protein surface and consists of a flexible loop followed by a long alpha-helix. This region diminished both the interaction with ribosome and cytotoxicity, but not cellular uptake. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure reveals that the active site is too small to accommodate two glutamate residues. Our structure suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins.