ribonuclease ribotoxin

Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities...

Ribotoxins are secreted ribonucleases that specifically target and cleave the universally conserved sarcin-ricin loop sequence of rRNA, which leads to inhibition of protein biosynthesis and subsequently to cell death. We have identified and characterized a secreted Ribo1 protein of plant pathogenic smut fungi. Heterologous expression in different model systems showed that smut Ribo1 has cytotoxic activity against bacteria, yeast, host and nonhost plants. Recombinant expression of Ribo1 in Nicotiana benthamiana induced plant cell death; however, an active site mutant induced cell death only when expressed as a secreted protein...

White button mushroom ( Agaricus bisporus (J.E. Lange) Imbach) is one of the widely consumed edible mushrooms. Indeed, A. bisporus fruiting bodies are a rich source of nutrients and bioactive molecules. In addition, several enzymes with biotechnological applications are found in A. bisporus (e.g., enzymes for lignocellulose degradation). Here, a novel ribotoxin-like protein (RL-P) from the edible mushroom A. bisporus was purified and characterized. This RL-P, named bisporitin, is a monomeric protein (17-kDa) exhibiting specific ribonucleolytic activity by releasing the α-fragment (hallmark of RL-Ps) when incubated with rabbit ribosomes...

Neofusicoccum parvum is a fungal plant pathogen of a wide range of hosts but knowledge about the virulence factors of N. parvum and host-pathogen interactions is rather limited. The molecules involved in the interaction between N. parvum and Eucalyptus are mostly unknown, so we used a multi-omics approach to understand pathogen-host interactions. We present the first comprehensive characterization of the in vitro secretome of N. parvum and a prediction of protein-protein interactions using a dry-lab non-targeted interactomics strategy...

Ageritin is a ribotoxin-like protein of biotechnological interest, belonging to a family of ribonucleases from edible mushrooms. Its enzymatic activity is explicated through the hydrolysis of a single phosphodiester bond, located in the sarcin/ricin loop of ribosomes. Unlike other ribotoxins, ageritin activity requires divalent cations (Zn2+ ). Here we investigated the conformational stability of ageritin in the pH range 4.0-7.4, using calorimetric and spectroscopic techniques. We observed a high protein thermal stability at all pHs with a denaturation temperature of 78 °C...

Ribotoxin-like proteins (RL-Ps) represent a novel specific ribonuclease family found in edible mushrooms and are able to inhibit protein synthesis. Here, we report the characterization and cytotoxic effects of four novel RL-Ps, named eryngitins, isolated from fruiting bodies of the king oyster mushroom (Pleurotus eryngii). These proteins induced formation of α-fragment from rabbit ribosomes, characteristic of their enzymatic action. The two 15 kDa eryngitins (3 and 4) are considerably more thermostable than the 21 kDa ones (1 and 2), however their overall structural features, as determined by far-UV CD spectrometry, are similar...

rRNA N-glycosylases (EC 3.2.2.22) remove a specific adenine (A4324 , rat 28S rRNA) in the sarcin ricin loop (SRL) involved into ribosome interaction with elongation factors, causing the inhibition of translation, for which they are known as plant 'ribosome inactivating proteins' (RIPs). However, protein synthesis inactivation could be the result of other enzymes, which often have rRNA as the target. In this scenario, Endo's assay is the most used method to detect the enzymes that are able to hydrolyze a phosphodiester bond or cleave a single N-glycosidic bond (rRNA N-glycosylases)...

Porcini are edible mushrooms widely used in cooking due to their extraordinary taste. Despite this, cases of food poisoning have been reported in the recent literature also for ingestion of porcini. Here, we report the isolation from Boletus edulis fruiting bodies of two novel ribotoxin-like proteins (RL-Ps), enzymes already studied in other organisms for their toxicity. These RL-Ps, named Edulitin 1 (16-kDa) and Edulitin 2 (14-kDa), show peculiar structural and enzymatic differences, which probably reflect their different bio-activities and a dose/time dependent toxicity (Edulitin 2) on normal and tumoral human cells...

Ageritin is a specific ribonuclease, extracted from the edible mushroom Cyclocybe aegerita (synonym Agrocybe aegerita ), which cleaves a single phosphodiester bond located within the universally conserved alpha-sarcin loop (SRL) of 23-28S rRNAs. This cleavage leads to the inhibition of protein biosynthesis, followed by cellular death through apoptosis. The structural and enzymatic properties show that Ageritin is the prototype of a novel specific ribonucleases family named 'ribotoxin-like proteins', recently found in fruiting bodies of other edible basidiomycetes mushrooms (e...

Fungi produce several toxins active against plants, animal or humans. Among them, ribotoxins are enzymes that specifically attack ribosomes irreparably compromising protein synthesis, useful as insecticides or as anticancer agents. Here, a novel ribotoxin from the edible mushroom Pleurotus ostreatus has been purified and characterized. This ribotoxin, named Ostreatin, is a specific ribonuclease releasing α-fragment when incubated with yeast or rabbit ribosomes. Ostreatin shows IC50 of 234 pM in rabbit reticulocyte lysate, and metal dependent endonuclease activity...

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes...

Entomopathogenic fungi are promising bio-pesticides. To facilitate infection, fungi recruit multiple virulence factors and deploy different molecular strategies to evade host immunity. Fungal ribotoxins are extracellular secreted ribonucleases (RNases) with ribotoxic cytotoxicity and insecticidal activity. However, it remains unclear whether they have further biological functions. Here we show that the entomopathogenic fungus Beauveria bassiana ribotoxin (Rib) contributes to fungal virulence by inhibiting insect host immunity...

Ageritin, a specific ribonuclease, damaging the largest rRNA in the highly conserved α-sarcin/ricin stem-loop (SRL) has been well characterized from edible mushroom Agrocybe aegerita. Given its peculiar characteristic, Ageritin is the prototype of a new ribotoxins family expressed in basidiomycetes. In this framework, we report the characterization of Met-Ageritin, an isoform of Ageritin with an additional N-terminal methionyl residue. This difference affects the enzymatic features of this toxin despite is able to release α-fragment when acting on yeast, rabbit or Trichoderma asperellum ribosomes...

Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin α-sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site...

Among the putative defense proteins that occur in fungi, one of the best studied is α-sarcin, produced by the mold Aspergillus giganteus. This protein is the most significant member of the ribotoxin family, which consists of extracellular rRNA ribonucleases that display cytotoxic activity toward animal cells. Ribotoxins are rRNA endonucleases that catalyze the hydrolysis of the phosphodiester bond between G4325 and A4326 from the rat 28S rRNA. The results of several experimental approaches have led to propose ribotoxins as insecticidal agents...

Fungi establish a complex network of biological interactions with other organisms in nature. In many cases, these involve the production of toxins for survival or colonization purposes. Among these toxins, ribotoxins stand out as promising candidates for their use in biotechnological applications. They constitute a group of highly specific extracellular ribonucleases that target a universally conserved sequence of RNA in the ribosome, the sarcin-ricin loop. The detailed molecular study of this family of toxic proteins over the past decades has highlighted their potential in applied research...

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin-ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids...

Metarhizium anisopliae is an entomopathogenic fungus relevant in biotechnology with applications like malaria vector control. Studies of its virulence factors are therefore of great interest. Fungal ribotoxins are toxic ribonucleases with extraordinary efficiency against ribosomes and suggested as potential insecticides. Here we describe this ribotoxin characteristic activity in M. anisopliae cultures. Anisoplin has been obtained as a recombinant protein and further characterized. It is structurally similar to hirsutellin A, the ribotoxin from the entomopathogen Hirsutella thompsonii...

Ribotoxins are cytotoxic members of the family of fungal extracellular ribonucleases best represented by RNase T1. They share a high degree of sequence identity and a common structural fold, including the geometric arrangement of their active sites. However, ribotoxins are larger, with a well-defined N-terminal β-hairpin, and display longer and positively charged unstructured loops. These structural differences account for their cytotoxic properties. Unexpectedly, the discovery of hirsutellin A (HtA), a ribotoxin produced by the invertebrate pathogen Hirsutella thompsonii, showed how it was possible to accommodate these features into a shorter amino acid sequence...

Restrictocin, a highly specific ribonuclease produced by Aspergillus restrictus, cleaves a single phosphodiester bond in a universally conserved stem and loop structure termed sarcin/ricin loop within the large ribosomal RNA of all organisms. In the current study, we demonstrate restrictocin to manifest anti-HIV-1 activity in two model cell systems. Using two mutants of restrictocin, we further show that the anti-HIV-1 activity of restrictocin is due to its specific ribonucleolytic activity. The study suggests that restrictocin is able to recognize region(s) within HIV-1 genome as its target...