The Biological Action and Structural Characterization of Eryngitin 3 and 4, Ribotoxin-like Proteins from Pleurotus eryngii Fruiting Bodies.

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. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition.

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.

Structure and Biological Properties of Ribosome-Inactivating Proteins and Lectins from Elder (Sambucus nigra L.) Leaves.

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.

Isolation, Characterization and Biological Action of Type-1 Ribosome-Inactivating Proteins from Tissues of Salsola soda L.

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 (IC50 = 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 (IC50 = 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.

Deciphering Molecular Determinants Underlying Penicillium digitatum's Response to Biological and Chemical Antifungal Agents by Tandem Mass Tag (TMT)-Based High-Resolution LC-MS/MS.

Penicillium digitatum is a widespread pathogen responsible for the postharvest decay of citrus, one of the most economically important crops worldwide. Currently, chemical fungicides are still the main strategy to control the green mould disease caused by the fungus. However, the increasing selection and proliferation of fungicide-resistant strains require more efforts to explore new alternatives acting via new or unexplored mechanisms for postharvest disease management. To date, several non-chemical compounds have been investigated for the control of fungal pathogens. In this scenario, understanding the molecular determinants underlying P. digitatum's response to biological and chemical antifungals may help in the development of safer and more effective non-chemical control methods. In this work, a proteomic approach based on isobaric labelling and a nanoLC tandem mass spectrometry approach was used to investigate molecular changes associated with P. digitatum's response to treatments with α-sarcin and beetin 27 (BE27), two proteins endowed with antifungal activity. The outcomes of treatments with these biological agents were then compared with those triggered by the commonly used chemical fungicide thiabendazole (TBZ). Our results showed that differentially expressed proteins mainly include cell wall-degrading enzymes, proteins involved in stress response, antioxidant and detoxification mechanisms and metabolic processes such as thiamine biosynthesis. Interestingly, specific modulations in response to protein toxins treatments were observed for a subset of proteins. Deciphering the inhibitory mechanisms of biofungicides and chemical compounds, together with understanding their effects on the fungal physiology, will provide a new direction for improving the efficacy of novel antifungal formulations and developing new control strategies.

Sequence, Structure, and Binding Site Analysis of Kirkiin in Comparison with Ricin and Other Type 2 RIPs.

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin.

Ebulin l Is Internalized in Cells by Both Clathrin-Dependent and -Independent Mechanisms and Does Not Require Clathrin or Dynamin for Intoxication.

Ebulin l is an A-B toxin, and despite the presence of a B chain, this toxin displays much less toxicity to cells than the potent A-B toxin ricin. Here, we studied the binding, mechanisms of endocytosis, and intracellular pathway followed by ebulin l and compared it with ricin. COS-1 cells and HeLa cells with inducible synthesis of a mutant dynamin (K44A) were used in this study. The transport of these toxins was measured using radioactively or fluorescently labeled toxins. The data show that ebulin l binds to cells to a lesser extent than ricin. Moreover, the expression of mutant dynamin does not affect the endocytosis, degradation, or toxicity of ebulin l. However, the inhibition of clathrin-coated pit formation by acidification of the cytosol reduced ebulin l endocytosis but not toxicity. Remarkably, unlike ricin, ebulin l is not transported through the Golgi apparatus to intoxicate the cells and ebulin l induces apoptosis as the predominant cell death mechanism. Therefore, after binding to cells, ebulin l is taken up by clathrin-dependent and -independent endocytosis into the endosomal/lysosomal system, but there is no apparent role for clathrin and dynamin in productive intracellular routing leading to intoxication.

Kirkiin: A New Toxic Type 2 Ribosome-Inactivating Protein from the Caudex of Adenia kirkii.

Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1 RIPs, single-chain enzymatic proteins, and type 2 RIPs, consisting of active A chains, similar to type 1 RIPs, linked to lectin B chains, which enable the rapid internalization of the toxin into the cell. For this reason, many type 2 RIPs are very cytotoxic, ricin, volkensin and stenodactylin being the most toxic ones. From the caudex of Adenia kirkii (Mast.) Engl., a new type 2 RIP, named kirkiin, was purified by affinity chromatography on acid-treated Sepharose CL-6B and gel filtration. The lectin, with molecular weight of about 58 kDa, agglutinated erythrocytes and inhibited protein synthesis in a cell-free system at very low concentrations. Moreover, kirkiin was able to depurinate mammalian and yeast ribosomes, but it showed little or no activity on other nucleotide substrates. In neuroblastoma cells, kirkiin inhibited protein synthesis and induced apoptosis at doses in the pM range. The biological characteristics of kirkiin make this protein a potential candidate for several experimental pharmacological applications both alone for local treatments and as component of immunoconjugates for systemic targeting in neurodegenerative studies and cancer therapy.

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.

Primary Sequence and 3D Structure Prediction of the Plant Toxin Stenodactylin.

Stenodactylin is one of the most potent type 2 ribosome-inactivating proteins (RIPs); its high toxicity has been demonstrated in several models both in vitro and in vivo. Due to its peculiarities, stenodactylin could have several medical and biotechnological applications in neuroscience and cancer treatment. In this work, we report the complete amino acid sequence of stenodactylin and 3D structure prediction. The comparison between the primary sequence of stenodactylin and other RIPs allowed us to identify homologies/differences and the amino acids involved in RIP toxic activity. Stenodactylin RNA was isolated from plant caudex, reverse transcribed through PCR and the cDNA was amplificated and cloned into a plasmid vector and further analyzed by sequencing. Nucleotide sequence analysis showed that stenodactylin A and B chains contain 251 and 258 amino acids, respectively. The key amino acids of the active site described for ricin and most other RIPs are also conserved in the stenodactylin A chain. Stenodactylin amino acid sequence shows a high identity degree with volkensin (81.7% for A chain, 90.3% for B chain), whilst when compared with other type 2 RIPs the identity degree ranges from 27.7 to 33.0% for the A chain and from 42.1 to 47.7% for the B chain.

Effect of an additional N-terminal methionyl residue on enzymatic and antifungal activities of Ageritin purified from Agrocybe aegerita fruiting bodies.

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. Met-Ageritin inhibits protein synthesis in vitro with an IC50 = 2.8 nM that is 21-fold lower than that of Ageritin, while not show endonuclease activity on DNA. Subsequently, we explored the antifungal activity of both isoforms against T. asperellum, pathogen for A. aegerita and Saccharomyces cerevisiae, used as eukaryotic model microorganism. The presence of an additional N-terminal methionyl residue in Met-Ageritin abolishes antifungal activity towards T. asperellum, while neither of two isoforms is able to inhibit the growth of S. cerevisiae. Overall, these data highlight the importance of the N-terminal region of this toxin that likely alters the conformational state of this enzyme considering the presence in this region of metal binding sites necessary for explicate enzymatic activity.

Ageritin, a Ribotoxin from Poplar Mushroom ( Agrocybe aegerita) with Defensive and Antiproliferative Activities.

Ribotoxins make up a group of extracellular rRNA endoribonucleases produced by ascomycetes that display cytotoxicity toward animal cells, having been proposed as insecticidal agents. Recently, the ribotoxin Ageritin has been isolated from the basidiomycetes Agrocybe aegerita (poplar mushroom), suggesting that ribotoxins are widely distributed among fungi. To gain insights into the protective properties of Ageritin against pathogens and its putative biotechnological applications, we have tested several biological activities of Ageritin, comparing them with those of the well-known ribotoxin α-sarcin, and we found that Ageritin displayed, in addition to the already reported activities, (i) antibacterial activity against Micrococcus lysodeikticus, (ii) activity against the tobacco mosaic virus RNA, (iii) endonuclease activity against a supercoiled plasmid, (iv) nuclease activity against genomic DNA, (v) cytotoxicity to COLO 320, HeLa, and Raji cells by promoting apoptosis, and (vi) antifungal activity against the green mold Penicillium digitatum. Therefore, Ageritin and α-sarcin can induce resistance not only to insects but also to viruses, bacteria, and fungi. The multiple biological activities of Ageritin could be exploited to improve resistance to different pathogens by engineering transgenic plants. Furthermore, the induction of cell death by different mechanisms turns these ribotoxins into useful tools for cancer therapy.

Formin Homology 2 Domain Containing 3 (FHOD3) Is a Genetic Basis for Hypertrophic Cardiomyopathy.

BACKGROUND: The genetic cause of hypertrophic cardiomyopathy remains unexplained in a substantial proportion of cases. Formin homology 2 domain containing 3 (FHOD3) may have a role in the pathogenesis of cardiac hypertrophy but has not been implicated in hypertrophic cardiomyopathy. OBJECTIVES: This study sought to investigate the relation between FHOD3 mutations and the development of hypertrophic cardiomyopathy. METHODS: FHOD3 was sequenced by massive parallel sequencing in 3,189 hypertrophic cardiomyopathy unrelated probands and 2,777 patients with no evidence of cardiomyopathy (disease control subjects). The authors evaluated protein-altering candidate variants in FHOD3 for cosegregation, clinical characteristics, and outcomes. RESULTS: The authors identified 94 candidate variants in 132 probands. The variants' frequencies were significantly higher in patients with hypertrophic cardiomyopathy (74 of 3,189 [2.32%]) than in disease control subjects (18 of 2,777 [0.65%]; p < 0.001) or in the gnomAD database (1,049 of 138,606 [0.76%]; p < 0.001). FHOD3 mutations cosegregated with hypertrophic cardiomyopathy in 17 families, with a combined logarithm of the odds score of 7.92, indicative of very strong segregation. One-half of the disease-causing variants were clustered in a small conserved coiled-coil domain (amino acids 622 to 655); odds ratio for hypertrophic cardiomyopathy was 21.8 versus disease control subjects (95% confidence interval: 1.3 to 37.9; p < 0.001) and 14.1 against gnomAD (95% confidence interval: 6.9 to 28.7; p < 0.001). Hypertrophic cardiomyopathy patients carrying (likely) pathogenic mutations in FHOD3 (n = 70) were diagnosed after age 30 years (mean 46.1 ± 18.7 years), and two-thirds (66%) were males. Of the patients, 82% had asymmetric septal hypertrophy (mean 18.8 ± 5 mm); left ventricular ejection fraction <50% was present in 14% and hypertrabeculation in 16%. Events were rare before age 30 years, with an annual cardiovascular death incidence of 1% during follow-up. CONCLUSIONS: FHOD3 is a novel disease gene in hypertrophic cardiomyopathy, accounting for approximately 1% to 2% of cases. The phenotype and the rate of cardiovascular events are similar to those reported in unselected cohorts. The FHOD3 gene should be routinely included in hypertrophic cardiomyopathy genetic testing panels.

Nueva mutación en el gen ALDH3A2 en un niño con síndrome de Sjögren-Larsson / A new mutation in the ALDH3A2 gene in a boy with Sjögren-Larsson syndrome

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Antifungal Activity of α-Sarcin against Penicillium digitatum: Proposal of a New Role for Fungal Ribotoxins.

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. In this work, we report that α-sarcin displays a strong antifungal activity against Penicillium digitatum, being able to enter into the cytosol where it inactivates the ribosomes, thus killing the cells and arresting the growth of the fungus. This is the first time that a ribotoxin has been found to display antifungal activity. Therefore, this protein could play, besides the already proposed insecticidal function, a role in nature as an antifungal agent.

Asociación de obesidad general y abdominal con hipertensión, dislipemia y presencia de prediabetes en el estudio PREDAPS / Association of general and abdominal obesity with hypertension, dyslipidemia and prediabetes in the PREDAPS study

Introducción y objetivos: Algunas medidas antropométricas muestran mayor capacidad que otras para discriminar la presencia de factores de riesgo cardiovascular. Este trabajo estima la magnitud de la asociación de diversos indicadores antropométricos de obesidad con hipertensión, dislipemia y prediabetes (glucemia basal o glucohemoglobina alteradas). Métodos: Análisis transversal de la información recogida en 2.022 sujetos del estudio PREDAPS (etapa basal). Se definió obesidad general como índice de masa corporal ≥ 30 kg/m2 y obesidad abdominal con 2 criterios: a) perímetro de cintura (PC) ≥ 102 cm en varones/PC ≥ 88 cm en mujeres, y b) índice cintura/estatura (ICE) ≥ 0,55. La magnitud de la asociación se estimó mediante regresión logística. Resultados: La hipertensión arterial mostró la asociación más alta con la obesidad general en mujeres (OR = 3,01; IC95%, 2,24-4,04) y con la obesidad abdominal según el criterio del ICE en varones (OR = 3,65; IC95%, 2,66-5,01). La hipertrigliceridemia y los valores bajos de colesterol unido a lipoproteínas de alta densidad mostraron la asociación más alta con obesidad abdominal según el criterio del ICE en mujeres (OR = 2,49; IC95%, 1,68-3,67 y OR = 2,70; IC95%, 1,89-3,86) y la obesidad general en varones (OR = 2,06; IC95%, 1,56-2,73 y OR = 1,68; IC95%, 1,21-2,33). La prediabetes mostró la asociación más alta con obesidad abdominal según el criterio del ICE en mujeres (OR = 2,48; IC95%, 1,85-3,33) y con obesidad abdominal según el criterio del PC en varones (OR = 2,33; IC95%, 1,75-3,08). Conclusiones: Los indicadores de obesidad abdominal mostraron la mayor asociación con la presencia de prediabetes. La relación de los indicadores antropométricos con hipertensión y con dislipemia mostró resultados heterogéneos (AU)

Introduction and objectives: Some anthropometric measurements show a greater capacity than others to identify the presence of cardiovascular risk factors. This study estimated the magnitude of the association of different anthropometric indicators of obesity with hypertension, dyslipidemia, and prediabetes (altered fasting plasma glucose and/or glycosylated hemoglobin). Methods: Cross-sectional analysis of information collected from 2022 participants in the PREDAPS study (baseline phase). General obesity was defined as body mass index ≥ 30 kg/m2 and abdominal obesity was defined with 2 criteria: a) waist circumference (WC) ≥ 102 cm in men/WC ≥ 88 cm in women, and b) waist-height ratio (WHtR) ≥ 0.55. The magnitude of the association was estimated by logistic regression. Results: Hypertension showed the strongest association with general obesity in women (OR, 3.01; 95%CI, 2.24-4.04) and with abdominal obesity based on the WHtR criterion in men (OR, 3.65; 95%CI, 2.66-5.01). Hypertriglyceridemia and low levels of high-density lipoprotein cholesterol showed the strongest association with abdominal obesity based on the WHtR criterion in women (OR, 2.49; 95%CI, 1.68-3.67 and OR, 2.70; 95%CI, 1.89-3.86) and with general obesity in men (OR, 2.06; 95%CI, 1.56-2.73 and OR, 1.68; 95%CI, 1.21-2.33). Prediabetes showed the strongest association with abdominal obesity based on the WHtR criterion in women (OR, 2.48; 95%CI, 1.85-3.33) and with abdominal obesity based on the WC criterion in men (OR, 2.33; 95%CI, 1.75-3.08). Conclusions: Abdominal obesity indicators showed the strongest association with the presence of prediabetes. The association of anthropometric indicators with hypertension and dyslipidemia showed heterogeneous results (AU)

Ebulin-RP, a novel member of the Ebulin gene family with low cytotoxicity as a result of deficient sugar binding domains.

BACKGROUND: Sambucus ebulus is a rich source of ribosome-inactivating proteins (RIPs) and RIP-related lectins generated from multiple genes. These proteins differ in their structure, enzymatic activity and sugar binding specificity. METHODS: We have purified and characterized ebulin-RP from S. ebulus leaves and determined the amino acid sequence by cDNA cloning. Cytotoxicity was studied in a variety of cancer cells and a comparative study of the ability of ebulin-RP to bind sugars using "in vitro" and "in silico" approaches was performed. RESULTS: Ebulin-RP is a novel heterodimeric type 2 RIP present in S. ebulus leaves together with the type 2 RIP ebulin l, which displayed rRNA N-glycosidase activity but unlike ebulin l, lacked functional sugar binding domains. As a consequence of changes in its B-chain, ebulin-RP displayed lower cytotoxicity than ebulin l towards cancer cells and induced apoptosis as the predominant pattern of cell death. CONCLUSIONS: Ebulin-RP is a novel member of the ebulin gene family with low cytotoxicity as a result of deficient sugar binding domains. Type 2 RIP genes from Sambucus have evolved to render proteins with different sugar affinities that may be related to different biological activities and could result in an advantage for the plant. GENERAL SIGNIFICANCE: The ebulin family of RIPs and lectins can serve as a good model for studying the evolutionary process which may have occurred in RIPs. The lack of cytotoxicity of ebulin-RP makes it a good candidate as a toxic moiety in the construction of immunotoxins and conjugates directed against specific targets.

Purification, characterization and cytotoxicity assessment of Ageritin: The first ribotoxin from the basidiomycete mushroom Agrocybe aegerita.

BACKGROUND: Several species belonging to Ascomycota phylum produce extracellular ribonucleases, known as ribotoxins, which exhibit RNase activity through the cleavage of a single phosphodiester bond, located at the universally conserved sarcin/ricin loop of the large rRNA leading to inhibition of protein biosynthesis. Clarifying the structure-function relationship in ribotoxins is interesting for their use in human tumour therapy and in construction of pest resistant transgenic plants. RESULTS: The ribotoxin Ageritin has been isolated for the first time from the Basidiomycetes class. The enzyme, characterized by means of its amino acid composition, N-terminal sequence and a circular dichroism, structurally differs from Ascomycota ribotoxin prototype, although it was able, as α-sarcin, to release a specific α-fragment. However, it does not display aspecific ribonucleolytic activity. Ageritin exerts cytotoxicity and cell death promoting effects towards CNS model cell lines (SK-N-BE(2)-C, U-251 and C6), as vinblastine, a plant alkaloid used in cancer therapy. Moreover, our results indicate that Ageritin initially activates caspase-8, whereas caspase-9 cleavage was not detected, demonstrating the involvement of an extrinsic apoptotic pathway. CONCLUSIONS: Our findings show that Ageritin is the earliest diverging member of the Ascomycota ribotoxin family, suggesting that ribotoxins are more widely distributed among fungi than previously believed. GENERAL SIGNIFICANCE: Ageritin, structurally different from the widely known Ascomycota ribotoxins, with promising anti-cancer properties vs. aggressive brain tumours, has been found from the basidiomycete fungus Agrocybe aegerita. Finally, this finding highlights that the ribotoxin family has divergent members in Basidiomycota phylum, whose structural and functional characterization can give new information on ribotoxin or ribonuclease superfamilies.