Fungal Ribotoxins

Fungal ribotoxins constitute a family of extracellular ribonucleases with exquisite specificity against rRNA (ribonucleic acid). They induce apoptotic death of cells after inhibiting protein translation. Ribosomes become functionally incompetent because ribotoxins cleave one single phosphodiester bond, located at a unique and universally conserved loop, needed for elongation factors function. As secreted proteins, ribotoxins need to cross the membrane of their target cells in order to exert their catalytic activity, and they do it without receptor mediation. Using lipid model systems, it has been shown that they are able to enter cells with membranes enriched in acidic phospholipids. Both membrane-interacting and ribosomal-recognition activities are characterised by distinct structural features. Even though the natural function of ribotoxins is not known yet, their production by entomopathogenic fungi has suggested their insecticidal role. After decades of detailed study, the biotechnological potential of ribotoxins in pest control and as antitumour agents is becoming evident

Identificación de riesgos geoambientales y su valoración en la zona de hundimiento del buque Prestige

Potential geological hazard assessment has been carried out in the area where the Prestige vessel was sunk using a broad database that comprises: multibeam, high and ultra-high resolution seismic profiles, gravity cores, onland seismicity stations and Ocean Bottom Seismometers (OBS). The main results of this study indicate that among the geologic factors that can be considered as potential hazards, four main categories can be differentiated based on their origin: morphologic, sedimentary, tectonic, and seismicity. Hazards of morphologic origin include steep gradients; the morphologic features suggest the occurrence of mass-wasting instabilities. Hazards of sedimentary origin also includes the occurrence of slope instability processes in form of single slides and a great variety of erosive and depositional gravity flows (debris and turbidity flows). Hazards of tectonic and seismic origin are important because the sinking area straddles the Calida Bank which is a structural seamount with a moderate tectonic activity that results in a latent seismicity of low to moderate magnitude. The interaction of these factors leads to consider to the risk as medium, and the degree of exposure of the bow and stern as high. Several general and specific recommendations are made in order to increase the geological and geophysics knowledgement in the Prestige sinking area and Spanish continental margins and deep sea areas. These recommendations also should be used to elaborate the options for reducing the hazard and loss

Fungal ribotoxins: structure, function and evolution

Ribotoxins are a family of fungal extracellular ribonucleases which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the universally conserved sarcin/ricin loop of the large rRNA. The subsequent inhibition of protein biosynthesis is followed by cell death via apoptosis. Ribotoxins are also able to interact with membranes containing acid phospholipids, their cytotoxicity being preferentially directed towards cells showing altered membrane permeability, e.g. transformed or virus infected cells. Many features of their cytotoxic action and their ribonucleolytic mechanism have been elucidated by comparison with other extracellular non toxic fungal RNases, best represented by RNase T1. The study of structure-function relationships in ribotoxins is of particular interest, since they are postulated as potential therapeutic agents against different human pathologies. The production of hypoallergenic variants with application in several Aspergillus-related allergic syndromes and the construction of immunotoxins against different carcinomas are promising examples of such potential therapeutic utilisation

Fungal ribotoxins: molecular dissection of a familyof natural killers

RNase T1 is the best known representative of a large family of ribonucleolytic proteins secreted by fungi, mostly Aspergillus and Penicillium species. Ribotoxins stand out among them by their cytotoxic character. They exert their toxic action by first entering the cells and then cleaving a single phosphodiester bond located within a universally conserved sequence of the large rRNA gene, known as the sarcin–ricin loop. This cleavage leads to inhibition of protein biosynthesis, followed by cellular death by apoptosis. Although no protein receptor has been found for ribotoxins, they preferentially kill cells showing altered membrane permeability, such as those that are infected with virus or transformed. Many steps of the cytotoxic process have been elucidated at the molecular level by means of a variety of methodological approaches and the construction and purification of different mutant versions of these ribotoxins. Ribotoxins have been used for the construction of immunotoxins, because of their cytotoxicity. Besides this activity, Aspf1, a ribotoxin produced by Aspergillus fumigatus, has been shown to be one of the major allergens involved in allergic aspergillosis-related pathologies. Protein engineering and peptide synthesis have been used in order to understand the basis of these pathogenic mechanisms as well as to produce hypoallergenic proteins with potential diagnostic and immunotherapeutic applications

The therapeutic potential of fungal ribotoxins

Ribotoxins constitute a family of toxic extracellular fungal RNases that exert a highly specific activity on a conserved region of the larger molecule of rRNA, known as the sarcin–ricin loop. This cleavage of a single phosphodiester bond inactivates the ribosome and leads to protein synthesis inhibition and cell death. In addition to this ribonucleolytic activity, ribotoxins can cross lipid membranes in the absence of any known protein receptor. This ability is due to their capacity to interact with acid phospholipid-containing membranes. Both activities together explain their cytotoxic character, being rather specific when assayed against some transformed cell lines. The determination of high-resolution structures of some ribotoxins, the characterization of a large number of mutants, and the use of lipid model vesicles and transformed cell lines have been the tools used for the study of their mechanism of action at the molecular level. The present knowledge suggests that wild-type ribotoxins or some modified variants might be used in human therapies. Production of hypoallergenic mutants and immunotoxins designed against specific tumors stand out as feasible alternatives to treat some human pathology in the mid-term future

Fungal extracellular ribotoxins as insecticidal agents

Fungal ribotoxins were discovered almost 50 years ago as extracellular ribonucleases (RNases) with antitumoral properties. However, the biological function of these toxic proteins has remained elusive. The discovery of the ribotoxin HtA, produced by the invertebrates pathogen H. thompsonii, revived the old proposal that insecticidal activity would be their long searched function. Unfortunately, HtA is rather singular among all ribotoxins known in terms of sequence and structure similarities. Thus, it was intriguing to answer the question of whether HtA is just an exception or, on the contrary, the paradigmatic example of the ribotoxins function. The work presented uses HtA and -sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells

α-sarcin and RNase T1 based immunoconjugates: the role of intracellular trafficking in cytotoxic efficiency

Toxins have been thoroughly studied for their use as therapeutic agents in search of an improvement in toxic efficiency together with a minimization of their undesired side effects. Different studies have shown how toxins can follow different intracellular pathways which are connected with their cytotoxic action inside the cells. The work herein presented describes the different pathways followed by the ribotoxin a-sarcin and the fungal RNase T1,as toxic domains of immunoconjugates with identical binding domain, the single chain variable fragment of a monoclonal antibody raised against the glycoprotein A33. According to the results obtained both immunoconjugates enter the cells via early endosomes and, while a-sarcin can translocate directly into the cytosol to exert its deathly action, RNase T1 follows a pathway that involves lysosomes and the Golgi apparatus. These facts contribute to explaining the different cytotoxicity observed against their targeted cells, and reveal how the innate properties of the toxic domain, apart from its catalytic features, can be a key factor to be considered for immunotoxin optimization

Role of the basic character of α-sarcin’s NH2-terminal β-hairpin in ribosome recognition and phospholipid interaction.

Ribotoxins are a family of toxic extracellular fungal RNases that first enter into the cells and then exert a highly specific ribonucleolytic activity on the larger rRNA molecule, leading to protein synthesis inhibition and cell death by apoptosis. α-Sarcin is the best characterized ribotoxin. Previous characterization of a deletion variant of this protein showed that its long NH2-terminal β-hairpin is essential for its cytotoxicity. Docking, enzymatic, and lipid-protein interaction studies suggested that this β-hairpin establishes specific interactions with ribosomal proteins and that it is a region involved in the interaction with cell membranes. Consequently, in order to assess the influence of the basic character of this NH2-terminal β-hairpin (there are 1 arginine and 4 lysines along its 16 residues) on the ribotoxins cytotoxic ability, five individual mutants substituting these 5 basic residues by glutamic acid were produced, purified to homogeneity, and characterized. Regarding ribosomal recognition, all mutants showed a diminished activity in a cell-free reticulocyte lysate, whereas the activity against an oligoribonucleotide mimicking the sarcin/ricin loop rRNA (SRL) or the homopolymer poly(A) remained unaffected, confirming that the mutated basic residues participate in electrostatic interactions with other ribosomal elements apart from this SRL. The study of the interaction with phospholipid vesicles showed that Lys 17, Arg 22, and, most importantly, Lys 14 and Lys 21, are crucial residues in the first stages of the aggregation phenomenon, where protein-vesicle and protein-protein interactions are required. The data obtained reveal that electrostatic interactions involving basic residues of the β-hairpin are required not only for establishing specific interactions with ribosomal regions other than the SRL but also to explain the ability of the protein to interact with acid phospholipid bilayers