Fungal ribotoxins: molecular dissection of a family of natural killers

Abstract

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.

Functional convergence of gliP and aspf1 in Aspergillus fumigatus pathogenicity

“…It functions by penetrating the cell membrane and cleaving a single phosphodiester bond of the 60S rRNA, which halts protein synthesis [34]. We found that mice infected with the Δ aspf1 mutant had prolonged survival, implicating Asp f 1 in A. fumigatus virulence.…”

Fungal extracellular ribotoxins as insecticidal agents

“…A few years later it was demonstrated that they inhibited protein biosynthesis by specifically cleaving a unique phosphodiester bond of the large rRNA fragment (Endo and Wool, 1982;Schindler and Davies, 1977) located at an evolutionarily conserved site with important roles in ribosome function. This observation prompted the development of their molecular characterization (Lacadena et al, 2007). Much later, more systematic analysis of fungal genomes revealed that ribotoxins were a more widespread group of proteins within the filamentous fungi than had been previously believed (Lin et al, 1995;Martínez-Ruiz et al, 1999b;Varga and Samson, 2008).…”

“…Much later, more systematic analysis of fungal genomes revealed that ribotoxins were a more widespread group of proteins within the filamentous fungi than had been previously believed (Lin et al, 1995;Martínez-Ruiz et al, 1999b;Varga and Samson, 2008). All this exhaustive work dealing with the characterization of their mechanism of action at the molecular level has not only helped to understand the basis of their antitumoral action (Lacadena et al, 2007) but also has established the principles to transform some of them into engineered chimeras with potential therapeutic uses (Carreras-Sangrà et al, 2008, 2012.…”

“…Fungal ribotoxins are extracellular and highly specific ribonucleases which behave as potent inhibitors of protein biosynthesis by being able to inactivate ribosomes from almost any organism Kao et al, 2001;Lacadena et al, 2007;Martínez-Ruiz et al, 2001). They cleave the larger rRNA component at a single phosphodiester bond located within the universally conserved sarcin/ricin loop (SRL) (Chan et al, 1983;Endo and Wool, 1982;Endo et al, 1983;Schindler and Davies, 1977), leading to complete inactivation of the ribosome and cell death by apoptosis (Olmo et al, 2001).…”

“…These studies have shown a high degree of conservation among ribotoxins, as most of those so far characterized display amino acid sequence similarities above 85% (Lacadena et al, 2007). Surprisingly, HtA is a much smaller protein (130 amino acids against 149/150 of the other known ribotoxins) which displays only 25% sequence identity with previously known family members but still contains the same elements of periodic secondary structure and identical arrangement of the active site residues (Boucias et al, 1998;Herrero-Galán et al, 2008;Martínez-Ruiz et al, 1999a).…”