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.

Key Concepts

  • Ribotoxins are extremely specific ribonucleases targeted against ribosomes.
  • Ribotoxins are produced by fungi, some of them entomopathogens.
  • They show a high degree of structural conservation, including the local arrangement of the active site residues.
  • Cleavage of a single rRNA phosphodiester bond leads to cell death by inhibiting translation.
  • Ribotoxins are cyclising RNases because they follow a general acid–base mechanism with production of a 2′,3′‐cyclic intermediate.
  • Ribotoxins must first enter their target cells to exert their lethal action.
  • Cell entrance is possible in cells with membranes enriched in acidic phospholipids and altered permeability.
  • Ribotoxins are optimal candidates to be employed as pest control agents and in antitumour immunotoxins.

Keywords: antitumoural; elongation factor; entomopathogen; fungal toxin; immunotoxin; insecticide; ribonuclease; ribosome; ribotoxin; sarcin

Figure 1. Representation of the three‐dimensional structure of representative fungal RNases. Diagrams showing the three‐dimensional structure of ribotoxins α‐sarcin (PDB ID: 1DE3), restrictocin (PDB ID: 1AQZ) and HtA (PDB ID: 2KAA), and two nontoxic fungal extracellular RNases from the same family: RNases T1 (PDB ID: 9RNT) and U2 (PDB ID:1RTU). Diagrams were generated using the Chimera software (Pettersen et al., ).
Figure 2. Representation of the active site arrangement of the most representative fungal RNases. The catalytic triad made of two His and one Glu residues is conserved in all proteins shown, as well as α‐sarcin Arg121, while a fifth residue, α‐sarcin Leu145, maintains its highly hydrophobic character (Phe or Leu). The position corresponding to α‐sarcin Tyr48 is also conserved except for HtA and anisoplin (not shown) where the equivalent position is occupied by an Asp residue (Asp40). Diagrams were generated using the Chimera software (Pettersen et al., ).
Figure 3. Catalytic mechanism of cyclising RNases. The catalytic mechanism of cyclic RNases such as ribotoxins against a dinucleotide substrate (ApA or GpA) is shown. A transphosphorylation process (in which the corresponding 2′,3′‐cyclic mononucleotide and adenosine are produced) is followed by hydrolysis of the cyclic nucleotide to produce the corresponding 3′‐mononucleotide. Side chains of residues corresponding to α‐sarcin His50, Glu96 and His137 are also shown, indicating at the bottom left corner of the figure their spatial location in the context of the whole protein three‐dimensional structure.
Figure 4. The substrate of ribotoxins. (a) Three‐dimensional structure of the large ribosomal subunit of Escherichia coli (PDB ID: 2AW4). The location of L1 and L7/L12 stalks (absent in this crystal) and E, P and A sites are indicated. Conserved proteins around the SRL (orange) appear in different colours: uL6 (green), uL11 (red) and uL14 (blue). Other ribosomal proteins appear in light grey. 23S (dark grey) and 5S (cyan) rRNAs are also shown; (b) SRL structure. The bulged G (red), the GAGA tetraloop (blue), the bond cleaved by α‐sarcin and the adenine depurinated by ricin are indicated. Diagrams were generated using the Chimera software (Pettersen et al., ).
Figure 5. Schematic representation of the translocation mechanism of α‐sarcin across the bilayer of negatively charged phospholipid vesicles. (a) Binding experiments reveal a strong ribotoxin–lipid vesicle interaction that causes vesicle aggregation (b) mediated by the formation of a vesicle dimer maintained by protein–protein associations. The N‐terminal stretch as well as some of the positively charged loops play a key role at this step. (c) Then, the β‐sheet region comprising residues 116–139, altogether with the Trp side chains (in pink), establishes a destabilising hydrophobic interaction with the membrane that leads to (d) protein internalisation.


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Further Reading

Carreras‐Sangrà N, Álvarez‐García E, Herrero‐Galán E, et al. (2008) The therapeutic potential of fungal ribotoxins. Current Pharmaceutical Biotechnology 9: 153–160.

Carreras‐Sangrà N, Tomé‐Amat J, García‐Ortega L, et al. (2012) Production and characterization of a colon cancer specific immunotoxin based on the fungal ribotoxin α‐sarcin. Protein Engineering Design and Selection 25: 425–435.

García‐Mayoral MF, Martínez‐del‐Pozo A, Campos‐Olivas R, et al. (2006) pH‐dependent conformational stability of the ribotoxin α‐sarcin and four active site charge substitution variants. Biochemistry 45: 13705–13718.

Gasset M, Mancheño JM, Lacadena J, et al. (1995) Spectroscopic characterization of the alkylated α‐sarcin cytotoxin: analysis of the structural requirements for the protein–lipid bilayer hydrophobic interaction. Biochimica et Biophysica Acta 1252: 43–52.

Herrero‐Galán E, García‐Ortega L, Olombrada M, et al. (2013) Hirsutellin A: a paradigmatic example of the insecticidal function of fungal ribotoxins. Insects 4: 339–356.

Martínez‐Ruiz A, García‐Ortega L, Kao R, et al. (2001) RNase U2 and α‐sarcin: a study of relationships. Methods in Enzymology 341: 335–351.

Masip M, Lacadena J, Mancheño JM, et al. (2001) Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin α‐sarcin. European Journal of Biochemistry 268: 6190–6196.

Pérez‐Cañadillas JM, Campos‐Olivas R, Lacadena J, et al. (1998) Characterization of pKa values and titration shifts in the cytotoxic ribonuclease a‐sarcin by NMR: relationship between electrostatic interactions, structure, and catalytic function. Biochemistry 37: 15865–15876.

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García‐Ortega, Lucía, Palacios‐Ortega, Juan, and Martínez‐del‐Pozo, Álvaro(Feb 2018) Fungal Ribotoxins. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0027741]