Fungal ribotoxins: Natural protein-based weapons against insects

Olombrada, Miriam; Martínez‐del‐Pozo, Álvaro; Medina, Pilar; Budia, F.; Gavilanes, José G.; Garcı́a-Ortega, Lucı́a

doi:10.1016/j.toxicon.2014.02.022

Cited by 37 publications

(43 citation statements)

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“…The main producer is Aspergillus, although the discovery of an entomopathogenic fungus, Hirsutella thompsonii, producing the ribotoxin hirsutellin A (HtA) suggests a wider distribution among fungi as well as their insecticide properties ( Martínez-Ruiz et al, 1999;Herrero-Galán et al, 2008;Olombrada et al, 2013Olombrada et al, , 2014a. Ribotoxins specifically cross some lipid barriers and then efficiently inactivate any kind of ribosome.…”

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“…As mentioned before, ribotoxins are extremely specific RNases against a unique element present in any kind of ribosome: the sarcin/ricin loop (SRL), a conserved sequence of about 35 nucleotides of the large ribosomal RNA (Schindler and Davies, 1977;Endo and Wool, 1982). The role of the SRL can be impaired by two types of toxins: N-glycosidades such as ricin, that depurinate one nucleotide (Stirpe and Battelli, 2006), and ribotoxins, that cleave the contiguous phosphodiester bond (Lacadena et al, 2007;Olombrada et al, 2014a). These modifications in the SRL, completely inactivate the mechanisms of correct positioning and GTPase activation of elongation factors that it is involved in, leading to protein biosynthesis inactivation and finally cell death (García-Ortega et al, 2010;Voorhees et al, 2010;Shi et al, 2012;Koch et al, 2015).…”

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Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin

Olombrada

Medina

Budia

et al. 2017

Biological Chemistry

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Abstract: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. Moreover, anisoplin shows the ribonucleolytic activity typical of ribotoxins and cytotoxicity against insect cells. How Metarhizium uses this toxin and possible applications are of interest.

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Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin

Olombrada

Medina

Budia

et al. 2017

Biological Chemistry

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“…HtA is considerably smaller than the rest of ribotoxins known but still contains the same elements of periodic secondary structure and an identical structural arrangement of the active site residues (Figure 1) (Herrero-Galán et al 2012a; HerreroGalán et al 2012b;Herrero-Galán et al 2008; Martínez-Ruiz et al 1999b; Viegas et al 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 HtA is indeed a well-known insecticidal protein (Boucias et al 1998;Liu et al 1995). Therefore, the characterization of HtA as a fungal ribotoxin not only proved that the unique abilities of ribotoxins can be accommodated into a shorter amino acid sequence (Herrero-Galán et al 2008), but also suggested that they might play an insecticidal role in nature Olombrada et al 2014a). Consequently, deciphering the distinct molecular features that enable rather different proteins like α-sarcin and HtA to show this identical and singular toxic behavior is of great interest and might be useful in the design and development of new and effective biotechnological tools for different applications like new biopesticides Olombrada et al 2014a), the study of ribosome-related diseases (De la Cruz et al 2015;Olombrada et al 2014b) or the construction of new immunotoxins (Tomé-Amat et al 2015).…”

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“…Therefore, the characterization of HtA as a fungal ribotoxin not only proved that the unique abilities of ribotoxins can be accommodated into a shorter amino acid sequence (Herrero-Galán et al 2008), but also suggested that they might play an insecticidal role in nature Olombrada et al 2014a). Consequently, deciphering the distinct molecular features that enable rather different proteins like α-sarcin and HtA to show this identical and singular toxic behavior is of great interest and might be useful in the design and development of new and effective biotechnological tools for different applications like new biopesticides Olombrada et al 2014a), the study of ribosome-related diseases (De la Cruz et al 2015;Olombrada et al 2014b) or the construction of new immunotoxins (Tomé-Amat et al 2015).The N-terminal β-hairpin of ribotoxins has been shown to modulate their catalytic activity García-Ortega et al 2002). Deletion variants of α-sarcin and Aspf1 (another well-known ribotoxin), in which this hairpin had been eliminated without affecting the overall three-dimensional structure of the proteins (García-Mayoral et al.…”

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“…Most of them show a high degree of conservation with sequence identities above 60% and even higher than 85% in many instances. The only exception known so far is hirsutellin A (HtA), another extracellular RNase produced by an invertebrate pathogen, the fungus Hirsutella thompsonii, which has been demonstrated to be a ribotoxin even though it only displays about 25% sequence identity with the previously known members of the family (Figure 1) (Boucias et al 1998;Herrero-Galán et al 2013;Herrero-Galán et al 2008;Martínez-Ruiz et al 1999b;Olombrada et al 2014a). HtA is considerably smaller than the rest of ribotoxins known but still contains the same elements of periodic secondary structure and an identical structural arrangement of the active site residues (Figure 1) (Herrero-Galán et al 2012a;HerreroGalán et al 2012b;Herrero-Galán et al 2008;Martínez-Ruiz et al 1999b;Viegas et al HtA is indeed a well-known insecticidal protein (Boucias et al 1998;Liu et al 1995).…”

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Involvement of loop 5 lysine residues and the N-terminal β-hairpin of the ribotoxin hirsutellin A on its insecticidal activity

Olombrada

Garcı́a-Ortega

Lacadena

et al. 2016

Biological Chemistry

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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. Examination of HtA Nterminal β-hairpin reveals differences in terms of length, charge, and spatial distribution.Consequently, four different HtA mutants were prepared and characterized. One of them was the result of deleting this hairpin [∆(8-15)] while the other three affected single Lys residues in its close spatial proximity (K115E, K118E, and K123E). The results obtained support the general conclusion that HtA active site would show a high degree of plasticity, being able to accommodate electrostatic and structural changes not suitable for the other previously known larger ribotoxins, as the variants described here only presented small differences in terms of ribonucleolytic activity and cytotoxicity against cultured insect cells. Keywords:Ribotoxins, hirsutellin A, Ribonucleases, rRNA, insecticidal. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 IntroductionRibotoxins are cytotoxic members of the family of fungal extracellular ribonucleases (RNases) best represented by RNase T1 (Yoshida 2001). They have been shown to be extremely toxic because they exert their ribonucleolytic activity just on a unique phosphodiester bond of the larger molecule of rRNA in the ribosome, leading to protein synthesis inhibition and cell death (Lacadena et al. 2007). This rRNA bond is unique because it is located at an evolutionarily conserved site, the sarcin-ricin loop (SRL), with essential roles in ribosome function (García-Ortega et al. 2010) and maturation (Lo et al. 2010), and it is also the target of the family of plant ribosome-inactivating proteins (RIPs), a group of glycosidases best represented by ricin (Nielsen and Boston 2001).In addition to their ribonucleolytic activity, fungal ribotoxins have the ability to cross lipid membranes in the absence of any known protein receptor, mainly due to their ability to interact with acid phospholipids Martínez-Ruiz et al. 2001;Oñaderra et al. 1993). This feature is the explanation of why these proteins display a remarkable but not highly specific antitumoral activity (Jennings et al. 1965;Lacadena et al. 2007;Olmo et al. 2001;Olson and Goerner 1965;Turnay et al. 1993).α-Sarcin is the most extensively characterized ribotoxin (Lacadena et al. 2007), but many others have been identified and/or characterized in different fungal spec...

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Fungal Ribotoxins

Garcı́a-Ortega

Palacios-Ortega

Martínez‐del‐Pozo

2018

Encyclopedia of Life Sciences

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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.

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Fungal ribotoxins: Natural protein-based weapons against insects

Cited by 37 publications

References 41 publications

Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin

Characterization of a new toxin from the entomopathogenic fungus Metarhizium anisopliae: the ribotoxin anisoplin

Involvement of loop 5 lysine residues and the N-terminal β-hairpin of the ribotoxin hirsutellin A on its insecticidal activity

Fungal Ribotoxins

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