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Hirsutellin A: A Paradigmatic Example of the Insecticidal Function of Fungal Ribotoxins.

Herrero-Galán E, García-Ortega L, Olombrada M, Lacadena J, Del Pozo ÁM, Gavilanes JG, Oñaderra M - Insects (2013)

Bottom Line: Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents.Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells.The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, 28040 Madrid, Spain. eherrero@cnb.csic.es.

ABSTRACT
The fungal pathogen Hirsutella thompsonii produces an insecticidal protein named hirsutellin A (HtA), which has been described to be toxic to several species of mites, insect larvae, and cells. On the other hand, on the basis of an extensive biochemical and structural characterization, HtA has been considered to be a member of the ribotoxins family. Ribotoxins are fungal extracellular ribonucleases, which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the large rRNA. Although ribotoxins were brought to light in the 1960s as antitumor agents, their biological function has remained elusive. Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents. Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells. The determination of high resolution structures, the characterization of a large number of mutants, and the toxicity assays against different cell lines have been the tools used for the study of the mechanism of action of ribotoxins at the molecular level. The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins.

No MeSH data available.


Related in: MedlinePlus

Three-dimensional structures of the ribotoxins α-sarcin and hirsutellin A (HtA) Spatial orientation of the N-terminal β-hairpin and loops 2 and 5 in HtA and α-sarcin. Positively charged residues are depicted. Color code is the same as in Figure 1. The diagrams were generated using MOLMOL [49].
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insects-04-00339-f003: Three-dimensional structures of the ribotoxins α-sarcin and hirsutellin A (HtA) Spatial orientation of the N-terminal β-hairpin and loops 2 and 5 in HtA and α-sarcin. Positively charged residues are depicted. Color code is the same as in Figure 1. The diagrams were generated using MOLMOL [49].

Mentions: From the clone of the cDNA of HtA, plasmid pTac-TacHtA was constructed for the expression of mature HtA in E. coli [30]. HtA was purified both from its natural source and also as a recombinant protein. Spectroscopic analysis determined that the fungal protein and the recombinant one were indistinguishable. The exhaustive characterization of both forms of HtA reveals an E coefficient (0.1%, 280 nm, 1 cm) of 2.00 for both proteins [30]. The mid-point of the thermal denaturation transition (Tm) determined by circular dichroism (CD), and differential scanning calorimetry (DSC) was 62 ºC. This value was 10 ºC higher than that reported for α-sarcin [30] but closer to 61 ºC and 59 ºC, the Tm values for the ribotoxins AspF1 and restrictocin, respectively [48]. In 2009, the elucidation of the three-dimensional structure of HtA in solution by nuclear magnetic resonance (NMR) [41] confirmed that the overall protein fold of ribotoxins is maintained in this smaller polypeptide chain, but some important differences apart from size-derived variations were observed. The structure consists of one α-helix, one helical turn, and seven β-strands that form a β-sheet and a N-terminal hairpin, with a characteristic α + β fold and a highly positively charged surface. The most relevant structural differences when compared to its larger homolog, α-sarcin, are the shorter lengths of loop 2 and the N-terminal β-hairpin, which is also less positively charged (Figure 3). This truncation and reduced charge of the N-terminal hairpin in HtA may be compensated by the extension and different orientation of its loop 5, which exhibits a higher amount of positively charged residues.


Hirsutellin A: A Paradigmatic Example of the Insecticidal Function of Fungal Ribotoxins.

Herrero-Galán E, García-Ortega L, Olombrada M, Lacadena J, Del Pozo ÁM, Gavilanes JG, Oñaderra M - Insects (2013)

Three-dimensional structures of the ribotoxins α-sarcin and hirsutellin A (HtA) Spatial orientation of the N-terminal β-hairpin and loops 2 and 5 in HtA and α-sarcin. Positively charged residues are depicted. Color code is the same as in Figure 1. The diagrams were generated using MOLMOL [49].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4553468&req=5

insects-04-00339-f003: Three-dimensional structures of the ribotoxins α-sarcin and hirsutellin A (HtA) Spatial orientation of the N-terminal β-hairpin and loops 2 and 5 in HtA and α-sarcin. Positively charged residues are depicted. Color code is the same as in Figure 1. The diagrams were generated using MOLMOL [49].
Mentions: From the clone of the cDNA of HtA, plasmid pTac-TacHtA was constructed for the expression of mature HtA in E. coli [30]. HtA was purified both from its natural source and also as a recombinant protein. Spectroscopic analysis determined that the fungal protein and the recombinant one were indistinguishable. The exhaustive characterization of both forms of HtA reveals an E coefficient (0.1%, 280 nm, 1 cm) of 2.00 for both proteins [30]. The mid-point of the thermal denaturation transition (Tm) determined by circular dichroism (CD), and differential scanning calorimetry (DSC) was 62 ºC. This value was 10 ºC higher than that reported for α-sarcin [30] but closer to 61 ºC and 59 ºC, the Tm values for the ribotoxins AspF1 and restrictocin, respectively [48]. In 2009, the elucidation of the three-dimensional structure of HtA in solution by nuclear magnetic resonance (NMR) [41] confirmed that the overall protein fold of ribotoxins is maintained in this smaller polypeptide chain, but some important differences apart from size-derived variations were observed. The structure consists of one α-helix, one helical turn, and seven β-strands that form a β-sheet and a N-terminal hairpin, with a characteristic α + β fold and a highly positively charged surface. The most relevant structural differences when compared to its larger homolog, α-sarcin, are the shorter lengths of loop 2 and the N-terminal β-hairpin, which is also less positively charged (Figure 3). This truncation and reduced charge of the N-terminal hairpin in HtA may be compensated by the extension and different orientation of its loop 5, which exhibits a higher amount of positively charged residues.

Bottom Line: Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents.Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells.The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, 28040 Madrid, Spain. eherrero@cnb.csic.es.

ABSTRACT
The fungal pathogen Hirsutella thompsonii produces an insecticidal protein named hirsutellin A (HtA), which has been described to be toxic to several species of mites, insect larvae, and cells. On the other hand, on the basis of an extensive biochemical and structural characterization, HtA has been considered to be a member of the ribotoxins family. Ribotoxins are fungal extracellular ribonucleases, which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the large rRNA. Although ribotoxins were brought to light in the 1960s as antitumor agents, their biological function has remained elusive. Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents. Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells. The determination of high resolution structures, the characterization of a large number of mutants, and the toxicity assays against different cell lines have been the tools used for the study of the mechanism of action of ribotoxins at the molecular level. The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins.

No MeSH data available.


Related in: MedlinePlus