The Acidic Ribosomal Stalk Proteins Are Not Required for the Highly Specific Inactivation Exerted by α-Sarcin of the Eukaryotic Ribosome

Olombrada, Miriam; Rodríguez-Mateos, María; Prieto, Daniel; Pla, Jesús; Remacha, Miguel; Martínez‐del‐Pozo, Álvaro; Gavilanes, José G.; Ballesta, Juan P. G.; Garcı́a-Ortega, Lucı́a

doi:10.1021/bi401470u

Cited by 10 publications

(13 citation statements)

References 25 publications

(62 reference statements)

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“…Ribotoxin cleavage was quantitated by poison primer extension (PPE) as described (García-Ortega et al 2010;Olombrada et al 2014b). Reverse transcription where dATP is substituted by ddATP was performed using the complementary sequence downstream the SRL in the 28S rRNA of O. cuniculus (5'-ACCAAATGTCTGAACCTGCGG-3').…”

Section: Ribonucleolytic Activity Assaysmentioning

confidence: 99%

“…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 2004;García-Ortega et al 2005b;García-Ortega et al 2002), retained their nonspecific ribonucleolytic activity as well as their ability to specifically cleave SRL-like oligonucleotides. However, these deletion mutants were not able to inactivate rabbit ribosomes with the high specificity and efficiency displayed by the wild-type proteins and, therefore, were much less cytotoxic (García-Ortega et al 2005b;García-Ortega et al 2002;Olombrada et al 2014b). Comparison of α-sarcin and HtA three-dimensional structures revealed major differences precisely in the sequence, length, and conformation of their Nterminal β-hairpins (Fig.…”

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confidence: 99%

“…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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confidence: 99%

“…After a 15 min incubation at room temperature of the sample with 1-50 nM of the different proteins in 23 mM Tris-HCl, 23 mM KCl, 6 mM EDTA pH 7.5, RNA was phenol-extracted, precipitated with isopropanol and visualized by ethidium bromide staining after electrophoresis on denaturing 2.4 % agarose gels as described (Herrero-Galán et al 2008;Lacadena et al 1994;Lacadena et al 1999). Ribotoxin cleavage was quantitated by poison primer extension (PPE) as described (García-Ortega et al 2010;Olombrada et al 2014b). Reverse transcription where dATP is substituted by ddATP was performed using the complementary sequence downstream the SRL in the 28S rRNA of O. cuniculus (5'-ACCAAATGTCTGAACCTGCGG-3').…”

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confidence: 99%

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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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Section: Ribonucleolytic Activity Assaysmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…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). In this regard, several studies have tried to specify the structural elements in the ribotoxin, the ribosome and the SRL responsible for this fine and efficient recognition (García-Ortega et al, 2002;García-Mayoral et al, 2005;Olombrada et al, 2014b).…”

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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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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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The Acidic Ribosomal Stalk Proteins Are Not Required for the Highly Specific Inactivation Exerted by α-Sarcin of the Eukaryotic Ribosome

Cited by 10 publications

References 25 publications

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

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

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

Fungal Ribotoxins

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