Tyr-48, a conserved residue in ribotoxins, is involved in the RNA-degrading activity of α-sarcin

Álvarez-García, Elisa; Garcı́a-Ortega, Lucı́a; Verdún, Yolanda; Bruix, Marta; Pozo, Álvaro Martı́nez del; Gavilanes, José G.

doi:10.1515/bc.2006.069

Cited by 18 publications

(26 citation statements)

References 31 publications

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“…These mutational studies have revealed the involvement of several residues, which are conserved among the different microbial extracellular RNases, in catalysis. Thus, it is well known that His137 and Glu96 are the only residues that are essential for the cleavage reaction performed by α-sarcin [35,45,[70][71][72][73][74][75], whereas His-50, Tyr-48, Arg-121, and Leu-145 mostly contribute to the stabilization of the transition state [45][46][47][48], as stated above. Most of these residues are located in the central β-sheet and their side-chains point towards the concave face of the protein structure where the substrate is supposed to dock [29].…”

Section: Structural Featuresmentioning

confidence: 85%

“…Mutational studies have also revealed that three other active site residues, Tyr48, Arg121, and Leu145, although not essential, appear to be determinants of the ribotoxin activity of α-sarcin [46][47][48]. Studies on the crystal structures of complexes of restrictocin with inhibitors led to the proposal that these ribotoxins may use base flipping to enable cleavage at the correct site of the SRL substrates [49].…”

Section: Ribonucleolytic Activitymentioning

confidence: 99%

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The Therapeutic Potential of Fungal Ribotoxins

Carreras-Sangrà

Álvarez-García²,

Herrero-Galán³

et al. 2008

CPB

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Ribotoxins constitute a family of toxic extracellular fungal RNases that exert a highly specific activity on a conserved region of the larger molecule of rRNA, known as the sarcin-ricin loop. This cleavage of a single phosphodiester bond inactivates the ribosome and leads to protein synthesis inhibition and cell death. In addition to this ribonucleolytic activity, ribotoxins can cross lipid membranes in the absence of any known protein receptor. This ability is due to their capacity to interact with acid phospholipid-containing membranes. Both activities together explain their cytotoxic character, being rather specific when assayed against some transformed cell lines. The determination of highresolution structures of some ribotoxins, the characterization of a large number of mutants, and the use of lipid model vesicles and transformed cell lines have been the tools used for the study of their mechanism of action at the molecular level. The present knowledge suggests that wild-type ribotoxins or some modified variants might be used in human therapies. Production of hypoallergenic mutants and immunotoxins designed against specific tumors stand out as feasible alternatives to treat some human pathology in the midterm future.3

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Section: Structural Featuresmentioning

confidence: 85%

Section: Ribonucleolytic Activitymentioning

confidence: 99%

The Therapeutic Potential of Fungal Ribotoxins

Carreras-Sangrà

Álvarez-García²,

Herrero-Galán³

et al. 2008

CPB

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“…After more than two decades of systematic studies, it has also become well-established that the activity of ribotoxins is extremely dependent on electrostatic interactions among their active site residues (Álvarez-García et al 2006;Masip et al 2001;Pérez-Cañadillas et al 1998;Pérez-Cañadillas et al 2000). His 42, Glu 66 and His 113 form the catalytic triad of HtA (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Spectroscopic characterization was performed following well-established procedures (Álvarez-García et al 2006;Álvarez-García et al 2009b;García-Ortega et al 2005a;García-Ortega et al 2001;García-Ortega et al 2002;Lacadena et al 1999;Martínez-Ruiz et al 2001). Absorbance measurements were carried out on a Beckman DU640 were also used to calculate their extinction coefficients (Table 1).…”

Section: Spectroscopic Characterizationmentioning

confidence: 99%

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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“…Cloning procedures, oligonucleotide site-directed mutagenesis, and bacterial manipulations were carried out as previously described [28,38,39]. The mutagenic primers used to substitute the mutated residues are shown in Table 1.…”

Section: Dna Manipulationsmentioning

confidence: 99%

Role of the basic character of α-sarcin’s NH2-terminal β-hairpin in ribosome recognition and phospholipid interaction

Álvarez-García

Martínez‐del‐Pozo

Gavilanes

2009

Archives of Biochemistry and Biophysics

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Ribotoxins are a family of toxic extracellular fungal RNases that first enter into the cells and then exert a highly specific ribonucleolytic activity on the larger rRNA molecule, leading to protein synthesis inhibition and cell death by apoptosis. α-Sarcin is the best characterized ribotoxin. Previous characterization of a deletion variant of this protein showed that its long NH 2 -terminal β-hairpin is essential for its cytotoxicity. Docking, enzymatic, and lipidprotein interaction studies suggested that this β-hairpin establishes specific interactions with ribosomal proteins and that it is a region involved in the interaction with cell membranes. Consequently, in order to assess the influence of the basic character of this NH 2 -terminal β-hairpin (there are 1 arginine and 4 lysines along its 16 residues) on the ribotoxins cytotoxic ability, five individual mutants substituting these 5 basic residues by glutamic acid were produced, purified to homogeneity, and characterized. Regarding ribosomal recognition, all mutants showed a diminished activity in a cell-free reticulocyte lysate, whereas the activity against an oligoribonucleotide mimicking the sarcin/ricin loop rRNA (SRL) or the homopolymer poly(A) remained unaffected, confirming that the mutated basic residues participate in electrostatic interactions with other ribosomal elements apart from this SRL. The study of the interaction with phospholipid vesicles showed that Lys 17, Arg 22, and, most importantly, Lys 14 and Lys 21, are crucial residues in the first stages of the aggregation phenomenon, where protein-vesicle and protein-protein interactions are required. The data obtained reveal that electrostatic interactions involving basic residues of the β-hairpin are required not only for establishing specific interactions with ribosomal regions other than the SRL but also to explain the ability of the protein to interact with acid phospholipid bilayers.

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Tyr-48, a conserved residue in ribotoxins, is involved in the RNA-degrading activity of α-sarcin

Cited by 18 publications

References 31 publications

The Therapeutic Potential of Fungal Ribotoxins

The Therapeutic Potential of Fungal Ribotoxins

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

Role of the basic character of α-sarcin’s NH2-terminal β-hairpin in ribosome recognition and phospholipid interaction

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