The cytotoxin α‐sarcin behaves as a cyclizing ribonuclease

Lacadena, Javier; Pozo, Álvaro Martı́nez del; Lacadena, Valle; Martı́nez-Ruiz, Antonio; Mancheño, José M.; Oñaderra, Mercedes; Gavilanes, José G.

doi:10.1016/s0014-5793(98)00137-9

Cited by 41 publications

(56 citation statements)

References 20 publications

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“…␣-Sarcin is a cytotoxic protein belonging to the ribotoxins, a family of ribosome-inactivating proteins (2,6,7,13,17,24,30). AFP is a highly basic polypeptide of 51 amino acids with a high content of cysteine, tyrosine, and lysine residues.…”

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confidence: 99%

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The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization

Theis

Wedde

Meyer

et al. 2003

Antimicrob Agents Chemother

103

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We investigated the inhibitory effects of the antifungal protein (AFP) from Aspergillus giganteus on the growth of several filamentous fungi. For this purpose, the MICs of AFP were determined and ranged from 0.1 g/ml for Fusarium oxysporum to 200 g/ml for Aspergillus nidulans. The antifungal activity of AFP was diminished in the presence of cations. We were able to show that incubation of AFP-sensitive fungi with the protein resulted in membrane permeabilization using an assay based on the uptake of the fluorescent dye SYTOX Green. No permeabilization by AFP could be detected at concentrations below the species-specific MIC. Furthermore, AFP-induced permeabilization could readily be detected after 5 min of incubation. Localization experiments with fluorescein isothiocyanate-labeled AFP and immunofluorescence staining with an AFP-specific antibody supported the observation that the protein interacts with membranes. After treatment of AFP-sensitive fungi with AFP, the protein was localized at the plasma membrane, whereas it was mainly detected inside the cells of AFP-resistant fungi. We conclude from these data that the growth-inhibitory effect of AFP is caused by permeabilization of the fungal membranes.The imperfect filamentous fungus Aspergillus giganteus was found to secrete two small basic proteins, namely, the antifungal protein (AFP) and ␣-sarcin (22). ␣-Sarcin is a cytotoxic protein belonging to the ribotoxins, a family of ribosome-inactivating proteins (2,6,7,13,17,24,30). AFP is a highly basic polypeptide of 51 amino acids with a high content of cysteine, tyrosine, and lysine residues. The isoelectric point was estimated to be 8.8; thus, the protein is positively charged under neutral conditions. AFP is folded into five highly twisted antiparallel strands, defining a small and compact structure with four stabilizing disulfide bridges (3,20,29).Only three small, basic antifungal proteins have been isolated from filamentous fungi to date: AFP from A. giganteus (22), PAF from Penicillium chrysogenum (18), and Anafp from Aspergillus niger (14). Recently, the naf gene was isolated from Penicillium nalgiovense and is identical to the paf gene from P. chrysogenum and encodes a protein with transient antifungal activity (8). All these antifungal proteins are secreted and inhibit the growth of numerous filamentous fungi without affecting bacteria and yeast. Most notably, they are significantly similar in regard to their structures, sizes, and basic characters. The PAF protein exhibits 42.6% sequence similarity with the AFP sequence (18).Not much is known about the modes of action of the antifungal proteins secreted by fungi. It has, however, been shown that defensins, a class of antimicrobial proteins which are quite similar to antifungal proteins in terms of their structures, sizes, and disulfide bridges, interact with anionic phospholipids of bacterial membranes (15). In addition, it has been found that some plant defensins have a receptor-mediated interaction with the fungal plasma membrane (27). However, noth...

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“…No effect on vesicles composed of zwitterionic phospholipids has been detected. It was assumed that the antifungal activity of AFP is related to its ability to interact with anionic phospholipids (13).…”

mentioning

confidence: 99%

The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization

Theis

Wedde

Meyer

et al. 2003

Antimicrob Agents Chemother

103

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“…However, they can also produce extensive non-specific digestion of almost any RNA when used at higher concentrations [9,41]. Ribotoxins follow the same general acid--base type endonucleolytic reaction scheme as the other members of the RNase T1 family [2,3,42,43]. of the RNase T1 family so far studied is implicit in the denomination of all these enzymes as cyclizing RNases [43].…”

Section: Ribonucleolytic Activitymentioning

confidence: 99%

“…Ribotoxins follow the same general acid--base type endonucleolytic reaction scheme as the other members of the RNase T1 family [2,3,42,43]. of the RNase T1 family so far studied is implicit in the denomination of all these enzymes as cyclizing RNases [43]. However, the catalytic efficiency of RNases T1 and U2 against naked RNA, homopolynucleotides, or dinucleotides is several orders of magnitude higher.…”

Section: Ribonucleolytic Activitymentioning

confidence: 99%

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The Therapeutic Potential of Fungal Ribotoxins

Carreras-Sangrà

Álvarez-García²,

Herrero-Galán³

et al. 2008

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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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Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin ?-sarcin

et al. 1999

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alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.

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The cytotoxin α‐sarcin behaves as a cyclizing ribonuclease

Cited by 41 publications

References 20 publications

The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization

The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization

The Therapeutic Potential of Fungal Ribotoxins

Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin ?-sarcin

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