Abstract
Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. alpha-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal beta-hairpin in ribotoxins and to the different length of their unstructured loops. The amino-terminal deletion mutant Delta(7-22) of alpha-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that alpha-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the beta-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells.
Highlights
Fungal extracellular ribonucleases are a diverse group of proteins, with RNase T1 being its best known representative
The results indicate that ␣-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the -hairpin of the protein
The three-dimensional structures of ␣-sarcin [17] and restrictocin [18], another member of the ribotoxin family, have been solved. This information has revealed that ribotoxins share a common structural core with the family of fungal extracellular nontoxic RNases, represented by RNase T1 [1], composed of a central antiparallel -sheet packed against a small ␣-helix and a conserved active site located on the other side
Summary
DNA Manipulations—All of the materials and reagents were molecular biology grade. Cloning procedures and bacteria manipulations were carried out according to standard methods [33], as described previously [27, 34]. The activity of the purified proteins against poly(A) was assayed in 15% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS and 0.3 mg/ml of the homopolynucleotide This zymogram method [34, 35, 45] was based on one previously described [46]. This assay, which was performed at two different pH values (4.5 and 7.0), is useful to detect the presence of other RNA degrading activities in the protein samples Volumograms of these bands (based on integrating all of the pixel intensities composing the spot) were obtained with the photo documentation system UVI-Tec (Cambridge, UK) and the software facility UVIsoft UVI band Windows Application V97.04. The reported values correspond to the averages of triplicate experiments
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