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A non-cytotoxic but ribonucleolytically specific ribotoxin variant: implication of tryptophan residues in the cytotoxicity of hirsutellin A.
- Source :
-
Biological chemistry [Biol Chem] 2012 May; Vol. 393 (6), pp. 449-56. - Publication Year :
- 2012
-
Abstract
- Ribotoxins are a family of toxic proteins that exert a highly specific cleavage at the universally conserved sarcin/ricin loop (SRL) of the larger rRNA molecule. Before this ribonucleolytic action, passage through the cell membrane is a necessary step for ribotoxin internalization and the limiting factor for cytotoxicity. Although extensive knowledge of their ribonucleolytic activity and substrate recognition has been accumulated, little is known about the mechanisms of cell entry of ribotoxins. Hirsutellin A (HtA) is a recently described member of this family, which accommodates the main abilities of previously characterized ribotoxins into a shorter sequence, but exhibits some differences regarding membrane interaction properties. This work investigates the contribution of tryptophan (Trp) residues 71 and 78 to both endoribonucleolytic activity and cellular toxicity of this ribotoxin. Substitution mutants W71F and W78F, as well as the double mutant W71/78F, were obtained and assayed against isolated ribosomes, synthetic SRL, and human tumor cells. The results provide evidence that cell membrane passage and internalization, as well as substrate-specific recognition, require the participation of the region involving both Trp 71 and Trp 78. Additionally, the mutant W71/78F is the first non-cytotoxic but specific ribosome-cleaving ribotoxin mutant obtained to date.
- Subjects :
- Cell Line, Tumor
Cell Membrane metabolism
Conserved Sequence
Cytotoxins genetics
Cytotoxins metabolism
Endoribonucleases chemistry
Fungal Proteins genetics
Fungal Proteins metabolism
Humans
Models, Molecular
Mutation
Protein Conformation
Protein Synthesis Inhibitors chemistry
Protein Synthesis Inhibitors metabolism
Protein Synthesis Inhibitors toxicity
Protein Transport
Ribonucleases genetics
Ribonucleases toxicity
Ricin chemistry
Substrate Specificity
Tryptophan genetics
Cytotoxins chemistry
Cytotoxins toxicity
Fungal Proteins chemistry
Fungal Proteins toxicity
RNA, Ribosomal metabolism
Ribonucleases chemistry
Ribonucleases metabolism
Tryptophan metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1437-4315
- Volume :
- 393
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 22628308
- Full Text :
- https://doi.org/10.1515/hsz-2011-0278