Assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin ?-sarcin

Antonio, C de; Pozo, Álvaro Martı́nez del; Mancheño, José M.; Oñaderra, Mercedes; Lacadena, Javier; Martínez‐Ruiz, Antonio; Pérez‐Cañadillas, José Manuel; Bruix, Marta; Gavilanes, José G.

doi:10.1002/1097-0134(20001115)41:3<350::aid-prot70>3.0.co;2-v

Cited by 28 publications

(38 citation statements)

References 48 publications

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“…CD spectrum of ␣-sarcin is dominated by the contribution of Trp 51 (a positive CD maximum of about 100 degrees ϫ cm 2 ϫ dmol Ϫ1 at 293 nm), whereas the contribution of Trp 4 is about 15 degrees ϫ cm 2 ϫ dmol Ϫ1 of residue in the range 285-293 nm (51). The calculated difference spectrum (wild type minus mutant; Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The variation in the tyrosine contribution is readily explained by the removal of Tyr 18 in the deletion mutant. The fluorescence emission of ␣-sarcin is dominated by the contribution of Trp 4 , because the other tryptophan residue of the molecule, Trp 51 , is strongly quenched in the WT protein (51). Thus, the increased Trp quantum yield in the ⌬(7-22) mutant could be attributed to local changes in the Trp 4 microenvironment upon deletion, which would be in agreement with the variation observed in the near UV CD spectrum.…”

Section: Resultsmentioning

confidence: 99%

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Deletion of the NH2-terminal β-Hairpin of the Ribotoxin α-Sarcin Produces a Nontoxic but Active Ribonuclease

Garcı́a-Ortega¹,

Masip²,

Mancheño³

et al. 2002

Journal of Biological Chemistry

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105

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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. ␣-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 ␤-hairpin in ribotoxins and to the different length of their unstructured loops. The aminoterminal deletion mutant ⌬(7-22) of ␣-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 ␣-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. 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Deletion of the NH2-terminal β-Hairpin of the Ribotoxin α-Sarcin Produces a Nontoxic but Active Ribonuclease

Garcı́a-Ortega¹,

Masip²,

Mancheño³

et al. 2002

Journal of Biological Chemistry

Self Cite

105

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“…The mean residue mass employed was 132.2 Da, calculated from their respective amino acid sequences. Peptides were dissolved in 50 mM sodium phosphate (with or without 30% TFE) (pH 7.0) at a concentration of 80 M. Ellipticity at 222 nm was also recorded as a function of different pH values (from 3.0 to 10.5) and salt concentrations (NaCl, CaCl 2 , or MgCl 2 ) in the range of 0 -200 mM (45).…”

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confidence: 99%

Structure-Activity Relationship of α Mating Pheromone from the Fungal Pathogen Fusarium oxysporum

Vitale

Partida-Hanon

Serrano

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellDuring sexual development ascomycete fungi produce two types of peptide pheromones termed a and ␣. The ␣ pheromone from the budding yeast Saccharomyces cerevisiae, a 13-residue peptide that elicits cell cycle arrest and chemotropic growth, has served as paradigm for the interaction of small peptides with their cognate G protein-coupled receptors. However, no structural information is currently available for ␣ pheromones from filamentous ascomycetes, which are significantly shorter and share almost no sequence similarity with the S. cerevisiae homolog. High resolution structure of synthetic ␣-pheromone from the plant pathogenic ascomycete Fusarium oxysporum revealed the presence of a central ␤-turn resembling that of its yeast counterpart. Disruption of the-fold by D-alanine substitution of the conserved central Gly 6 -Gln 7 residues or by random sequence scrambling demonstrated a crucial role for this structural determinant in chemoattractant activity. Unexpectedly, the growth inhibitory effect of F. oxysporum ␣-pheromone was independent of the cognate G protein-coupled receptors Ste2 and of the central ␤-turn but instead required two conserved Trp 1 -Cys 2 residues at the N terminus. These results indicate that, despite their reduced size, fungal ␣-pheromones contain discrete functional regions with a defined secondary structure that regulate diverse biological processes such as polarity reorientation and cell division.

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“…With regard to the protein regions involved in the interaction with membranes, the use of water-soluble synthetic peptides and mutant variants of α-sarcin within the main β-sheet of this protein suggested that this region (residues 116-139) would be directly involved in this interaction [76][77][78].…”

Section: Structural Featuresmentioning

confidence: 99%

“…The study of the mentioned mutants suggested that it would be located within the hydrophobic core of the phospholipid bilayer once the protein-lipid complexes were formed [46,78]. Within this same idea, mutants affecting α-sarcin active site residue Arg121 (R121K and R121Q),…”

Section: Structural Featuresmentioning

confidence: 99%

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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Assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin ?-sarcin

Cited by 28 publications

References 48 publications

Deletion of the NH2-terminal β-Hairpin of the Ribotoxin α-Sarcin Produces a Nontoxic but Active Ribonuclease

Deletion of the NH2-terminal β-Hairpin of the Ribotoxin α-Sarcin Produces a Nontoxic but Active Ribonuclease

Structure-Activity Relationship of α Mating Pheromone from the Fungal Pathogen Fusarium oxysporum

The Therapeutic Potential of Fungal Ribotoxins

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