The solubility of the ribotoxin alpha-sarcin, produced as a recombinant protein in Escherichia coli, is increased in the presence of thioredoxin

Garcı́a-Ortega, Lucı́a; Lacadena, Javier; Lacadena, Valle; Masip, Manuel; Antonio, C de; Martı́nez-Ruiz, Antonio; Pozo, Álvaro Martı́nez del

doi:10.1046/j.1472-765x.2000.00714.x

Cited by 18 publications

(18 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Protein Production and Purification-BL21(DE3) cotransformed with pT-Trx and the corresponding ␣-sarcin mutant plasmid were used to produce and purify the mutant as described for the wild-type protein (34,38). Fungal wild-type ␣-sarcin was produced and purified according to methods previously reported (10,34).…”

Section: Methodsmentioning

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

105

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

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

105

View full text Add to dashboard Cite

show abstract

“…E. coli BL21 (DE3) cells, previously cotransformed with a thioredoxin-producing plasmid (pT-Trx) and the corresponding plasmid (pINPGαSH137Q), were used to produce the catalytically inactive -sarcin H137Q mutant, also as previously described (García-Ortega et al, 2000;Lacadena et al, 1994Lacadena et al, , 1995Lacadena et al, , 1999. This mutant retains the structural features of the wild-type protein, as well as its ability to interact with membranes, but lacks the characteristic ribonucleolytic activity of ribotoxins (Lacadena et al, 1995(Lacadena et al, , 1999.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

“…This mutant retains the structural features of the wild-type protein, as well as its ability to interact with membranes, but lacks the characteristic ribonucleolytic activity of ribotoxins (Lacadena et al, 1995(Lacadena et al, , 1999. SDS-PAGE of proteins, Western blots, protein hydrolysis, amino acid analysis, and spectroscopic characterization were performed according to standardized procedures described before (García-Ortega et al, 2000, 2002Lacadena et al, 1994;Martínez-Ruiz et al, 2001). According to all these criteria, the three proteins used in this study were purified to homogeneity and retained their structural and functional properties.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

Fungal extracellular ribotoxins as insecticidal agents

Olombrada¹,

Herrero-Galán²,

Tello³

et al. 2013

Insect Biochemistry and Molecular Biology

View full text Add to dashboard Cite

Fungal ribotoxins were discovered almost 50 years ago as extracellular ribonucleases (RNases) with antitumoral properties. However, the biological function of these toxic proteins has remained elusive. The discovery of the ribotoxin HtA, produced by the invertebrates pathogen H. thompsonii, revived the old proposal that insecticidal activity would be their long searched function. Unfortunately, HtA is rather singular among all ribotoxins known in terms of sequence and structure similarities. Thus, it was intriguing to answer the question of whether HtA is just an exception or, on the contrary, the paradigmatic example of the ribotoxins function. The work presented uses HtA and -sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells.

show abstract

“…Protein production and purification E. coli BL21 (DE3) cells cotransformed with a thioredoxin-producing plasmid (pT-Trx) and the corresponding α-sarcin mutant plasmids were used to produce the different proteins studied, as previously described [38,40,41].…”

Section: Dna Manipulationsmentioning

confidence: 99%

“…Protein purification included ion exchange and molecular exclusion chromatographies [28]. PAGE of proteins, Western blot immunodetection, protein hydrolysis, and amino acid analysis were performed according to standard procedures [38,41].…”

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

View full text Add to dashboard Cite

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.

show abstract

The solubility of the ribotoxin alpha-sarcin, produced as a recombinant protein in Escherichia coli, is increased in the presence of thioredoxin

Cited by 18 publications

References 23 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

Fungal extracellular ribotoxins as insecticidal agents

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

Contact Info

Product

Resources

About