Publications (24)58.2 Total impact
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Article: Production and characterization of scFvA33T1, an immunoRNase targeting colon cancer cells.
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ABSTRACT: Within the last 10 years, the use of different RNases as therapeutic agents for various diseases has been pursued. Furthermore, the advancements of recombinant technology have allowed the assembly of proteins with different functions. In this regard, immunoribonucleases (immunoRNases) stand out as some of the most promising therapeutic candidates given their enzymatic and non-mutagenic character. Accordingly, the work reported here describes fusing RNase T1, one of the most studied members of the microbial RNase family, to the single-chain variable fragment (scFv) of a monoclonal antibody that targets the glycoprotein A33 antigen (GPA33) from human colon cancer cells. A heterologous production system, which employs the yeast Pichia pastoris, has been optimized to produce this immunoRNase (scFvA33T1) with yields of ∼ 5-10 mg · L(-1). The purified protein appears to be correctly folded as it retains its antigen specificity and ribonucleolytic activity. Finally, it also shows specific binding to, internalization into and toxicity against GPA33-positive cell lines compared with the control, GPA33-negative cells. Overall, it can be concluded that scFvA33T1 is a promising therapeutic fusion protein with the additional advantage that presumably it can be produced and purified in large amounts using an easily scalable yeast-based system.FEBS Journal 07/2012; 279(17):3022-32. · 3.79 Impact Factor -
Article: Production and characterization of a colon cancer-specific immunotoxin based on the fungal ribotoxin α-sarcin.
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ABSTRACT: A single-chain fusion protein that directed the cytolytic activity of α-sarcin to A33 tumor antigen expressing cells was constructed and shown to effectively kill targeted cells. Glycoprotein A33 (GPA33) is a well-known colon cancer marker and a humanized antibody against it was used to target the α-sarcin. The fungal ribotoxin α-sarcin is one of the most potent and specific toxins known. It is small, protease resistant, thermostable and highly efficient towards the inactivation of ribosomes. This work describes the production and characterization of an immunotoxin resulting from fusing the single-chain variable fragment (scFv) of the monoclonal antibody that targets GPA33 to fungal α-sarcin. This chimeric protein (scFvA33αsarcin), produced in Pichia pastoris and purified in high yield was proven to be properly folded, active, specific and stable. It showed high specific toxicity against GPA33-positive tumoral cell lines providing scientific evidence to sustain that scFvA33αsarcin is a good immunotherapeutic candidate against GPA33-positive colon carcinomas.Protein Engineering Design and Selection 06/2012; 25(8):425-35. · 2.94 Impact Factor -
Article: A non-cytotoxic but ribonucleolytically specific ribotoxin variant: implication of tryptophan residues in the cytotoxicity of hirsutellin A.
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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.Biological Chemistry 05/2012; 393(6):449-56. · 2.96 Impact Factor -
Article: Implication of an Asp residue in the ribonucleolytic activity of hirsutellin A reveals new electrostatic interactions at the active site of ribotoxins.
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ABSTRACT: Ribotoxins are fungal extracellular ribonucleases that specifically cleave ribosomes leading to cell-death via apoptosis. α-Sarcin is the ribotoxin studied in deepest detail, and therefore constitutes the referential protein for the whole family. It has been demonstrated that ribotoxin activity depends on a very precise structural microenvironment in which electrostatic interactions among residues in the active site are of the highest importance. Hirsutellin A (HtA) has been recently described as the smallest ribotoxin known to date, encompassing all the abilities of previously characterized members of this family into a shorter sequence. Comparison of HtA and α-sarcin three-dimensional structures suggested that residues presumably forming the catalytic triad of HtA would be His 42, Glu 66, and His 113. Within this same idea, the presence of an Asp residue (Asp 40) in a position equivalent to α-sarcin Tyr 48 is highlighted as a novelty in this field. In this work, substitution mutants H42Q, E66Q and H113Q, as well as double and triple mutants in all possible combinations, are studied regarding their ribonucleolytic activity and cytotoxicity. Implication of these three residues in the ribotoxin activity of HtA is confirmed, though none of them is strictly essential for ribosomal cleavage. Studies with mutants D40N and D40N/E66Q demonstrate an important role for Asp 40 in the activity of HtA and establish a new set of electrostatic interactions different from the one described for already known ribotoxins.Biochimie 08/2011; 94(2):427-33. · 3.02 Impact Factor -
Article: Involvement of the amino‐terminal β‐hairpin of the Aspergillus ribotoxins on the interaction with membrances and nonspecific ribonuclease activity
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ABSTRACT: Ribotoxins are a family of potent cytotoxic proteins from Aspergillus whose members display a high sequence identity (85% for about 150 amino acid residues). The three-dimensional structures of two of these proteins, α-sarcin and restrictocin, are known. They interact with phospholipid bilayers, according to their ability to enter cells, and cleave a specific phosphodiester bond in the large subunit of ribosome thus inhibiting protein biosynthesis. Two nonconservative sequence changes between these proteins are located at the amino-terminal β-hairpin of α-sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of α-sarcin, Lys 11 and Thr 20, have been substituted with the equivalent amino acids in restrictocin. The single mutants (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopic characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid-perturbing activities of the three mutants and restrictocin have been evaluated and compared with those of α-sarcin. These proteins exhibit the same ability to specifically inactivate ribosomes, although they show different activity against nonspecific substrate analogs such as poly(A). The mutant variant K11L and restrictocin display a lower phospholipid-interacting ability correlated with a decreased cytotoxicity. The results obtained are interpreted in terms of the involvement of the amino-terminal β-hairpin in the interaction with both membranes and polyadenylic acid.Protein Science 12/2008; 10(8):1658 - 1668. · 2.80 Impact Factor -
Article: The insecticidal protein hirsutellin A from the mite fungal pathogen Hirsutella thompsonii is a ribotoxin.
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ABSTRACT: The mite fungal pathogen Hirsutella thompsonii produces a single polypeptide chain, insecticidal protein named hirsutellin A (HtA) that is composed of 130 amino acid residues. This protein has been purified from its natural source and produced as a recombinant protein in Escherichia coli. Spectroscopic analysis has determined that the two protein forms are indistinguishable. HtA specifically inactivates ribosomes and produces the alpha-fragment characteristic of ribotoxin activity on rRNA. Behaving as a cyclizing ribonuclease, HtA specifically cleaves oligonucleotides that mimick the sarcin/ricin loop of the ribosome, as well as selected polynucleotides and dinucleosides. HtA interacts with phospholipid membranes as do other ribotoxins. As a consequence of its ribonuclease activity and its ability to interact with cell membranes, HtA exhibits cytotoxic activity on human tumor cells. On the basis of these results, HtA is considered to be a member of the ribotoxin group of proteins, although it is significantly smaller (130 aa) than all known ribotoxins that are composed of 149/150 amino acids. Ribotoxins are members of a larger family of fungal ribonucleases whose members of smaller size (100/110 aa) are not cytotoxic. Thus, the characterization of the fungal ribotoxin HtA represents an important milestone in the study of the diversity and the function of fungal ribonucleases.Proteins Structure Function and Bioinformatics 08/2008; 72(1):217-28. · 3.39 Impact Factor -
Article: The therapeutic potential of fungal ribotoxins.
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ABSTRACT: 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 high-resolution 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 mid-term future.Current pharmaceutical biotechnology 07/2008; 9(3):153-60. · 3.40 Impact Factor -
Article: Fungal ribotoxins: molecular dissection of a family of natural killers.
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ABSTRACT: RNase T1 is the best known representative of a large family of ribonucleolytic proteins secreted by fungi, mostly Aspergillus and Penicillium species. Ribotoxins stand out among them by their cytotoxic character. They exert their toxic action by first entering the cells and then cleaving a single phosphodiester bond located within a universally conserved sequence of the large rRNA gene, known as the sarcin-ricin loop. This cleavage leads to inhibition of protein biosynthesis, followed by cellular death by apoptosis. Although no protein receptor has been found for ribotoxins, they preferentially kill cells showing altered membrane permeability, such as those that are infected with virus or transformed. Many steps of the cytotoxic process have been elucidated at the molecular level by means of a variety of methodological approaches and the construction and purification of different mutant versions of these ribotoxins. Ribotoxins have been used for the construction of immunotoxins, because of their cytotoxicity. Besides this activity, Aspf1, a ribotoxin produced by Aspergillus fumigatus, has been shown to be one of the major allergens involved in allergic aspergillosis-related pathologies. Protein engineering and peptide synthesis have been used in order to understand the basis of these pathogenic mechanisms as well as to produce hypoallergenic proteins with potential diagnostic and immunotherapeutic applications.FEMS Microbiology Reviews 04/2007; 31(2):212-37. · 10.96 Impact Factor -
Article: Anomalous electrophoretic behavior of a very acidic protein: ribonuclease U2.
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ABSTRACT: Ribonuclease U2 is a low-molecular-weight acidic protein with three disulfide bridges. This protein displays an anomalous electrophoretic behavior on standard SDS-PAGE. The electrophoretic mobility of the nonreduced protein roughly corresponds to its molecular mass while the migration of the reduced protein would be in accordance with the expected molecular mass of the protein dimer. This study reveals that the protein does not bind SDS under the SDS-PAGE conditions, its electrophoretic mobility being only determined by its electrostatic charge and hydrodynamic properties. In addition, the nonreduced protein cannot be blotted to a membrane. Unfolding of the protein upon reduction of its disulfide bridges enables electrotransference to membranes due to a restricted diffusion along the electrophoresis gel.Electrophoresis 10/2005; 26(18):3407-13. · 3.30 Impact Factor -
Article: Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding.
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ABSTRACT: Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.FEBS Journal 06/2005; 272(10):2536-44. · 3.79 Impact Factor -
Article: Dissecting structural and electrostatic interactions of charged groups in alpha-sarcin. An NMR study of some mutants involving the catalytic residues.
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ABSTRACT: The cytotoxic ribonuclease alpha-sarcin is the best characterized member of the ribotoxin family. Ribotoxins share a common structural core, catalytic residues, and active site topology with members of the broader family of nontoxic microbial extracellular RNases. They are, however, much more specific in their biological action. To shed light on the highly specific alpha-sarcin activity, we have evaluated the structural and electrostatic interactions of its charged groups, by combining the structural and pK(a) characterization by NMR of several variants with theoretical calculations based on the Tanford-Kirkwood and Poisson-Boltzmann models. The NMR data reveal that the global conformation of wild-type alpha-sarcin is preserved in the H50Q, E96Q, H137Q, and H50/137Q variants, and that His137 is involved in an H-bond that is crucial in maintaining the active site structure and in reinforcing the stability of the enzyme. The loss of this H-bond in the H137Q and H50/137Q variants modifies the local structure of the active site. The pK(a) values of active site groups H50, E96, and H137 in the four variants have been determined by two-dimensional NMR. The catalytic dyad of E96 and H137 is not sensitive to charge replacements, since their pK(a) values vary less than +/-0.3 pH unit with respect to those of the wild type. On the contrary, the pK(a) of His50 undergoes drastic changes when compared to its value in the intact protein. These amount to an increase of 0.5 pH unit or a decrease of 1.1 pH units depending on whether a positive or negative charge is substituted at the active site. The main determinants of the pK(a) values of most of the charged groups in alpha-sarcin have been established by considering the NMR results in conjunction with those derived from theoretical pK(a) calculations. With regard to the active site residues, the H50 pK(a) is chiefly influenced by electrostatic interactions with E96 and H137, whereas the effect of the low dielectric constant and the interaction with R121 appear to be the main determinants of the altered pK(a) value of E96 and H137. Charge-charge interactions and an increased level of burial perturb the pK(a) values of the active site residues of alpha-sarcin, which can account for its reduced ribonucleolytic activity and its high specificity.Biochemistry 12/2003; 42(45):13122-33. · 3.42 Impact Factor -
Article: Dissecting Structural and Electrostatic Interactions of Charged Groups in α-Sarcin. An NMR Study of Some Mutants Involving the Catalytic Residues†
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ABSTRACT: The cytotoxic ribonuclease α-sarcin is the best characterized member of the ribotoxin family. Ribotoxins share a common structural core, catalytic residues, and active site topology with members of the broader family of nontoxic microbial extracellular RNases. They are, however, much more specific in their biological action. To shed light on the highly specific α-sarcin activity, we have evaluated the structural and electrostatic interactions of its charged groups, by combining the structural and pKa characterization by NMR of several variants with theoretical calculations based on the Tanford−Kirkwood and Poisson−Boltzmann models. The NMR data reveal that the global conformation of wild-type α-sarcin is preserved in the H50Q, E96Q, H137Q, and H50/137Q variants, and that His137 is involved in an H-bond that is crucial in maintaining the active site structure and in reinforcing the stability of the enzyme. The loss of this H-bond in the H137Q and H50/137Q variants modifies the local structure of the active site. The pKa values of active site groups H50, E96, and H137 in the four variants have been determined by two-dimensional NMR. The catalytic dyad of E96 and H137 is not sensitive to charge replacements, since their pKa values vary less than ±0.3 pH unit with respect to those of the wild type. On the contrary, the pKa of His50 undergoes drastic changes when compared to its value in the intact protein. These amount to an increase of 0.5 pH unit or a decrease of 1.1 pH units depending on whether a positive or negative charge is substituted at the active site. The main determinants of the pKa values of most of the charged groups in α-sarcin have been established by considering the NMR results in conjunction with those derived from theoretical pKa calculations. With regard to the active site residues, the H50 pKa is chiefly influenced by electrostatic interactions with E96 and H137, whereas the effect of the low dielectric constant and the interaction with R121 appear to be the main determinants of the altered pKa value of E96 and H137. Charge−charge interactions and an increased level of burial perturb the pKa values of the active site residues of α-sarcin, which can account for its reduced ribonucleolytic activity and its high specificity.10/2003; -
Article: RNase U2 and α-sarcin: A study of relationships.
Methods in enzymology 01/2001; 341C:335-351. · 1.90 Impact Factor -
Article: Assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin α‐sarcin
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ABSTRACT: α-Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called α-fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp-51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp-51 by Phe. Substitution of each Trp residue results in a 4 °C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9°C lower. Trp-51 is responsible for most of the near-UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescence emission. The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein–vesicle interaction. Proteins 2000;41:350–361. © 2000 Wiley-Liss, Inc.Proteins Structure Function and Bioinformatics 09/2000; 41(3):350 - 361. · 3.39 Impact Factor -
Article: Role of histidine‐50, glutamic acid‐96, and histidine‐137 in the ribonucleolytic mechanism of the ribotoxin α‐sarcin
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ABSTRACT: α-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 dif- ferent 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 α-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 α-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 α-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of α-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by α-sarcin. Proteins 1999;37:474–484. ©1999 Wiley-Liss, Inc.Proteins Structure Function and Bioinformatics 12/1999; 37(3):474 - 484. · 3.39 Impact Factor -
Article: Characterization of pKa Values and Titration Shifts in the Cytotoxic Ribonuclease α-Sarcin by NMR. Relationship between Electrostatic Interactions, Structure, and Catalytic Function†
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ABSTRACT: The electrostatic behavior of titrating groups in α-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0−8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson−Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the α-sarcin−2‘-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, 4.8, and 6.8 in the α-sarcin−2‘-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe−His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in α-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.10/1998; -
Article: 1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease α‐Sarcin
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ABSTRACT: The ribosome-inactivating protein -Sarcin (S) is a 150-residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure-dynamics-function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of S on the basis of homonuclear (1H-1H) and heteronuclear (1H-15N) two-dimensional correlation spectra of a uniformly 15N-labeled sample, and two selectively 15N-labeled (Tyr and Phe) samples, as well as a single three-dimensional experiment. The secondary structure of S, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N-terminal β-hairpin, a short -helical segment, and a C-terminal β-sheet of five short strands arranged in a+1,+1,+ 1, + 1 topology, connected by long loops in which the 13 Pro residues are located.Protein Science 04/1996; 5(5):969 - 972. · 2.80 Impact Factor -
Article: Fungal ribotoxins: molecular dissection of a familyof natural killers
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ABSTRACT: RNase T1 is the best known representative of a large family of ribonucleolytic proteins secreted by fungi, mostly Aspergillus and Penicillium species. Ribotoxins stand out among them by their cytotoxic character. They exert their toxic action by first entering the cells and then cleaving a single phosphodiester bond located within a universally conserved sequence of the large rRNA gene, known as the sarcin–ricin loop. This cleavage leads to inhibition of protein biosynthesis, followed by cellular death by apoptosis. Although no protein receptor has been found for ribotoxins, they preferentially kill cells showing altered membrane permeability, such as those that are infected with virus or transformed. Many steps of the cytotoxic process have been elucidated at the molecular level by means of a variety of methodological approaches and the construction and purification of different mutant versions of these ribotoxins. Ribotoxins have been used for the construction of immunotoxins, because of their cytotoxicity. Besides this activity, Aspf1, a ribotoxin produced by Aspergillus fumigatus, has been shown to be one of the major allergens involved in allergic aspergillosis-related pathologies. Protein engineering and peptide synthesis have been used in order to understand the basis of these pathogenic mechanisms as well as to produce hypoallergenic proteins with potential diagnostic and immunotherapeutic applications. -
Article: Structural basis for the catalytic mechanism and substrate specificity of the ribonuclease α-sarcin
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ABSTRACT: α-Sarcin is a ribosome-inactivating protein which selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA. The solution structure of α-sarcin has been determined on the basis of 1898 distance and angular experimental constraints from NMR spectroscopy. It reveals a catalytic mechanism analogous to that of the T1 family of ribonucleases while its exquisite specificity resides in the contacts provided by its distinctive loops.FEBS Letters. -
Article: Characterization of a natural larger form of the antifungal protein (AFP) from Aspergillus giganteus
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ABSTRACT: Two major proteins, α-sarcin and an antifungal polypeptide (AFP), are secreted by the mould Aspergillus giganteus MDH 18894 when it is cultured for 70–80 h. A third major protein is also found in the extracellular medium at 48–60 h, but it disappears as the culture proceeds. This protein has been isolated and characterized in terms of apparent molecular mass, electrophoretic and chromatographic behaviour, NH2-terminal primary structure, amino acid content, spectroscopical features, reactivity against anti-AFP antibodies, and antifungal activity. Based on the obtained results it would be an extracellular inactive precursor form of AFP, designated as the large form of AFP (lf-AFP). Its amino acid composition is identical to that of AFP but containing six extra residues. NH2-terminal sequence analysis of the first eight amino acid residues of this polypeptide revealed that the extra residues can be perfectly accommodated within the DNA-deduced sequence of the precursor form of AFP. Its alignment with precursor sequences of different proteins, secreted by a variety of Aspergillus spp., reveals the existence of a common tetrapeptide at the carboxy-terminal end of their leader peptides. This sequence would be Ile/Leu-Xaa-Yaa-Arg, being mostly Xaa and Yaa an acid residue (Asp/Glu) and alanine, respectively. The presence of lf-AFP as an extracellular protein would be in perfect agreement with the existence of this tetrapeptide motif, that can be involved in the protein secretion mechanisms of filamentous fungi.Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology.
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Institutions
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1996–2012
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Complutense University of Madrid
- • Facultad de Ciencias Químicas
- • Departamento de Bioquímica y Biología Molecular II
Madrid, Madrid, Spain
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2003
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Spanish National Research Council
- Institute of Physical Chemistry "Rocasolano"
Madrid, Madrid, Spain
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