Ramiro Téllez-Sanz

Universidad de Almería, Unci, Andalusia, Spain

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Publications (13)46.87 Total impact

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    ABSTRACT: The glutathione S-transferase from Plasmodium falciparum presents distinct features which are absent from mammalian GST isoenzyme counterparts. Most apparent among these are the ability to tetramerize and the presence of a flexible loop. The loop, situated between the 113-119 residues, has been reported as necessary for the tetramerization process. In this article, we report that a residue outside of this loop, Asn112, is key to the process - to the point where the single Asn112Leu mutation prevents tetramerization altogether. We propose that a structural pattern involving the interaction of the Asn112 and Lys117 residues from two neighboring subunits plays a role in keeping the tetramer structure stable. We also report that, for the tetramerization of the wild-type PfGST to occur, phosphate or pyrophosphate anions must be present. In other words, tetramerization is a phosphate- or pyrophosphate-induced process. Furthermore, the presence of magnesium reinforces this induction. We present experimental evidence for these claims as well as a preliminary calorimetric and kinetic study of the dimeric Asn112Leu PfGST mutant. We also propose a putative binding site for phosphate or pyrophosphate anions through a comparative structural analysis of PfGST and pyrophosphatases from several organisms. Our results highlight the differences between PfGST and the human isoenzymes, which make the parasite enzyme a suitable antimalarial target.
    Biochimica et Biophysica Acta 05/2014; · 4.66 Impact Factor
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    ABSTRACT: Figure optionsDownload full-size imageDownload high-quality image (485 K)Download as PowerPoint slide
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 01/2014; · 3.73 Impact Factor
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    ABSTRACT: Over the last decade an increasing number of studies have been published reporting on the inhibitory potency or selectivity that several types of ligands show against human galectin-3 (hGal-3). The reason for this interest lies in the many important roles galectins play both in intra and extra-cellular functions. Among galectins, galectin-3 stands out because it is the only known member of its subfamily in mammals, is small and monomeric but capable of aggregating, and is known to be involved in a large number of disease processes, from cancer to heart failure. These characteristics and roles make hGal-3 an ideal target for drugs. Since it binds β-galactosides, like the rest of the galectin family of proteins, the search and design of potent and at the same time selective inhibitors for it is not an easy task. Herein we discuss the chemical features of the most potent inhibitors described so far, as well as the structural basis of their exhibited selectivity, in order to shed light on the rational design of drugs against this target.
    Current Medicinal Chemistry 06/2013; · 3.72 Impact Factor
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    ABSTRACT: Human glutathione S-transferase P1-1 (hGST P1-1) is involved in cell detoxification processes through the conjugation of its natural substrate, reduced glutathione (GSH), with xenobiotics. GSTs are known to be overexpressed in tumors, and naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are effective cancer chemopreventive compounds. To identify and characterize the potential inhibitory mechanisms of GST P1-1 induced by isothiocyanate conjugates, we studied the binding of GST P1-1 and some cysteine mutants to the BITC-SG conjugate as well as to the synthetic S-(N-benzylcarbamoylmethyl)glutathione conjugate (BC-SG). We report here the inactivation of GST P1-1 through the covalent modification of two Cys47 residues per dimer and one Cys101. The evidence has been compiled by isothermal titration calorimetry (ITC) and electrospray ionization mass spectrometry (ESI-MS). ITC experiments suggest that the BITC-SG conjugate generates adducts with Cys47 and Cys101 at physiological temperatures through a corresponding kinetic process, in which the BITC moiety is covalently bound to these enzyme cysteines through an S-thiocarbamoylation reaction. ESI-MS analysis of the BITC-SG incubated enzymes indicates that although the Cys47 in each subunit is covalently attached to the BITC ligand moiety, only one of the Cys101 residues in the dimer is so attached. A plausible mechanism is given for the emergence of inactivation through the kinetic processes with both cysteines. Likewise, our molecular docking simulations suggest that steric hindrance is the reason why only one Cys101 per dimer is covalently modified by BITC-SG. No covalent inactivation of GST P1-1 with the BC-SG inhibitor has been observed. The affinities and inhibitory potencies for both conjugates are high and very similar, but slightly lower for BC-SG. Thus, we conclude that the presence of the sulfur atom from the isothiocyanate moiety in BITC-SG is crucial for its irreversible inhibition of GST P1-1.
    ChemBioChem 06/2012; 13(11):1594-604. · 3.74 Impact Factor
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    ABSTRACT: The binding properties of two electroactive glutathione-ferrocene conjugates that consist in glutathione attached to one or both of the cyclopentadienyl rings of ferrocene (GSFc and GSFcSG), to Schistosoma japonica glutathione S-transferase (SjGST) were studied by spectroscopy fluorescence, isothermal titration calorimetry (ITC) and differential pulse voltammetry (DPV). Such ferrocene conjugates resulted to be competitive inhibitors of glutathione S-transferase with an increased binding affinity relative to the natural substrate glutathione (GSH). We found that the conjugate having two glutathione units (GSFcSG) exhibits an affinity for SjGST approximately two orders of magnitude higher than GSH. Furthermore, it shows negative cooperativity with the affinity for the second binding site two orders of magnitude lower than that for the first one. We propose that the reason for such negative cooperativity is steric since, i) the obtained thermodynamic parameters do not indicate profound conformational changes upon GSFcSG binding and ii) docking studies have shown that, when bound, part of the first bound ligand invades the second site due to its large size. In addition, voltammetric measurements show a strong decrease of the peak current upon binding of ferrocene-glutathione conjugates to SjGST and provide very similar K values than those obtained by ITC. Moreover, the sensing ability, expressed by the sensitivity parameter shows that GSFcSG is much more sensitive than GSFc, for the detection of SjGST.
    Biochimie 09/2011; 94(2):541-50. · 3.14 Impact Factor
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    ABSTRACT: dUTPase (deoxyuridine 5'-triphosphate nucleotide hydrolase) is an enzyme responsible for maintaining low levels of intracellular dUTP and thus prevents uracil incorporation into DNA by DNA polymerases during replication and repair processes. The thermodynamics of binding for both dUTP and dUMP (deoxyuridine 5'-monophosphate) to the D80A mutant form of Trypanosoma cruzi dUTPase have been investigated by fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry. In the presence of magnesium, approximately a 30-fold decrease in the value of the k(cat) and a 15-fold increase in the K(m) for dUTP hydrolysis was calculated while a 5-fold decrease was observed in the affinity for dUMP. In the absence of magnesium, the affinity for dUTP binding was similar for both enzymes while that for dUMP was lowered 3-fold as a consequence of the mutation. Calorimetric titrations in several buffers with different ionization heats rendered similar proton exchanges during the binding of dUMP. Thus, apparently the side chain of Asp 80 does not seem to vary its protonation state during the binding process. The enthalpy change values for the D80A mutant hardly change with temperature and, in addition, were Mg(2+) independent. We conclude that the D80A mutation induces only a slight conformational change in the active site yet results in a significant alteration of nucleotide binding and modifies the ability of the enzyme to discriminate between dUTP and dUMP when magnesium is present.
    Biochimie 09/2007; 89(8):972-80. · 3.14 Impact Factor
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    ABSTRACT: Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme preventing a deleterious incorporation of uracil into DNA, has been thought of as a novel target for anticancer and antiviral drug design. The interaction of Plasmodium falciparum dUTPase (PfdUTPase) with deoxyuridine derivatives (dU, dUMP, dUDP and dUpNHpp) has been studied thermodynamically by both isothermal titration and differential scanning calorimetry. ITC shows no cooperativity for the binding of these derivatives. Dependencies in the binding thermodynamic parameters (enthalpy, entropy and Gibbs energy changes) with the number of phosphate groups in the nucleotide are obtained, and from the heat capacity changes no significant conformational changes upon binding are inferred. DSC shows PfdUTPase trimer is very stable but denatures irreversibly, with a more complex denaturation profile than other homologous trimeric dUTPases. The presence of magnesium ions does not influence the denaturation profile, while the presence of deoxyuridine derivatives increases the stability. The increase depends upon nucleotide concentration and type, with dUDP having the greater effect.
    Biochimica et Biophysica Acta 08/2007; 1774(7):936-45. · 4.66 Impact Factor
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    ABSTRACT: The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 A resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix alpha2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix alpha2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.
    Protein Science 06/2006; 15(5):1093-105. · 2.74 Impact Factor
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    ABSTRACT: We have investigated the binding of 2'-deoxyuridine 5'-monophosphate (2'-dUMP) to Leishmania major deoxyuridine 5'-triphosphate nucleotide hydrolase (dUTPase) by isothermal titration microcalorimetry under different experimental conditions. Binding to dimeric L. major dUTPase is a non-cooperative process, with a stoichiometry of 1 molecule of 2'-dUMP per subunit. The utilization of buffers with different ionization enthalpies has allowed us to conclude that the formation of the 2'-dUMP-dUTPase complex, at pH 7.5 and 30 degrees C, is accompanied by the uptake of 0.33 +/- 0.05 protons per dUTPase subunit from the buffer media. Moreover, 2'-dUMP shows a moderate affinity for the enzyme, and binding is enthalpically driven across the temperature range studied. Besides, whereas DeltaG degrees remains practically invariant as a function of temperature, both DeltaH and DeltaS degrees decrease with increasing temperature. The TS and TH were 23.4 and 13.6 degrees C, respectively. The temperature dependence of the enthalpy change yields a heat capacity change of DeltaCp degrees = -618.1 +/- 126.4 cal x mol(-1) x K(-1), a value low enough to discard major conformational changes, in agreement with the fitting model. An interpretation of this value in terms of solvent-accessible surface areas is provided.
    Biochimica et Biophysica Acta 11/2004; 1702(1):33-40. · 4.66 Impact Factor
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    ABSTRACT: There has been some speculation about the salt independence of Schistosoma japonicum glutathione S-transferase (Sj26GST, EC. 2.5.1.18), but this aspect has not been carefully studied before. To establish the basis for a further development of this dependence, we have performed a methodical study of the influence of some important ions and their concentration on the binding properties of glutathione to Sj26GST by means of isothermal calorimetry and fluorescence quenching. Salts like NaCl, Na(2)SO(4) and MgSO(4) do not change practically the affinity of the protein for its substrate, whilst MgCl(2) has the effect of decreasing the affinity as its concentration rises. However, the enthalpy change is not affected by all the salts studied, and so, the entropy change is the causal factor in dropping the affinity. We also looked at the conformational stability of the protein under different conditions to check the structural changes they provide, and found that the unfolding parameters are practically not affected by the salt concentration. We discuss the results in terms of the chaotropic nature of the ions implied.
    International Journal of Biological Macromolecules 02/2003; 31(4-5):155-62. · 2.60 Impact Factor
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    R Téllez-Sanz, L García-Fuentes, C Barón
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    ABSTRACT: Isothermal titration microcalorimetry has been used to measure changes in enthalpy and heat capacity for binding of lisinopril to the angiotensin I-converting enzyme (ACE; EC 3.4.15.1) and to its apoenzyme at pH 7.5 over a temperature range of 15-30 degrees C. Calorimetric measurements indicate that lisinopril binds to two sites in the monomer of both holo- and apo-ACE. Binding of lisinopril to both systems is enthalpically unfavorable and, thus, is dominated by a large positive entropy change. The enthalpy change of binding is strongly temperature-dependent for both holo- and apo-ACE, arising from a large heat capacity change of binding equal to -2.4 +/- 0.2 kJ/K/mol of monomeric holo-ACE) and to -1.9 +/- 0.2 kJ/K/mol of monomeric apo-ACE), respectively. The negative values of deltaCp for both systems are consistent with burial of a large non-polar surface area upon binding. Although the binding of lisinopril to holo- and apo-ACE is favored by entropy changes, this is more positive for the holoenzyme. Thus, the interaction between Zn2+ and lisinopril results in a higher affinity of the holoenzyme for this drug due to a more favorable entropic contribution.
    FEBS Letters 02/1998; 423(1):75-80. · 3.58 Impact Factor
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    ABSTRACT: Isothermal titration microcalorimetry and equilibrium dialysis have been used to characterize the binding of 2'-deoxycytidine 5'-monophosphate (dCMP) to the Asn229Asp mutant of Lactobacillus casei recombinant thymidylate synthase at pH 7.4 over a temperature range of 15 degrees C to 35 degrees C. Equilibrium dialysis analysis shows that dCMP binds to two sites in the dimer of both wild-type and mutant thymidylate synthase. A concomitant net uptake of protons with binding of dCMP to both enzymes, was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The change in protonation for binding of dCMP to wild-type enzyme is lower than that obtained for binding of this nucleotide to TS N229D, which suggests that the pK value of Asp-229 is increased upon dCMP binding to the mutant enzyme. At 25 degrees C, although the binding of dCMP to wild-type and N229D TS is favoured by both enthalpy and entropy changes, the enthalpy change is more negative for the mutant protein. Thus, the substitution of Asn 229 for Asp results in a higher affinity of TS for dCMP due to a more favourable enthalpic contribution. The Gibbs energy change of binding of dCMP to the mutant enzyme is weakly temperature-dependent, because of the enthalpy-entropy compensation arising from a negative heat capacity change of binding equal to -0.83 +/- 0.02 kJ K(-1) per mol of dCMP bound.
    FEBS Letters 07/1997; 409(3):385-90. · 3.58 Impact Factor
  • Progress in Biophysics and Molecular Biology 01/1996; 65. · 2.91 Impact Factor