Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear Fe(III)... more Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear Fe(III)M(II) center (M(II) = Fe, Mn, Zn) in the active site and are able to catalyze the hydrolysis of a variety of phosphoric acid esters. The dinuclear complex [(H(2)O)Fe(III)(μ-OH)Zn(II)(L-H)](ClO(4))(2) (2) with the ligand 2-[N-bis(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N'-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H(2)L-H) has recently been prepared and is found to closely mimic the coordination environment of the Fe(III)Zn(II) active site found in red kidney bean PAP (Neves et al. J. Am. Chem. Soc. 2007, 129, 7486). The biomimetic shows significant catalytic activity in hydrolytic reactions. By using a variety of structural, spectroscopic, and computational techniques the electronic structure of the Fe(III) center of this biomimetic complex was determined. In the solid state the electronic ground state reflects the rhombically distorted Fe(III)N(2)O(4) octahedron with a dominant tetragonal compression aligned along the μ-OH-Fe-O(phenolate) direction. To probe the role of the Fe-O(phenolate) bond, the phenolate moiety was modified to contain electron-donating or -withdrawing groups (-CH(3), -H, -Br, -NO(2)) in the 5-position. The effects of the substituents on the electronic properties of the biomimetic complexes were studied with a range of experimental and computational techniques. This study establishes benchmarks against accurate crystallographic structural information using spectroscopic techniques that are not restricted to single crystals. Kinetic studies on the hydrolysis reaction revealed that the phosphodiesterase activity increases in the order -NO(2) ←Br ←H ←CH(3) when 2,4-bis(dinitrophenyl)phosphate (2,4-bdnpp) was used as substrate, and a linear free energy relationship is found when log(k(cat)/k(0)) is plotted against the Hammett parameter σ. However, nuclease activity measurements in the cleavage of double stranded DNA showed that the complexes containing the electron-withdrawing -NO(2) and electron-donating -CH(3) groups are the most active while the cytotoxic activity of the biomimetics on leukemia and lung tumoral cells is highest for complexes with electron-donating groups.
This paper reports on the synthesis and characterization of two new ternary copper(II) complexes:... more This paper reports on the synthesis and characterization of two new ternary copper(II) complexes: [Cu(doxycycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (1) and [Cu(tetracycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (2). These compounds exhibit a distorted tetragonal geometry around copper, which is coordinated to two bidentate ligands, 1,10-phenanthroline and tetracycline or doxycyline, a water molecule, and a perchlorate ion weakly bonded in the axial positions. In both compounds, copper(II) binds to tetracyclines via the oxygen of the hydroxyl group and oxygen of the amide group at ring A and to 1,10-phenanthroline via its two heterocyclic nitrogens. We have evaluated the binding of the new complexes to DNA, their capacity to cleave it, their cytotoxic activity, and uptake in tumoral cells. The complexes bind to DNA preferentially by the major groove, and then cleave its strands by an oxidative mechanism involving the generation of ROS. The cleavage of DNA was inhi...
We investigated the heat shock response of the adenylate cyclase deficient mutant cr-1 (crisp) of... more We investigated the heat shock response of the adenylate cyclase deficient mutant cr-1 (crisp) of Neurospora crassa. This strain was observed to be much more resistant to a lethal temperature of 50 degrees C than the wild type. This constitutive thermotolerance was absent in cr-1 conidiospores raised on cyclic AMP (cAMP, 2.5 mM) supplemented solid medium, but was partially restored when the conidiospores were germinated at 30 degrees C, a temperature which fails to induce thermotolerance in the wild-type strain. Two other crisp-like Neurospora mutants, cr-2 and cr-3 which, in contrast to cr-1, contain normal levels of cAMP, did not exhibit the thermotolerance phenomenon observed for cr-1. A cr-1, pe, fl (crisp-microconidial) strain also lacked the ability to tolerate a lethal heat treatment. Our results demonstrate that the adenylate cyclase deficient cr-1 mutant of Neurospora crassa expresses a constitutive thermotolerant phenotype as a consequence of its primary genetic defect: low levels of cAMP.
ABSTRACT This work demonstrates the efficient photocleavage of DNA induced by a terbium complex. ... more ABSTRACT This work demonstrates the efficient photocleavage of DNA induced by a terbium complex. Mechanistic studies and DNA groove binding dependence were performed to better understand the photo-cleavage reaction, suggesting the presence of oxygen and carbon-centered radicals in the strand scission event. Furthermore, kinetic analyses were also achieved. The DNA photocleavage promoted by the title complex proceeds with a kobs of ~ 10.2 h− 1, a reaction rate useful in molecular biology practices, with a t1/2 of 4 min. This complex emerges as a new example of lanthanide compound with DNA cleavage ability by a non-hydrolytic mechanism.
The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacteri... more The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhi...
The role of nitric oxide (NO) as a major regulator of plant physiological functions has become in... more The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the biotin switch technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed.
We have previously demonstrated the stimulation of adenylyl cyclase by guanine nucleotides in the... more We have previously demonstrated the stimulation of adenylyl cyclase by guanine nucleotides in the plasma membranes of the aquatic fungus Blastocladiella emersonii. The existence of a G-protein in these membranes similar to Gs, the G-protein responsible for hormonal stimulation of adenylyl cyclase in mammals, was shown. In this report the crude membranes were further analyzed and a polypeptide of ~
The nuclear receptor superfamily comprises a group of transcriptional regulators involved in a wi... more The nuclear receptor superfamily comprises a group of transcriptional regulators involved in a wide variety of physiological responses. Rev-erb beta is a member of a growing subfamily of orphan nuclear receptors that bind DNA with high affinity either as monomers or as hetero- or homodimers. DNA bending assays, high-resolution footprinting, molecular modeling, and site-directed mutagenesis were used to analyze the structural features of the interaction between the DNA-binding domain (DBD) of the nuclear receptor Rev-erb beta and its DNA target sites. The results obtained point to the involvement of a carboxyl-terminal sequence adjacent to the second zinc finger of the Rev-erb beta DBD in protein-DNA interaction as a monomer or in protein-DNA and protein-protein interactions as a homodimer. They also provide insight about the amino acid residues directly involved in protein-protein contacts.
Rev-erbβ is a member of the nuclear receptor superfamily, which includes a group of transcription... more Rev-erbβ is a member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, vitamin D, retinoic acids, and other lipophilic molecules. The Rev-erb nuclear receptors exist at least in two forms, namely α and β , with a high degree of evolutionary conservation at the level of the DNA binding domain.
Transferrin hepato-specific gene enhancer, associated with the liver-enriched HNF-3α transcriptio... more Transferrin hepato-specific gene enhancer, associated with the liver-enriched HNF-3α transcriptional factor and ubiquitous proteins, is a complex molecular edifice maintained through DNA-protein and protein-protein interactions. As a first step to understand the mechanisms responsible for its organization and activity, we have analyzed the interaction of the DNA binding domain of HNF-3α (HDBD) with a specific DNA segment present in the
Hydrolysis of DNA is of increasing importance in biotechnology and medicine. In this Letter, we p... more Hydrolysis of DNA is of increasing importance in biotechnology and medicine. In this Letter, we present the DNA-cleavage potential of metal-free hydroxylamines and oximes as new members of nucleic acid cleavage agents.
The search for efficient artificial nucleases is a consequence of the huge efforts towards DNA se... more The search for efficient artificial nucleases is a consequence of the huge efforts towards DNA sequencing by genome projects around the world. In the present study, we describe the activity of the Cu(II)–BMXD complex (BMXD=3,6,9,17,20,23-hexaazatricyclo[23.3.1.111,15]triaconta-1(29),11(30),12,14,25,27-hexaene) as a DNA cleavage agent. The rate constant for the DNA backbone cleavage reaction at room temperature (25°C) and pH 7.0 was estimated to be
The reaction of FeSO4·7H2O with the ligand HPClNOL (1-(bis-pyridin-2-ylmethyl-amino)-3-chloroprop... more The reaction of FeSO4·7H2O with the ligand HPClNOL (1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol results in the complex [Fe(HPClNOL)(SO4)]2-μ-oxo·6H2O 1, in which the iron ions are coordinated by two pyridines, one amine and one alcohol group from the ligand HPClNOL, an oxo bridge and monodentate sulfate ion. The complex is highly soluble in water and potentiometric titration experiments reveal four protonation/deprotonation equilibria, with pKa values
Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-V... more Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and
The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's ... more The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo-and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells.
As propriedades antitumorais de complexos de Pt II e as características favoráveis das tetracicli... more As propriedades antitumorais de complexos de Pt II e as características favoráveis das tetraciclinas levaram-nos a estudar compostos de Pt II da tetraciclina (tc) e doxiciclina (dox) como candidatos a agentes antitumorais. [PtCl 2 (dox)], 1, é mais potente que [PtCl 2 (tc)], 2, em inibir o crescimento de células de leucemia mielóide crônica. Os compostos 1 e 2 formam um complexo ternário com o ADN com valores de K iguais a 3,85×10 4 e 5,43×10 4 , respectivamente. Os complexos deslocam o brometo de etídeo dos sítios no ADN, o que indica um mecanismo intercalativo. Ambos os complexos diminuem a mobilidade eletroforética e a temperatura de fusão do ADN. Após a incubação das células com 1 e 2, o ADN foi extraído e os adutos formados foram quantificados. Na concentração em que os compostos são citotóxicos contra células cancerosas, eles não afetam a viabilidade de macrófagos. A velocidade de entrada do complexo da doxiciclina nas células é três vezes maior do que a do complexo da tetraciclina e isto parece ser determinante para a sua maior atividade. The antitumoral properties of Pt II compounds and the favorable characteristics of tetracyclines led us to study Pt II compounds of tetracycline (tc) and doxycycline (dox) as candidates for anticancer agents. [PtCl 2 (dox)], 1, is more potent than [PtCl 2 (tc)], 2, in inhibiting the growth of chronic myelogenous leukemia cells. Compounds 1 and 2 form a ternary complex with DNA with K values of 3.85×10 4 and 5.43×10 4 , respectively. The compounds displace ethidium bromide from DNA sites, which points to an intercalative mechanism. Both complexes decrease DNA eletrophoretic mobility and melting temperature. After incubation of cells with 1 and 2, the DNA was extracted and the adducts formed were quantified. At the concentration that the compounds are cytotoxic to cancer cells, they do not affect macrophages viability. The rate of uptake of the doxycycline complex in cells is three times higher than that of the tetracycline complex and this seems to be determinant for its higher activity.
Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a na... more Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42 degrees C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC(2), pNPC(4), pNPC(10), pNPC(12), pNPC(14), pNPC(16), pNPC(18)). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95 degrees C, 77% of the initial activity was retained.
Os estudos sobre enzimas têm mostrado que existe uma relação clara entre a evolução enzimática e ... more Os estudos sobre enzimas têm mostrado que existe uma relação clara entre a evolução enzimática e a propriedade de promiscuidade catalítica que elas podem apresentar. Com isto, o desenvolvimento de sistemas biomiméticos capazes de atuar de modo promíscuo, de forma similar a algumas enzimas, mostrou-se um novo desafio para os químicos bioinorgânicos. Neste trabalho, reportamos a estrutura de raios X e os estudos em solução e de promiscuidade catalítica do complexo [(bpbpmp)Fe III (m-OAc) 2 Fe II ](ClO 4 ), (1), contendo o ligante binucleante não-simétrico 2-{[(2-hidroxibenzil)(2-piridilmetil)aminometil]-4-metil-6-[bis(2-piridilmetil)aminometil]}fenol (H 2 bpbpmp). Estudos de titulações potenciométrica e espectrofotométrica e cinéticos mostraram que esse composto de coordenação gera espécies ativas capazes de clivar o dsDNA (DNA de fita dupla) hidroliticamente, com um aumento de 1,9×10 8 na taxa de reação em comparação com a reação não catalisada, e capazes de oxidar o 3,5-di-terc-butilcatecol (3,5-dtbc), com k cat = 1,16 × 10 -2 s -1 e K M = 7,1×10 -4 mol L -1 . Assim, o complexo 1 apresenta atividades de hidrolase e oxidoredutase, sendo um interessante modelo sintético para estudos de promiscuidade catalítica.
The new homodinuclear complexes, ½Cu II 2 ðHLdtbÞðl-OCH 3 ÞðClO 4 Þ 2 (1) and ½Cu II 2 ðLdtbÞðl-O... more The new homodinuclear complexes, ½Cu II 2 ðHLdtbÞðl-OCH 3 ÞðClO 4 Þ 2 (1) and ½Cu II 2 ðLdtbÞðl-OCH 3 ÞðBPh 4 Þ (2), with the unsymmetrical N 5 O 2 donor ligand (H 2 Ldtb) -{2-[N,N-Bis(2-pyridylmethyl)aminomethyl]-6-[N 0 ,N 0 -(3,5-di-tert-butylbenzyl-2-hydroxy)(2-pyridylmethyl)]aminomethyl}-4-methylphenol have been synthesized and characterized in the solid state by X-ray crystallography.In both cases the structure reveals that the complexes have a common {Cu II (l-phenoxo)(l-OCH 3 )Cu II } structural unit.Magnetic susceptibility studies of 1 and 2 reveal J values of À38.3 cm À1 and À2.02 cm À1 , respectively, and that the degree of antiferromagnetic coupling is strongly dependent on the coordination geometries of the copper centers within the dinuclear {Cu II (l-OCH 3 )(l-phenolate)Cu II } structural unit.Solution studies in dichloromethane, using UV-Visible spectroscopy and electrochemistry, indicate that under these experimental conditions the first coordination spheres of the Cu II centers are maintained as observed in the solid state structures, and that both forms can be brought into equilibrium ([Cu 2 (HLdtb)(l-OCH 3 )] 2+ = [Cu 2 (Ldtb)(l-OCH 3 )] + + H + ) by adjusting the pH with Et 3 N (Ldtb 2À is the deprotonated form of the ligand).On the other hand, potentiometric titration studies of 1 in an ethanol/water mixture (70:30 V/V; I = 0.1 M KCl) show three titrable protons, indicating the dissociation of the bridging CH 3 O À group.The catecholase activity of 1 and 2 in methanol/water buffer (30:1 V/V) demonstrates that the deprotonated form is the active species in the oxidation of 3,5di-tert-butylcatechol and that the reaction follows Michaelis-Menten behavior with k cat = 5.33 · 10 À3 s À1 and K M = 3.96 · 10 À3 M. Interestingly, 2 can be electrochemically oxidized with E 1/2 = 0.27 V vs.Fc + /Fc (Fc + /Fc is the redox pair ferrocinium/ferrocene), a redox potential which is believed to be related to the formation of a phenoxyl radical.Since these complexes are redox active species, we analyzed their activity toward the nucleic acid DNA, a macromolecule prone to oxidative damage.Interestingly these complexes promoted DNA cleavage following an oxygen dependent pathway.
Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear Fe(III)... more Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear Fe(III)M(II) center (M(II) = Fe, Mn, Zn) in the active site and are able to catalyze the hydrolysis of a variety of phosphoric acid esters. The dinuclear complex [(H(2)O)Fe(III)(μ-OH)Zn(II)(L-H)](ClO(4))(2) (2) with the ligand 2-[N-bis(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N'-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H(2)L-H) has recently been prepared and is found to closely mimic the coordination environment of the Fe(III)Zn(II) active site found in red kidney bean PAP (Neves et al. J. Am. Chem. Soc. 2007, 129, 7486). The biomimetic shows significant catalytic activity in hydrolytic reactions. By using a variety of structural, spectroscopic, and computational techniques the electronic structure of the Fe(III) center of this biomimetic complex was determined. In the solid state the electronic ground state reflects the rhombically distorted Fe(III)N(2)O(4) octahedron with a dominant tetragonal compression aligned along the μ-OH-Fe-O(phenolate) direction. To probe the role of the Fe-O(phenolate) bond, the phenolate moiety was modified to contain electron-donating or -withdrawing groups (-CH(3), -H, -Br, -NO(2)) in the 5-position. The effects of the substituents on the electronic properties of the biomimetic complexes were studied with a range of experimental and computational techniques. This study establishes benchmarks against accurate crystallographic structural information using spectroscopic techniques that are not restricted to single crystals. Kinetic studies on the hydrolysis reaction revealed that the phosphodiesterase activity increases in the order -NO(2) ←Br ←H ←CH(3) when 2,4-bis(dinitrophenyl)phosphate (2,4-bdnpp) was used as substrate, and a linear free energy relationship is found when log(k(cat)/k(0)) is plotted against the Hammett parameter σ. However, nuclease activity measurements in the cleavage of double stranded DNA showed that the complexes containing the electron-withdrawing -NO(2) and electron-donating -CH(3) groups are the most active while the cytotoxic activity of the biomimetics on leukemia and lung tumoral cells is highest for complexes with electron-donating groups.
This paper reports on the synthesis and characterization of two new ternary copper(II) complexes:... more This paper reports on the synthesis and characterization of two new ternary copper(II) complexes: [Cu(doxycycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (1) and [Cu(tetracycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (2). These compounds exhibit a distorted tetragonal geometry around copper, which is coordinated to two bidentate ligands, 1,10-phenanthroline and tetracycline or doxycyline, a water molecule, and a perchlorate ion weakly bonded in the axial positions. In both compounds, copper(II) binds to tetracyclines via the oxygen of the hydroxyl group and oxygen of the amide group at ring A and to 1,10-phenanthroline via its two heterocyclic nitrogens. We have evaluated the binding of the new complexes to DNA, their capacity to cleave it, their cytotoxic activity, and uptake in tumoral cells. The complexes bind to DNA preferentially by the major groove, and then cleave its strands by an oxidative mechanism involving the generation of ROS. The cleavage of DNA was inhi...
We investigated the heat shock response of the adenylate cyclase deficient mutant cr-1 (crisp) of... more We investigated the heat shock response of the adenylate cyclase deficient mutant cr-1 (crisp) of Neurospora crassa. This strain was observed to be much more resistant to a lethal temperature of 50 degrees C than the wild type. This constitutive thermotolerance was absent in cr-1 conidiospores raised on cyclic AMP (cAMP, 2.5 mM) supplemented solid medium, but was partially restored when the conidiospores were germinated at 30 degrees C, a temperature which fails to induce thermotolerance in the wild-type strain. Two other crisp-like Neurospora mutants, cr-2 and cr-3 which, in contrast to cr-1, contain normal levels of cAMP, did not exhibit the thermotolerance phenomenon observed for cr-1. A cr-1, pe, fl (crisp-microconidial) strain also lacked the ability to tolerate a lethal heat treatment. Our results demonstrate that the adenylate cyclase deficient cr-1 mutant of Neurospora crassa expresses a constitutive thermotolerant phenotype as a consequence of its primary genetic defect: low levels of cAMP.
ABSTRACT This work demonstrates the efficient photocleavage of DNA induced by a terbium complex. ... more ABSTRACT This work demonstrates the efficient photocleavage of DNA induced by a terbium complex. Mechanistic studies and DNA groove binding dependence were performed to better understand the photo-cleavage reaction, suggesting the presence of oxygen and carbon-centered radicals in the strand scission event. Furthermore, kinetic analyses were also achieved. The DNA photocleavage promoted by the title complex proceeds with a kobs of ~ 10.2 h− 1, a reaction rate useful in molecular biology practices, with a t1/2 of 4 min. This complex emerges as a new example of lanthanide compound with DNA cleavage ability by a non-hydrolytic mechanism.
The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacteri... more The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhi...
The role of nitric oxide (NO) as a major regulator of plant physiological functions has become in... more The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the biotin switch technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed.
We have previously demonstrated the stimulation of adenylyl cyclase by guanine nucleotides in the... more We have previously demonstrated the stimulation of adenylyl cyclase by guanine nucleotides in the plasma membranes of the aquatic fungus Blastocladiella emersonii. The existence of a G-protein in these membranes similar to Gs, the G-protein responsible for hormonal stimulation of adenylyl cyclase in mammals, was shown. In this report the crude membranes were further analyzed and a polypeptide of ~
The nuclear receptor superfamily comprises a group of transcriptional regulators involved in a wi... more The nuclear receptor superfamily comprises a group of transcriptional regulators involved in a wide variety of physiological responses. Rev-erb beta is a member of a growing subfamily of orphan nuclear receptors that bind DNA with high affinity either as monomers or as hetero- or homodimers. DNA bending assays, high-resolution footprinting, molecular modeling, and site-directed mutagenesis were used to analyze the structural features of the interaction between the DNA-binding domain (DBD) of the nuclear receptor Rev-erb beta and its DNA target sites. The results obtained point to the involvement of a carboxyl-terminal sequence adjacent to the second zinc finger of the Rev-erb beta DBD in protein-DNA interaction as a monomer or in protein-DNA and protein-protein interactions as a homodimer. They also provide insight about the amino acid residues directly involved in protein-protein contacts.
Rev-erbβ is a member of the nuclear receptor superfamily, which includes a group of transcription... more Rev-erbβ is a member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, vitamin D, retinoic acids, and other lipophilic molecules. The Rev-erb nuclear receptors exist at least in two forms, namely α and β , with a high degree of evolutionary conservation at the level of the DNA binding domain.
Transferrin hepato-specific gene enhancer, associated with the liver-enriched HNF-3α transcriptio... more Transferrin hepato-specific gene enhancer, associated with the liver-enriched HNF-3α transcriptional factor and ubiquitous proteins, is a complex molecular edifice maintained through DNA-protein and protein-protein interactions. As a first step to understand the mechanisms responsible for its organization and activity, we have analyzed the interaction of the DNA binding domain of HNF-3α (HDBD) with a specific DNA segment present in the
Hydrolysis of DNA is of increasing importance in biotechnology and medicine. In this Letter, we p... more Hydrolysis of DNA is of increasing importance in biotechnology and medicine. In this Letter, we present the DNA-cleavage potential of metal-free hydroxylamines and oximes as new members of nucleic acid cleavage agents.
The search for efficient artificial nucleases is a consequence of the huge efforts towards DNA se... more The search for efficient artificial nucleases is a consequence of the huge efforts towards DNA sequencing by genome projects around the world. In the present study, we describe the activity of the Cu(II)–BMXD complex (BMXD=3,6,9,17,20,23-hexaazatricyclo[23.3.1.111,15]triaconta-1(29),11(30),12,14,25,27-hexaene) as a DNA cleavage agent. The rate constant for the DNA backbone cleavage reaction at room temperature (25°C) and pH 7.0 was estimated to be
The reaction of FeSO4·7H2O with the ligand HPClNOL (1-(bis-pyridin-2-ylmethyl-amino)-3-chloroprop... more The reaction of FeSO4·7H2O with the ligand HPClNOL (1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol results in the complex [Fe(HPClNOL)(SO4)]2-μ-oxo·6H2O 1, in which the iron ions are coordinated by two pyridines, one amine and one alcohol group from the ligand HPClNOL, an oxo bridge and monodentate sulfate ion. The complex is highly soluble in water and potentiometric titration experiments reveal four protonation/deprotonation equilibria, with pKa values
Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-V... more Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and
The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's ... more The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo-and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells.
As propriedades antitumorais de complexos de Pt II e as características favoráveis das tetracicli... more As propriedades antitumorais de complexos de Pt II e as características favoráveis das tetraciclinas levaram-nos a estudar compostos de Pt II da tetraciclina (tc) e doxiciclina (dox) como candidatos a agentes antitumorais. [PtCl 2 (dox)], 1, é mais potente que [PtCl 2 (tc)], 2, em inibir o crescimento de células de leucemia mielóide crônica. Os compostos 1 e 2 formam um complexo ternário com o ADN com valores de K iguais a 3,85×10 4 e 5,43×10 4 , respectivamente. Os complexos deslocam o brometo de etídeo dos sítios no ADN, o que indica um mecanismo intercalativo. Ambos os complexos diminuem a mobilidade eletroforética e a temperatura de fusão do ADN. Após a incubação das células com 1 e 2, o ADN foi extraído e os adutos formados foram quantificados. Na concentração em que os compostos são citotóxicos contra células cancerosas, eles não afetam a viabilidade de macrófagos. A velocidade de entrada do complexo da doxiciclina nas células é três vezes maior do que a do complexo da tetraciclina e isto parece ser determinante para a sua maior atividade. The antitumoral properties of Pt II compounds and the favorable characteristics of tetracyclines led us to study Pt II compounds of tetracycline (tc) and doxycycline (dox) as candidates for anticancer agents. [PtCl 2 (dox)], 1, is more potent than [PtCl 2 (tc)], 2, in inhibiting the growth of chronic myelogenous leukemia cells. Compounds 1 and 2 form a ternary complex with DNA with K values of 3.85×10 4 and 5.43×10 4 , respectively. The compounds displace ethidium bromide from DNA sites, which points to an intercalative mechanism. Both complexes decrease DNA eletrophoretic mobility and melting temperature. After incubation of cells with 1 and 2, the DNA was extracted and the adducts formed were quantified. At the concentration that the compounds are cytotoxic to cancer cells, they do not affect macrophages viability. The rate of uptake of the doxycycline complex in cells is three times higher than that of the tetracycline complex and this seems to be determinant for its higher activity.
Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a na... more Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42 degrees C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC(2), pNPC(4), pNPC(10), pNPC(12), pNPC(14), pNPC(16), pNPC(18)). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95 degrees C, 77% of the initial activity was retained.
Os estudos sobre enzimas têm mostrado que existe uma relação clara entre a evolução enzimática e ... more Os estudos sobre enzimas têm mostrado que existe uma relação clara entre a evolução enzimática e a propriedade de promiscuidade catalítica que elas podem apresentar. Com isto, o desenvolvimento de sistemas biomiméticos capazes de atuar de modo promíscuo, de forma similar a algumas enzimas, mostrou-se um novo desafio para os químicos bioinorgânicos. Neste trabalho, reportamos a estrutura de raios X e os estudos em solução e de promiscuidade catalítica do complexo [(bpbpmp)Fe III (m-OAc) 2 Fe II ](ClO 4 ), (1), contendo o ligante binucleante não-simétrico 2-{[(2-hidroxibenzil)(2-piridilmetil)aminometil]-4-metil-6-[bis(2-piridilmetil)aminometil]}fenol (H 2 bpbpmp). Estudos de titulações potenciométrica e espectrofotométrica e cinéticos mostraram que esse composto de coordenação gera espécies ativas capazes de clivar o dsDNA (DNA de fita dupla) hidroliticamente, com um aumento de 1,9×10 8 na taxa de reação em comparação com a reação não catalisada, e capazes de oxidar o 3,5-di-terc-butilcatecol (3,5-dtbc), com k cat = 1,16 × 10 -2 s -1 e K M = 7,1×10 -4 mol L -1 . Assim, o complexo 1 apresenta atividades de hidrolase e oxidoredutase, sendo um interessante modelo sintético para estudos de promiscuidade catalítica.
The new homodinuclear complexes, ½Cu II 2 ðHLdtbÞðl-OCH 3 ÞðClO 4 Þ 2 (1) and ½Cu II 2 ðLdtbÞðl-O... more The new homodinuclear complexes, ½Cu II 2 ðHLdtbÞðl-OCH 3 ÞðClO 4 Þ 2 (1) and ½Cu II 2 ðLdtbÞðl-OCH 3 ÞðBPh 4 Þ (2), with the unsymmetrical N 5 O 2 donor ligand (H 2 Ldtb) -{2-[N,N-Bis(2-pyridylmethyl)aminomethyl]-6-[N 0 ,N 0 -(3,5-di-tert-butylbenzyl-2-hydroxy)(2-pyridylmethyl)]aminomethyl}-4-methylphenol have been synthesized and characterized in the solid state by X-ray crystallography.In both cases the structure reveals that the complexes have a common {Cu II (l-phenoxo)(l-OCH 3 )Cu II } structural unit.Magnetic susceptibility studies of 1 and 2 reveal J values of À38.3 cm À1 and À2.02 cm À1 , respectively, and that the degree of antiferromagnetic coupling is strongly dependent on the coordination geometries of the copper centers within the dinuclear {Cu II (l-OCH 3 )(l-phenolate)Cu II } structural unit.Solution studies in dichloromethane, using UV-Visible spectroscopy and electrochemistry, indicate that under these experimental conditions the first coordination spheres of the Cu II centers are maintained as observed in the solid state structures, and that both forms can be brought into equilibrium ([Cu 2 (HLdtb)(l-OCH 3 )] 2+ = [Cu 2 (Ldtb)(l-OCH 3 )] + + H + ) by adjusting the pH with Et 3 N (Ldtb 2À is the deprotonated form of the ligand).On the other hand, potentiometric titration studies of 1 in an ethanol/water mixture (70:30 V/V; I = 0.1 M KCl) show three titrable protons, indicating the dissociation of the bridging CH 3 O À group.The catecholase activity of 1 and 2 in methanol/water buffer (30:1 V/V) demonstrates that the deprotonated form is the active species in the oxidation of 3,5di-tert-butylcatechol and that the reaction follows Michaelis-Menten behavior with k cat = 5.33 · 10 À3 s À1 and K M = 3.96 · 10 À3 M. Interestingly, 2 can be electrochemically oxidized with E 1/2 = 0.27 V vs.Fc + /Fc (Fc + /Fc is the redox pair ferrocinium/ferrocene), a redox potential which is believed to be related to the formation of a phenoxyl radical.Since these complexes are redox active species, we analyzed their activity toward the nucleic acid DNA, a macromolecule prone to oxidative damage.Interestingly these complexes promoted DNA cleavage following an oxygen dependent pathway.
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