Showing papers in "Journal of Biological Inorganic Chemistry in 2013"
TL;DR: Conditional stability constants for the binding of KP1019 and KP1339 to sites I and II of HSA were determined, indicating that both Ru(III) compounds bind to both sites with moderately strong affinity.
Abstract: Indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019) and its Na(+) analogue (KP1339) are two of the most prominent non-platinum antitumor metal complexes currently undergoing clinical trials. After intravenous administration, they are known to bind to human serum albumin (HSA) in a noncovalent manner. To elucidate their HSA binding sites, displacement reactions with the established site markers warfarin and dansylglycine as well as bilirubin were monitored by spectrofluorimetry, ultrafiltration-UV-vis spectrophotometry, and/or capillary zone electrophoresis. Conditional stability constants for the binding of KP1019 and KP1339 to sites I and II of HSA were determined, indicating that both Ru(III) compounds bind to both sites with moderately strong affinity (log K(1)' = 5.3-5.8). No preference for either binding site was found, and similar results were obtained for both metal complexes, demonstrating low influence of the counter ion on the binding event.
123 citations
TL;DR: Combining a bioactive compound such as KTZ and a metal in a single molecule results in a synergy that can translate into improved activity and/or selectivity against parasites, and complexation of KTZ with RuII in compounds 3–5 produces a marked enhancement of the activity toward promastigote and intracellular amastigotes of Leishmania major.
Abstract: In our ongoing search for new metal-based chemotherapeutic agents against leishmaniasis and Chagas disease, six new ruthenium-ketoconazole (KTZ) complexes have been synthesized and characterized, including two octahedral coordination complexes-cis,fac-[Ru(II)Cl2(DMSO)3(KTZ)] (1) and cis-[Ru(II)Cl2(bipy)(DMSO)(KTZ)] (2) (where DMSO is dimethyl sulfoxide and bipy is 2,2'-bipyridine)-and four organometallic compounds-[Ru(II)(η(6)-p-cymene)Cl2(KTZ)] (3), [Ru(II)(η(6)-p-cymene)(en)(KTZ)][BF4]2 (4), [Ru(II)(η(6)-p-cymene)(bipy)(KTZ)][BF4]2 (5), and [Ru(II)(η(6)-p-cymene)(acac)(KTZ)][BF4] (6) (where en is ethylenediamine and acac is acetylacetonate); the crystal structure of 3 is described. The central hypothesis of our work is that combining a bioactive compound such as KTZ and a metal in a single molecule results in a synergy that can translate into improved activity and/or selectivity against parasites. In agreement with this hypothesis, complexation of KTZ with Ru(II) in compounds 3-5 produces a marked enhancement of the activity toward promastigotes and intracellular amastigotes of Leishmania major, when compared with uncomplexed KTZ, or with similar ruthenium compounds not containing KTZ. Importantly, the selective toxicity of compounds 3-5 toward the leishmania parasites, in relation to human fibroblasts and osteoblasts or murine macrophages, is also superior to the selective toxicities of the individual constituents of the drug. When tested against Trypanosoma cruzi epimastigotes, some of the organometallic complexes displayed activity and selectivity comparable to those of free KTZ. A dual-target mechanism is suggested to account for the antiparasitic properties of these complexes.
67 citations
TL;DR: In this review, the recent literature on dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) enzymes is summarized, with an emphasis on structure–function studies that provide insight into the catalytic mechanism.
Abstract: In this review, we summarize the recent literature on dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) enzymes, with an emphasis on structure–function studies that provide insight into the catalytic mechanism. Crystallographic data have also provided insight into residues that might be involved in substrate and hence inhibitor recognition and binding. These data have led to the design and synthesis of several new DapE inhibitors, which are described along with what is known about how inhibitors interact with the active site of DapE enzymes, including the efficacy of a moderately strong DapE inhibitor.
57 citations
TL;DR: Functional evidence supported the hypothesis that various types of bacteria can induce or aggravate metabolic stone disease, particularly the CaOx type.
Abstract: Our previous report showed that uropathogenic bacteria, e.g., Escherichia coli, are commonly found inside the nidus of calcium oxalate (CaOx) kidney stones and may play pivotal roles in stone genesis. The present study aimed to prove this new hypothesis by direct examining CaOx lithogenic activities of both Gram-negative and Gram-positive bacteria. CaOx was crystallized in the absence (blank control) or presence of 105 CFU/ml E. coli, Klebsiella pneumoniae, Staphylococcus aureus, or Streptococcus pneumoniae. Fragmented red blood cell membranes and intact red blood cells were used as positive and negative controls, respectively. The crystal area and the number of aggregates were measured to initially screen for effects of bacteria on CaOx crystal growth and aggregation. The data revealed that all the bacteria tested dramatically increased the crystal area and number of crystal aggregates. Validation assays (spectrophotometric oxalate-depletion assay and an aggregation–sedimentation study) confirmed their promoting effects on both growth (20.17 ± 3.42, 17.55 ± 2.27, 16.37 ± 1.38, and 21.87 ± 0.85 % increase, respectively) and aggregation (57.45 ± 2.08, 51.06 ± 5.51, 55.32 ± 2.08, and 46.81 ± 3.61 % increase, respectively) of CaOx crystals. Also, these bacteria significantly enlarged CaOx aggregates, with the diameter greater than the luminal size of distal tubules, implying that tubular occlusion might occur. Moreover, these bacterial effects were dose-dependent and specific to intact viable bacteria, not intact dead or fragmented bacteria. In summary, intact viable E. coli, K. pneumoniae, S. aureus, and S. pneumoniae had significant promoting effects on CaOx crystal growth and aggregation. This functional evidence supported the hypothesis that various types of bacteria can induce or aggravate metabolic stone disease, particularly the CaOx type.
55 citations
TL;DR: Western blotting analysis shows that [Ru(dmp)2(adppz)](ClO4)2 induces apoptosis in BEL-7402 cells through activation of caspase 3, caspases 7, and procaspase 7 and ROS-mediated mitochondrial dysfunction pathways.
Abstract: Three new ruthenium(II) complexes—[Ru(bpy)2(adppz)](ClO4)2, [Ru(dmb)2(adppz)](ClO4)2, and [Ru(dmp)2(adppz)](ClO4)2 (bpy is 2,2′-bipyridine, adppz is 7-aminodipyrido[3,2-a:2′,3′-c]phenazine, dmb is 4,4′-dimethyl-2,2′-bipyridine, and dmp is 2,9-dimethyl-1,10-phenanthroline)—were synthesized. [Ru(dmp)2(adppz)](ClO4)2 exhibits higher cytotoxicity than cisplatin toward A549, MG-63, and SKBR-3 cells. The apoptosis and cellular uptake were studied by fluorescence microscopy. [Ru(dmp)2(adppz)](ClO4)2 enhances the level of reactive oxygen species (ROS) and decreases the mitochondrial membrane potential. These complexes induce cell cycle arrest in S phase in BEL-7402 cells, and inhibit the antiproliferation of SKBR-3 cells at G0/G1 phase. Western blotting analysis shows that [Ru(dmp)2(adppz)](ClO4)2 induces apoptosis in BEL-7402 cells through activation of caspase 3, caspase 7, and procaspase 7 and ROS-mediated mitochondrial dysfunction pathways.
52 citations
TL;DR: Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies and showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion.
Abstract: Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies. In complex 1, the ligand 2-hydroxy-1-naphthaldehydethiosemicarbazone coordinated as a monobasic tridentate donor, whereas in complexes 2 and 3, the ligands salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hydroxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone coordinated as a dibasic tridentate donor. The DNA binding ability of the complexes in calf thymus DNA was explored by absorption and emission titration experiments. The antioxidant property of the new complexes was evaluated to test their free-radical scavenging ability. In vitro cytotoxicity assays were performed for the new complexes in A549 and HepG2 cell lines. The new compounds overcome cisplatin resistance in the A549 cell line and they were also active in the HepG2 cell line. The cellular uptake study showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion.
49 citations
TL;DR: It is concluded from this study that 2,2′-dipyridylamine as an ancillary ligand with a hydrogen-bonding motif has a better binding constant and antitumor activity than the other anCillary ligands and so it has significant potential for the design of new metal-based drugs.
Abstract: Four new ruthenium(II) polypyridyl complexes—[Ru(phen)2(7-F-dppz)]2+ (7-F-dppz is 7-fluorodipyrido[3,2-a:2′,3′-c]phenazine, phen is 1,10-phenanthroline), [Ru(bpy)2(7-F-dppz)]2+(2) (bpy is 2,2′-bipyridine), [Ru(dmb)2(7-F-dppz)]2+ (dmb is 4,4′-dimethyl-2,2′-bipyridine), and [Ru(hdpa)2(7-F-dppz)]2+ (hdpa is 2,2′-dipyridylamine)—have been synthesized and characterized. Their DNA binding behavior has been explored by various spectroscopic titrations and viscosity measurements, which indicated that all the complexes bind to calf thymus DNA by means of intercalation with different binding strengths. The light switching properties of these complexes have been evaluated, and their antimicrobial activities have been investigated. Photoinduced DNA cleavage studies have been performed. All the complexes exhibited efficient photocleavage of pBR322 DNA on irradiation. The cytotoxicity of these complexes has been evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with various tumor cell lines. Cellular uptake was studied by flow cytometry and confocal microscopy. Flow cytometry experiments showed that these complexes induced apoptosis of HeLa cell lines.
49 citations
TL;DR: Derivative X-ray absorption spectra suggest that mineral complexation in the transfer region of ferritin-expressing grains is quite different from that in wild-type grain, which may explain why the raised levels of minerals transported to the developing grain accumulate within the crease region of the transgenic grain.
Abstract: We have used synchrotron-based X-ray fluorescence and absorption techniques to establish both metal distribution and complexation in mature wheat grains. In planta, extended X-ray absorption fine structure (EXAFS) spectroscopy reveals iron phytate and zinc phytate structures in aleurone cells and in modified aleurone cells in the transfer region of the grain: iron is coordinated octahedrally by six oxygen atoms and fewer than two phosphorous atoms. Zinc is coordinated tetrahedrally by four oxygen atoms and approximately 1.5 phosphorus atoms in an asymmetric coordination shell. We also present evidence of modified complexation of both metals in transgenic grain overexpressing wheat ferritin. For zinc, there is a consistent doubling of the number of complexing phosphorus atoms. Although there is some EXAFS evidence for iron phytate in ferritin-expressing grain, there is also evidence of a structure lacking phosphorus. This change may lead to an excess of phosphorus within the storage regions of grain, and in turn to the demonstrated increased association of phosphorus with zinc in ferritin-expressing grains. Derivative X-ray absorption spectra also suggest that mineral complexation in the transfer region of ferritin-expressing grains is quite different from that in wild-type grain. This may explain why the raised levels of minerals transported to the developing grain accumulate within the crease region of the transgenic grain.
47 citations
TL;DR: This study contributes to the understanding of the functional role of the CIAPIN1 domain in the anamorsin family, suggesting that only the [2Fe–2S] cluster bound to the CX8CX2CXC motif is indispensable in the electron transfer chain assembling cytosolic Fe/S proteins.
Abstract: The eukaryotic anamorsin protein family, which has recently been proposed to be part of an electron transfer chain functioning in the early steps of cytosolic iron–sulfur (Fe/S) protein biogenesis, is characterized by a largely unstructured domain (CIAPIN1) containing two conserved cysteine-rich motifs (CX8CX2CXC and CX2CX7CX2C) whose Fe/S binding properties and electronic structures are not well defined. Here, we found that (1) each motif in human anamorsin is able to bind independently a [2Fe–2S] cluster through its four cysteine residues, the binding of one cluster mutually excluding the binding of the second, (2) the reduced [2Fe–2S]+ clusters exhibit a unique electronic structure with considerable anisotropy in their coordination environment, different from that observed in reduced, plant-type and vertebrate-type [2Fe–2S] ferredoxin centers, (3) the reduced cluster bound to the CX2CX7CX2C motif reveals an unprecedented valence localization-to-delocalization transition as a function of temperature, and (4) only the [2Fe–2S] cluster bound to the CX8CX2CXC motif is involved in the electron transfer with its physiological protein partner Ndor1. The unique electronic properties of both [2Fe–2S] centers can be interpreted by considering that both cysteine-rich motifs are located in a highly unstructured and flexible protein region, whose local conformational heterogeneity can induce anisotropy in metal coordination. This study contributes to the understanding of the functional role of the CIAPIN1 domain in the anamorsin family, suggesting that only the [2Fe–2S] cluster bound to the CX8CX2CXC motif is indispensable in the electron transfer chain assembling cytosolic Fe/S proteins.
47 citations
TL;DR: The results suggest the impact of residue 41 on the conformation of cytochrome c influences its ability to act in both of its physiological roles, electron transport and caspase activation.
Abstract: Cytochrome c is a highly conserved protein, with 20 residues identical in all eukaryotic cytochromes c. Gly-41 is one of these invariant residues, and is the position of the only reported naturally occurring mutation in cytochrome c (human G41S). The basis, if any, for the conservation of Gly-41 is unknown. The mutation of Gly-41 to Ser enhances the apoptotic activity of cytochrome c without altering its role in mitochondrial electron transport. Here we have studied additional residue 41 variants and determined their effects on cytochrome c functions and conformation. A G41T mutation decreased the ability of cytochrome c to induce caspase activation and decreased the redox potential, whereas a G41A mutation had no impact on caspase induction but the redox potential increased. All residue 41 variants decreased the pK
a of a structural transition of oxidized cytochrome c to the alkaline conformation, and this correlated with a destabilization of the interaction of Met-80 with the heme iron(III) at physiological pH. In reduced cytochrome c the G41T and G41S mutations had distinct effects on a network of hydrogen bonds involving Met-80, and in G41T the conformational mobility of two Ω-loops was altered. These results suggest the impact of residue 41 on the conformation of cytochrome c influences its ability to act in both of its physiological roles, electron transport and caspase activation.
46 citations
TL;DR: The citrate ligand has a significantly extended structure compared with previously reported ligands co-crystallized with urease and thus represents a unique and promising scaffold for the design of new, highly active, stable, selective inhibitors.
Abstract: Urease, the enzyme that catalyses the hydrolysis of urea, is a virulence factor for a large number of ureolytic bacterial human pathogens. The increasing resistance of these pathogens to common antibiotics as well as the need to control urease activity to improve the yield of soil nitrogen fertilization in agricultural applications has stimulated the development of novel classes of molecules that target urease as enzyme inhibitors. We report on the crystal structure at 1.50-A resolution of a complex formed between citrate and urease from Sporosarcina pasteurii, a widespread and highly ureolytic soil bacterium. The fit of the ligand to the active site involves stabilizing interactions, such as a carboxylate group that binds the nickel ions at the active site and several hydrogen bonds with the surrounding residues. The citrate ligand has a significantly extended structure compared with previously reported ligands co-crystallized with urease and thus represents a unique and promising scaffold for the design of new, highly active, stable, selective inhibitors.
TL;DR: It is suggested that magnetite changes microtubule dynamics and polymerization through two paths: changing the secondary and tertiary structure of tubulin and binding to either tubulin dimer or tau protein and preventing tau–tubulin interaction.
Abstract: In recent decades, considerable efforts have been made to understand the mechanism of memory, cognition, and relevant neurodegenerative diseases in the human brain. Several studies have shown the importance of microtubule proteins in the memory mechanism and memory dysfunction. Microtubules possess dynamicity, which is essential for functions of neuronal networks. Microtubule-associated proteins, i.e., tau, play vital roles in microtubule stability. On the other hand, the ferromagnetic mineral magnetite (Fe3O4) has been detected in the normal human brain, and elevated levels of magnetite are also observed in the brains of Alzheimer’s disease patients. Therefore, we propose that a relationship between microtubule organization in axons and brain magnetite nanoparticles is possible. In this study we found alterations of microtubule polymerization in the presence of increasing concentrations of magnetite through transmission electron microscopy images and a turbidimetry method. Structural changes of microtubule and tau protein, as an essential microtubule-associated protein for tubulin assembly, were detected via circular dichroism spectroscopy, intrinsic fluorescence, and 8-anilino-1-naphthalenesulfonic acid fluorometry. We predicted three possible binding sites on tau protein and one possible binding site on tubulin dimer for magnetite nanoparticles. Magnetite also causes the morphology of PC12 cells to change abnormally and cell viability to decrease. Finally, we suggest that magnetite changes microtubule dynamics and polymerization through two paths: (1) changing the secondary and tertiary structure of tubulin and (2) binding to either tubulin dimer or tau protein and preventing tau–tubulin interaction.
TL;DR: The crystal structure of AoCO4 diverges from that of enzymes belonging to the conventional tyrosinase family in several ways, particularly around the central α-helical core region, which has been reported to be typical of the oxy form of type 3 copper enzymes.
Abstract: Catechol oxidases (EC 1.10.3.1) catalyse the oxidation of o-diphenols to their corresponding o-quinones. These oxidases contain two copper ions (CuA and CuB) within the so-called coupled type 3 copper site as found in tyrosinases (EC 1.14.18.1) and haemocyanins. The crystal structures of a limited number of bacterial and fungal tyrosinases and plant catechol oxidases have been solved. In this study, we present the first crystal structure of a fungal catechol oxidase from Aspergillus oryzae (AoCO4) at 2.5-A resolution. AoCO4 belongs to the newly discovered family of short-tyrosinases, which are distinct from other tyrosinases and catechol oxidases because of their lack of the conserved C-terminal domain and differences in the histidine pattern for CuA. The sequence identity of AoCO4 with other structurally known enzymes is low (less than 30 %), and the crystal structure of AoCO4 diverges from that of enzymes belonging to the conventional tyrosinase family in several ways, particularly around the central α-helical core region. A diatomic oxygen moiety was identified as a bridging molecule between the two copper ions CuA and CuB separated by a distance of 4.2–4.3 A. The UV/vis absorption spectrum of AoCO4 exhibits a distinct maximum of absorbance at 350 nm, which has been reported to be typical of the oxy form of type 3 copper enzymes.
TL;DR: Combining the findings of this study and those of recent animal and human studies confirms that blood iron isotopic patterns may serve as a novel compound biomarker of iron metabolism to assess impairments in regulation of intestinal iron absorption in individuals or population groups.
Abstract: Persistent impairments in the regulation of intestinal iron absorption result in iron deficiency or iron accumulation in the long term. Diagnosis remains difficult unless pathological symptoms develop as iron absorption varies strongly between meals and days. Variations in the natural iron isotopic composition of whole blood have recently been suggested as a novel parameter to assess long-term differences in intestinal absorption efficiency between individuals. In this study, baseline blood samples collected in two previous conventional iron absorption studies in Swiss and Thai women using stable isotope tracers were reanalyzed by multicollector inductively coupled plasma mass spectrometry. The natural iron isotopic compositions obtained were compared with fractional absorption from the test meals observed in these earlier trials. Correlations of natural blood iron isotopic composition and fractional absorption from the test meals were found to be highly significant in both cohorts (for Swiss women, r = 0.40, P = 0.01, n = 38; for Thai women, r = 0.57, P < 0.01, n = 24), with the blood of both ethnicities clearly differing in iron isotopic composition (P < 0.001). Combining the findings of this study and those of recent animal and human studies confirms that blood iron isotopic patterns may serve as a novel compound biomarker of iron metabolism to assess impairments in regulation of intestinal iron absorption in individuals or population groups.
TL;DR: Insight is provided into the use of PMIDA coated CoO nanoparticles as antigen delivery vehicles by upregulating IFN-γ and TNF-α and induce an anticancer immune response by activating macrophages.
Abstract: The purpose of this study is to evaluate the prospect of using surface modified cobalt oxide(CoO) nanoparticles as carriers of cancerantigens to human macrophages. N-Phosnomethyliminodiacetic acid (PMIDA) was used for surface modification to overcome the toxic effect of CoO nanoparticles. Here, the phosphonate group of the PMIDA acts as a surface-anchoring agent and the remaining –COOH groups bind nonspecifically with tumor associated antigens. This modification allows the conjugation of human oral carcinoma (KB) cell lysate (CL) as an antigen with PMIDA coated CoO nanoparticles (CL–PMIDA–CoO). Particle characterization was performed by dynamic light scattering, atomic force microscopy, and scanning electron microscopy studies. Fourier transform IR spectroscopy was used to investigate conjugation of the protein with nanoparticles. Protein encapsulation was confirmed by protein gel electrophoresis. Active uptake of antigen-conjugated nanoparticles by macrophages was confirmed by fluorescence microscopy. The antitumor activity of the nanocomplex pulsed macrophages was investigated on a human oral carcinoma cell line (KB) in vitro. The modified nanocomplexes upregulate IFN-γ and TNF-α and induce an anticancer immune response by activating macrophages. The use of TNF-α inhibitor confirmed the ability of the CL–PMIDA–CoO nanocomplex to stimulate TNF-α mediated immunostimulation. CL–PMIDA–CoO nanoparticles efficiently increased the CD4+ population. Thus, our findings provide insight into the use of PMIDA coated CoO nanoparticles as antigen delivery vehicles.
TL;DR: Higher basal ATP level in cisplatin-resistant cells, which appears to be a consequence of enhanced mitochondrial ATP production, may represent a survival mechanism established during development of resistance.
Abstract: Decreased cellular accumulation of cisplatin is a frequently observed mechanism of resistance to the drug. Beside passive diffusion, several cellular proteins using ATP hydrolysis as an energy source are assumed to be involved in cisplatin transport in and out of the cell. This investigation aimed at clarifying the contribution of intracellular ATP as an indicator of energy-dependent transport to cisplatin resistance using the A2780 human ovarian adenocarcinoma cell line and its cisplatin-resistant variant A2780cis. Depletion of intracellular ATP with oligomycin significantly decreased cellular platinum accumulation (measured by flameless atomic absorption spectrometry) in sensitive but not in resistant cells, and did not affect cisplatin efflux in both cell lines. Inhibition of Na+,K+-ATPase with ouabain reduced platinum accumulation in A2780 cells but to a lesser extent compared with oligomycin. Western blot analysis revealed lower expression of Na+,K+-ATPase α1 subunit in resistant cells compared with sensitive counterparts. The basal intracellular ATP level (determined using a bioluminescence-based assay) was significantly higher in A2780cis cells than in A2780 cells. Our results highlight the importance of ATP-dependent transport, among other processes mediated by Na+,K+-ATPase, for cisplatin influx in sensitive cells. Cellular platinum accumulation in resistant cells is reduced and less dependent on energy sources, which may partly result from Na+,K+-ATPase downregulation. Our data suggest the involvement of other ATP-dependent processes beside those regulated by Na+,K+-ATPase. Higher basal ATP level in cisplatin-resistant cells, which appears to be a consequence of enhanced mitochondrial ATP production, may represent a survival mechanism established during development of resistance.
TL;DR: The binding of VIVO2+ to human serum transferrin (hTF) at the FeIII binding sites is addressed and the one that yields the lowest calculated heats of formation and minimum deviations from the experimental values of the 51V and 14N A tensor components is the structure that includes CO32− as a synergistic anion.
Abstract: The binding of VIVO2+ to human serum transferrin (hTF) at the FeIII binding sites is addressed. Geometry optimization calculations were performed for the binding of VIVO2+ to the N-terminal lobe of hTF (hTFN), and indicate that in the presence of CO3
2− or HCO3
−, VIV is bound to five atoms in a distorted geometry. The structures of VIVO–hTFN species optimized at the semiempirical level were also used to calculate the 51V and 14N A tensors by density functional theory methods, and were compared with the reported experimental values. Globally, of all the calculated VIVO–hTF structures, the one that yields the lowest calculated heats of formation and minimum deviations from the experimental values of the 51V and 14N A tensor components is the structure that includes CO3
2− as a synergistic anion. In this structure the V=O bond length is approximately 1.6 A, and the vanadium atom is also coordinated to the phenolate oxygen atom of Tyr188 (at approximately 1.9 A), the aspartate oxygen atom of Asp63 (at approximately 1.9 A), the His249 Nτ atom (at approximately 2.1 A), and a carbonate oxygen atom (at approximately 1.8 A). The Tyr95 phenolic ocygen atom is approximately 3.3 A from the metal center, and thus is very weakly bound to VIV. All of these oxygen atoms are able to establish dipolar interactions with groups of the protein.
TL;DR: In this paper, a database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL, which is a family of metalloenzymes that are capable of hydrolyzing β-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics.
Abstract: Metallo-β-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing β-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK a values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn(2+) center is formed only once the substrate is present.
TL;DR: A mechanism of copper accumulation by the enzyme as well as an approach to changing the selectivity of TyrBm towards l-dopa production are proposed.
Abstract: Tyrosinase belongs to the type 3 copper enzyme family, containing a dinuclear copper center, CuA and CuB. It is mainly responsible for melanin production in a wide range of organisms. Although copper ions are essential for the activity of tyrosinase, the mechanism of copper uptake is still unclear. We have recently determined the crystal structure of tyrosinase from Bacillus megaterium (TyrBm) and revealed that this enzyme has tighter binding of CuA in comparison with CuB. Investigating copper accumulation in TyrBm, we found that the presence of copper has a more significant effect on the diphenolase activity. By decreasing the concentration of copper, we increased the diphenolase to monophenolase activity ratio twofold. Using a rational design approach, we identified five variants having an impact on copper uptake. We have found that a major role of the highly conserved Asn205 residue is to stabilize the orientation of the His204 imidazole ring in the binding site, thereby promoting the correct coordination of CuB. Further investigation of these variants revealed that Phe197, Met61, and Met184, which are located at the entrance to the binding site, not only play a role in copper uptake, but are also important for enhancing the diphenolase activity. We propose a mechanism of copper accumulation by the enzyme as well as an approach to changing the selectivity of TyrBm towards l-dopa production.
TL;DR: The combined results demonstrate the importance of both free ferrihydrite, a natural environmental form of iron and a model for an insoluble form of partly denatured ferritin called hemosiderin, and caged Ferritin iron minerals as bacterial iron sources.
Abstract: Metabolism of iron derived from insoluble and/or scarce sources is essential for pathogenic and environmental microbes. The ability of Pseudomonas aeruginosa to acquire iron from exogenous ferritin was assessed; ferritin is an iron-concentrating and antioxidant protein complex composed of a catalytic protein and caged ferrihydrite nanomineral synthesized from Fe(II) and O2 or H2O2. Ferritin and free ferrihydrite supported growth of P. aeruginosa with indistinguishable kinetics and final culture densities. The P. aeruginosa PAO1 mutant (ΔpvdDΔpchEF), which is incapable of siderophore production, grew as well as the wild type when ferritin was the iron source. Such data suggest that P. aeruginosa can acquire iron by siderophore-independent mechanisms, including secretion of small-molecule reductant(s). Protease inhibitors abolished the growth of the siderophore-free strain on ferritins, with only a small effect on growth of the wild type; predictably, protease inhibitors had no effect on growth with free ferrihydrite as the iron source. Proteolytic activity was higher with the siderophore-free strain, suggesting that the role of proteases in the degradation of ferritin is particularly important for iron acquisition in the absence of siderophores. The combined results demonstrate the importance of both free ferrihydrite, a natural environmental form of iron and a model for an insoluble form of partly denatured ferritin called hemosiderin, and caged ferritin iron minerals as bacterial iron sources. Ferritin is also revealed as a growth promoter of opportunistic, pathogenic bacteria such a P. aeruginosa in diseased tissues such as the cystic fibrotic lung, where ferritin concentrations are abnormally high.
TL;DR: The results suggest that vanadyl complexes may upregulate PPARγ by suppressing PParγ degradation, and thus stimulate adiponectin expression and multimerization.
Abstract: Vanadium compounds are promising agents in the therapeutic treatment of diabetes mellitus, but their mechanism of action has not been fully elucidated. The current work investigated the effects of vanadyl acetylacetonate, VO(acac)2, on peroxisome-proliferator-activated receptor γ (PPARγ) and adiponectin, which are important targets of antidiabetic drugs. The experimental results revealed that vanadyl complexes increased the expression and multimerization of adiponectin in differentiated rat adipocytes. VO(acac)2 caused activation of p38 mitogen-activated protein kinase (MAPK) and AMP-activated protein kinase (AMPK) and elevation of PPARγ levels. The specific inhibitors SB203580 (p38 MAPK inhibitor) and T0070907 (PPARγ inhibitor) decreased the expression of adiponectin; however, compound C (AMPK inhibitor) did not significantly reduce the expression of adiponectin. In addition, vanadyl complexes induced protein–protein interaction between PPARγ and a vanadium-binding chaperone, heat shock protein 60 kDa. Overall, our results suggest that vanadyl complexes may upregulate PPARγ by suppressing PPARγ degradation, and thus stimulate adiponectin expression and multimerization. The present work has provided new insights into the mechanism of the antidiabetic actions of vanadium compounds.
TL;DR: It is demonstrated that in the disulfide-containing wild-type protein cyanide ligation is fivefold faster for one of the two heme orientations compared with the other isomer, which is attributed to the lower stability of the distal His64–iron bond and reduced steric hindrance at the bottom of the cavity for heme sliding in the A conformer.
Abstract: Neuroglobin (Ngb) is a globin present in the brain and retina of mammals. This hexacoordinated hemoprotein binds small diatomic molecules, albeit with lower affinity compared with other globins. Another distinctive feature of most mammalian Ngb is their ability to form an internal disulfide bridge that increases ligand affinity. As often seen for prosthetic heme b containing proteins, human Ngb exhibits heme heterogeneity with two alternative heme orientations within the heme pocket. To date, no details are available on the impact of heme orientation on the binding properties of human Ngb and its interplay with the cysteine oxidation state. In this work, we used 1H NMR spectroscopy to probe the cyanide binding properties of different Ngb species in solution, including wild-type Ngb and the single (C120S) and triple (C46G/C55S/C120S) mutants. We demonstrate that in the disulfide-containing wild-type protein cyanide ligation is fivefold faster for one of the two heme orientations (the A isomer) compared with the other isomer, which is attributed to the lower stability of the distal His64–iron bond and reduced steric hindrance at the bottom of the cavity for heme sliding in the A conformer. We also attribute the slower cyanide reactivity in the absence of a disulfide bridge to the tighter histidine–iron bond. More generally, enhanced internal mobility in the CD loop bearing the disulfide bridge hinders access of the ligand to heme iron by stabilizing the histidine–iron bond. The functional impact of heme disorder and cysteine oxidation state on the properties of the Ngb ligand is discussed.
TL;DR: The results show that CuNS has a greater ability to stabilize G-quadruplex DNA over calf-thymus DNA, and they showed significant potential for antineoplastic effects.
Abstract: A novel naphthalene-2,3-diamine-2-salicylaldehyde (NS) ligand and its mononuclear copper(II) complex (CuNS) have been synthesized and structurally characterized. The UV–vis absorption and emission spectra of NS showed obvious changes on addition of Cu2+ solution. The interaction of the compounds with calf thymus DNA and G-quadruplex DNA were investigated by spectroscopic methods and thermal melting assay. The nucleolytic cleavage activity of the compounds was investigated on double-stranded circular pBR322 plasmid DNA and G-quadruplex DNA by electrophoretic mobility shift assay. The results show that CuNS has a greater ability to stabilize G-quadruplex DNA over calf-thymus DNA. The cytotoxicity of the compounds toward HpeG2 cancer cells was also studied, and they showed significant potential for antineoplastic effects.
TL;DR: The results indicate that both complexes show some degree of binding to DNA in an intercalative mode, resulting in intrinsic binding constants of (2.88 ± 0.4) × 104 and (5.38 ±-0.8) –104 for 1 and 2, respectively.
Abstract: Two new platinum(II) complexes, [Pt(bpy)(pip)](NO3)2 (1) and [Pt(bpy)(hpip)](NO3)2·2H2O (2) (bpy is 2,2′-bypyridine; pip is 2-phenylimidazo[4,5-f][1,10]phenanthroline; hpip is 2-(2-hydroxyphenyl) imidazo[4,5-f][1,10]phenanthroline), have been synthesized and fully characterized by CHN analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 1H-NMR spectroscopy, IR spectroscopy (attenuated total reflection), and UV–vis spectroscopy. The DNA-binding behaviors of both complexes have been studied by spectroscopic methods and viscosity measurements, and their ability to inhibit DNA transcription was measured. The results indicate that both complexes show some degree of binding to DNA in an intercalative mode, resulting in intrinsic binding constants of (2.88 ± 0.4) × 104 and (5.38 ± 0.8) × 104 for 1 and 2, respectively. The comparatively observed difference in the DNA-binding affinities of the two complexes can be reasonably explained by the presence of intramolecular hydrogen bonding between the ortho phenolic group and the nitrogen atom of the imidazole ring. The extended coplanarity of the hpip ligand due to intramolecular hydrogen bonding may lead to an enhancement of the DNA-binding affinity of the hpip complex. In addition, the complexes can promote photocleavage of pUC19 DNA on irradiation as revealed by the spectroscopic and viscometric measurements, with 2 promoting cleavage of pUC19 DNA at lower concentration. Moreover, increasing concentrations of both complexes inhibited DNA transcription, and as expected 2 was shown to be a better antitumor agent than 1.
TL;DR: Water-soluble conjugates between dopamine-derived melanin and bovine serum albumin (BSA) were synthesized to understand the interaction between the pigment and this metal ion, and appear to be good models for the natural pigment.
Abstract: Elucidating the structure and biosynthesis of neuromelanin (NM) would be an important step towards understanding its putative role in the pathogenesis of Parkinson’s disease. A useful complement to studies aimed at unraveling the origin and properties of this essentially insoluble natural substance is the preparation of synthetic derivatives that resemble NM. With this aim in mind, water-soluble conjugates between dopamine-derived melanin and bovine serum albumin (BSA) were synthesized. Melanin–BSA adducts were prepared with both eumelanic oligomers obtained through the oxidative polymerization of dopamine and pheomelanic oligomers obtained under the same conditions from dopamine and cysteine. Iron ions were added during the synthesis to understand the interaction between the pigment and this metal ion, as the NM in neurons in several human brain regions contains significant amounts of iron. The structures of the conjugates were analyzed by 1H NMR spectroscopy and controlled proteolysis/MS experiments. The binding of iron(III) ions was evaluated by ICP analysis and EPR spectroscopy. The EPR signal from bound iron(III) indicated high-spin octahedral sites and, as also seen for NM, the signal is coupled to a signal from a radical associated with the melanic components of the conjugates. However, the intensity of the EPR signal from iron suggested a reduced fraction of the total iron, indicating that most of the iron is strongly coupled in clusters within the matrix. The amount of paramagnetic, mononuclear iron(III) was greater in the pheomelanin–BSA conjugates, suggesting that iron clustering is reduced in the sulfur-containing pigment. Thus, the melanin–BSA conjugates appear to be good models for the natural pigment.
TL;DR: This work finds that when overexpressed, most of the 17 Drosophila Zip and ZnT genes modify the zinc toxicity phenotypes in a manner consistent with their predicted zinc transport activity, and can reconcile that activity with the cellular localization of an enhanced green fluorescent protein tagged version of the protein.
Abstract: Members of the ZIP (SLC39A) and ZnT (SLC30A) families of transmembrane domain proteins are predicted to transport the essential transition metal zinc across membranes, regulating cellular zinc content and distribution via uptake and efflux at the outer plasma and organellar membranes. Twenty-four ZIP and ZnT proteins are encoded in mammalian genomes, raising questions of whether all actually transport zinc, whether several function together in the same tissues/cell types, and how the activity of these transporters is coordinated. To address these questions, we have taken advantage of the ability to manipulate several genes simultaneously in targeted cell types in Drosophila. Previously we reported zinc toxicity phenotypes caused by combining overexpression of a zinc uptake gene, dZip42C.1, with suppression of a zinc efflux gene, dZnT63C. Here we show that these phenotypes can be used as a sensitized in vivo system to detect subtle alterations in zinc transport activity that would be buffered in healthy cells. Using two adult tissues, the fly eye and midline (thorax/abdomen), we find that when overexpressed, most of the 17 DrosophilaZip and ZnT genes modify the zinc toxicity phenotypes in a manner consistent with their predicted zinc transport activity. In most cases, we can reconcile that activity with the cellular localization of an enhanced green fluorescent protein tagged version of the protein. Additionally, targeted suppression of each gene by RNA interference reveals several of the fly Zip and ZnT genes are required in the eye, indicating that numerous independent zinc transport genes are acting together in a single tissue.
TL;DR: complex 1 exhibited the highest interaction ability and was found to be the most potent antitumor agent among the four complexes, suggesting that these complexes bind to DNA in an intercalative mode.
Abstract: Four novel oxovanadium(IV) complexes—[VO(PAHN)(phen)] (1; PAHN is 4-pyridinecarboxylic acid, 2-[(2-hydroxy)-1-naphthalenylene] hydrazide, phen is 1,10-phenanthroline), [VO(PAHN)(bpy)] (2; bpy is 2,2′-bipyridine), [VO(PAH)(phen)] (3; PAH is 4-pyridinecarboxylic acid, 2-[(2-hydroxy)-1-phenyl]methylene hydrazide), and [VO(PAH)(bpy)] (4)—have been synthesized and characterized by elemental analysis, UV–vis spectroscopy, electrospray ionization mass spectrometry, IR spectroscopy, 1H-NMR spectroscopy, and 13C-NMR spectroscopy. Their interactions with calf thymus DNA were investigated. The results suggest that these complexes bind to DNA in an intercalative mode. All four complexes exhibited highly cytotoxic activity against tumor cells (SH-SY5Y, MCF-7, and SK-N-SH), with 50 % inhibitory concentrations of the same order of magnitude as for cisplatin or of lower order of magnitude. Complex 1 exhibited the highest interaction ability and was found to be the most potent antitumor agent among the four complexes. It can cause G2/M phase arrest of the cell cycle, induces significant apoptosis in SK-N-SH cells, and displays typical morphological apoptotic characteristics. In addition, their hydroxyl radical scavenging properties have been tested, and complex 1 was the best inhibitor.
TL;DR: The data indicate that the higher cytotoxicity of 1 and 2 originates mainly from their efficient cellular accumulation, different effects at the level of cell cycle regulation, and reduced propensity for DNA adduct repair, and that direct analogues of conventional cisplatin-containing halogeno-substituted 7-azaindoles offer much promise for the design of novel therapeutic agents.
Abstract: The cisplatin analogues cis-[PtCl2(3ClHaza)2] (1) and cis-[PtCl2(3IHaza)2] (2) (3ClHaza and 3IHaza are 3-chloro-7-azaindole and 3-iodo-7-azaindole, respectively) are quite toxic to ovarian tumor cells, with moderately better IC50 values than for cisplatin in the cisplatin-sensitive cell line A2780. We investigated potential factors which might be involved in the mechanism underlying the cytotoxic effects of 1 and 2 and compared these factors with those involved in the mechanism underlying the effects of conventional cisplatin. Our data indicate that the higher cytotoxicity of 1 and 2 originates mainly from their efficient cellular accumulation, different effects at the level of cell cycle regulation, and reduced propensity for DNA adduct repair. Studies of their reactivity toward cellular components reveal efficient binding to DNA, which is typically required for an active platinum drug. Further results suggest that 1 and 2 are capable of circumventing resistance to cisplatin induced by alterations in cellular accumulation and DNA repair. Hence, the latter two factors appear to be responsible for differences in the toxicity of 1 or 2, and cisplatin in tumor cells. The results of this work reinforce the idea that direct analogues of conventional cisplatin-containing halogeno-substituted 7-azaindoles offer much promise for the design of novel therapeutic agents.
TL;DR: The structure of the oxidized form of PHM complexed with hydrogen peroxide is determined and the geometry of the observed side-on coordinated peroxide ligand in L3CuM(II)O22− is in good agreement with the results of a hybrid quantum mechanical–molecular mechanical optimization of this species.
Abstract: Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine α-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction—the hydroxylation of peptidylglycine substrates at the Cα position of the terminal glycine. The hydroxylation reaction is copper- and O2-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests that O2 is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (CuH and CuM). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key intermediates in the PHM cycle, we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.98-A-resolution structure (hydro)peroxide binds solely to CuM in a slightly asymmetric side-on mode. The O–O interatomic distance of the copper-bound ligand is 1.5 A, characteristic of peroxide/hydroperoxide species, and the Cu–O distances are 2.0 and 2.1 A. Density functional theory calculations using the first coordination sphere of the CuM active site as a model system show that the computed energies of the side-on L3CuM(II)–O22− species and its isomeric, end-on structure L3CuM(I)–O2·− are similar, suggesting that both these intermediates are significantly populated within the protein environment. This observation has important mechanistic implications. The geometry of the observed side-on coordinated peroxide ligand in L3CuM(II)O22− is in good agreement with the results of a hybrid quantum mechanical–molecular mechanical optimization of this species.
TL;DR: X-ray fluorescence imaging of single cultured A549 cells treated with 2 or 4 showed that, in both cases, the distribution of ruthenium and iodine was identical, indicating that the Ru–N bonds in the anionic complexes remained intact after incubation in culture medium and subsequent cellular uptake and processing.
Abstract: Analogues of KP1019 containing iodinated indazole ligands were prepared to investigate the biological fate of the Ru–N-heterocycle bond in this class of anticancer agents. The new complexes, 5-iodoindazolium trans-tetrachloridobis(5-iodoindazole)ruthen(III)ate (1) and 5-iodoindazolium trans-tetrachlorido(dimethyl sulfoxide)(5-iodoindazole)ruthen(III)ate (3), were characterized by elemental analysis, mass spectrometry and UV–vis spectrophotometry. Tetramethylammonium salts of these complexes (2 and 4) were synthesized and characterized in a similar manner. Half-maximum inhibitory concentrations of 2 and 4 with regard to A549 cells at 24 h were determined on the basis of the dose–response curves derived from real-time cell adhesion impedance measurements and were shown to be in the same range as those determined for KP1019 and NAMI-A using the same method. X-ray fluorescence imaging of single cultured A549 cells treated with 2 or 4 showed that, in both cases, the distribution of ruthenium and iodine was identical, indicating that the Ru–N bonds in the anionic complexes remained intact after incubation in culture medium and subsequent cellular uptake and processing.