Papers by Merce Capdevila
Histidine-Rich C-Terminal Tail of Mycobacterial GroEL1 and Its Copper Complex─The Impact of Point Mutations
Inorganic Chemistry
Metallomics, 2020
The tiny contribution of cadmium (Cd) to the composition of the earth’s crust contrasts with its ... more The tiny contribution of cadmium (Cd) to the composition of the earth’s crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic t...
Molecular Biology and Evolution, 2021
To investigate novel patterns and processes of protein evolution, we have focused in the metallot... more To investigate novel patterns and processes of protein evolution, we have focused in the metallothioneins (MTs), a singular group of metal-binding, cysteine-rich proteins that, due to their high degree of sequence diversity, still represents a “black hole” in Evolutionary Biology. We have identified and analyzed more than 160 new MTs in nonvertebrate chordates (especially in 37 species of ascidians, 4 thaliaceans, and 3 appendicularians) showing that prototypic tunicate MTs are mono-modular proteins with a pervasive preference for cadmium ions, whereas vertebrate and cephalochordate MTs are bimodular proteins with diverse metal preferences. These structural and functional differences imply a complex evolutionary history of chordate MTs—including de novo emergence of genes and domains, processes of convergent evolution, events of gene gains and losses, and recurrent amplifications of functional domains—that would stand for an unprecedented case in the field of protein evolution.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
CCDC 1535610: Experimental Crystal Structure Determination
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Three novel dinuclear Cu(II) complexes based on a N,N,O-chelating salphen-like ligand scaffold an... more Three novel dinuclear Cu(II) complexes based on a N,N,O-chelating salphen-like ligand scaffold and bearing varying aromatic substituents (−H, −Cl, and −Br) have been synthesized and characterized. The experimental and computational data obtained suggest that all three complexes exist in the dimeric form in the solid state and adopt the same conformation. The mass spectrometry and electron paramagnetic resonance results indicate that the dimeric structure coexists with the monomeric form in solution upon solvent (dimethyl sulfoxide and water) coordination. The three synthesized Cu(II) complexes exhibit high potentiality as ROS generators, with the Cu(II)/Cu(I) redox potential inside the biological redox window, and thus being able to biologically undergo Cu(II)/Cu(I) redox cycling. The formation of ROS is one of the most promising reported cell death mechanisms for metal complexes to offer an inherent selectivity to cancer cells. In vitro cytotoxic studies in two different cancer cell lines (HeLa and MCF7) and in a normal fibroblast cell line show promising selective cytotoxicity for cancer cells (IC 50 about 25 μM in HeLa cells, which is in the range of cisplatin and improved with respect to carboplatin), hence placing this N,N,O-chelating salphen-like metallic core as a promising scaffold to be explored in the design of future tailor-made Cu(II) cytotoxic compounds.
Frontiers in Cell and Developmental Biology, 2021
Chordate Oikopleura dioica probably is the fastest evolving metazoan reported so far, and thereby... more Chordate Oikopleura dioica probably is the fastest evolving metazoan reported so far, and thereby, a suitable system in which to explore the limits of evolutionary processes. For this reason, and in order to gain new insights on the evolution of protein modularity, we have investigated the organization, function and evolution of multi-modular metallothionein (MT) proteins in O. dioica. MTs are a heterogeneous group of modular proteins defined by their cysteine (C)-rich domains, which confer the capacity of coordinating different transition metal ions. O. dioica has two MTs, a bi-modular OdiMT1 consisting of two domains (t-12C and 12C), and a multi-modular OdiMT2 with six t-12C/12C repeats. By means of mass spectrometry and spectroscopy of metal-protein complexes, we have shown that the 12C domain is able to autonomously bind four divalent metal ions, although the t-12C/12C pair –as it is found in OdiMT1– is the optimized unit for divalent metal binding. We have also shown a direct r...
Molecular Biology and Evolution, 2020
Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxificatio... more Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxification, and therefore, MTs have been crucial for the adaptation of the living beings to variable situations of metal bioavailability. The evolution of MTs is, however, not yet fully understood, and to provide new insights into it, we have investigated the MTs in the diverse classes of Mollusks. We have shown that most molluskan MTs are bimodular proteins that combine six domains—α, β1, β2, β3, γ, and δ—in a lineage-specific manner. We have functionally characterized the Neritimorpha β3β1 and the Patellogastropoda γβ1 MTs, demonstrating the metal-binding capacity of the new γ domain. Our results have revealed a modular organization of mollusk MT, whose evolution has been impacted by duplication, loss, and de novo emergence of domains. MTs represent a paradigmatic example of modular evolution probably driven by the structural and functional requirements of metal binding.
Biomolecules, 2019
Model systems constituted by proteins and unsaturated lipid vesicles were used to gain more insig... more Model systems constituted by proteins and unsaturated lipid vesicles were used to gain more insight into the effects of the propagation of an initial radical damage on protein to the lipid compartment. The latter is based on liposome technology and allows measuring the trans unsaturated fatty acid content as a result of free radical stress on proteins. Two kinds of sulfur-containing proteins were chosen to connect their chemical reactivity with membrane lipid transformation, serum albumins and metallothioneins. Biomimetic systems based on radiation chemistry were used to mimic the protein exposure to different kinds of free radical stress and Raman spectroscopy to shed light on protein structural changes caused by the free radical attack. Among the amino acid residues, Cys is one of the most sensitive residues towards the attack of free radicals, thus suggesting that metal-Cys clusters are good interceptors of reactive species in metallothioneins, together with disulfides moieties i...
Journal of Inorganic Biochemistry, 2019
Reactive oxygen species (ROS) formation appears as one of the most promising pathways to induce c... more Reactive oxygen species (ROS) formation appears as one of the most promising pathways to induce cell death. The interesting Cu(II)/Cu(I) redox pair has been reported to biologically generate ROS and induce cell damage. Simple metal complexes, such as cisplatin, sometimes offer even better properties than others highly accurately synthesized, which imply considerable time and economical efforts. This work relies on the synthesis and characterisation of four existing Cu(II) complexes bearing N-donor ligands, previously used for a totally different intend, but tested now for anticancer purposes. Furthermore, a relationship between their coordinating features, i.e. their redox behaviour, with their biological activity have been inferred to further understand the medicinal role of the Cu(II)/Cu(I) redox pair. Cytotoxicity studies and interactions towards DNA have been assessed, studying both covalent and non-covalent modes of binding via mass spectrometry (MS), UV-Vis and fluorescence, evaluating the cleaving properties of the assayed compounds, as well as their capacity to generate ROS inside the cells. The role of the ligand for one of the complexes has been evaluated by a computational approach. The idea of using "old" complexes for "novel" anticancer purposes can offer promising results in the future, being a simple but interesting approach to study, as we demonstrate here for most of the complexes analysed, showing a non-expected "new" and beneficial role.
Metallomics, 2019
MT3 can bind Pb2+ to form a variety of metallated Pb-MT3 species.
Inorganic Chemistry, 2018
Two new squaramide-based platinum(II) complexes C1 and C2 have been synthesized and fully charact... more Two new squaramide-based platinum(II) complexes C1 and C2 have been synthesized and fully characterized. Their photoresponse has been assessed and is discussed. A remarkable enhancement in the DNA binding activity has been observed for both complexes, up on irradiation. For C2, the release of Pt(II) provoked by its irradiation has been studied. The response of C2 has been found to be regulated by the presence of oxygen. In vitro cytotoxicity tests show an enhancement in the activity of complex C2 after selective irradiation under hypoxic conditions. Resulting Pt(II) species have been isolated and characterized by various analytical methods establishing this type of squaramido-based complexes as a proof of concept for new Pt(II) photocages.
Journal of inorganic biochemistry, Jan 27, 2017
The presence of a conserved cysteine residue in the C-terminal amino acid sequences of plant frat... more The presence of a conserved cysteine residue in the C-terminal amino acid sequences of plant frataxins differentiates these frataxins from those of other kingdoms and may be key in frataxin assembly and function. We report a full study on the ability of Arabidopsis (AtFH) and Zea mays (ZmFH-1 and ZmFH-2) frataxins to assemble into disulfide-bridged dimers by copper-driven oxidation and to revert to monomers by chemical reduction. We monitored the redox assembly-disassembly process by electrospray ionization mass spectrometry, electrophoresis, UV-Vis spectroscopy, and fluorescence measurements. We conclude that plant frataxins AtFH, ZmFH-1 and ZmFH-2 are oxidized by Cu2+ and exhibit redox cysteine monomer - cystine dimer interexchange. Interestingly, the tendency to interconvert is not the same for each protein. Through yeast phenotypic rescue experiments, we show that plant frataxins are important for plant survival under conditions of excess copper, indicating that these proteins m...
Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology, Jan 3, 2017
Iron metabolism is an important subject of study for undergraduate students of chemistry and bioc... more Iron metabolism is an important subject of study for undergraduate students of chemistry and biochemistry. Relevant laboratory exercises are scarce in the literature but would be very helpful in assisting students grasp key concepts. The experiment described here deals with different iron release mechanisms of two protagonists in iron metabolism: serum transferrin (Tf) and lactoferrin (Lf). Despite having very similar structures and iron-binding sites, Tf releases practically all its iron at pH 5.5 while Lf requires a significantly lower pH of 3. This difference in behavior is directly related to their respective biological functions as Tf blood-borne iron into the cell, while Lf competes with pathogens to sequester iron in biological fluids at more acidic pHs. During this experiment, the students will carry out iron loading and unloading on both human Lf and Tf and monitor the iron release at different pHs using UV-Vis spectroscopy. With this simple approach, the students will dis...
Protein Engineering Design and Selection, 1999
The β domain of mouse metallothionein 1 (βMT) was synthesized in Escherichia coli cells grown in ... more The β domain of mouse metallothionein 1 (βMT) was synthesized in Escherichia coli cells grown in the presence of copper or cadmium. Homogenous preparations of Cu-βMT and Cd-βMT were used to characterize the corresponding in vivo-conformed metal-clusters, and to compare them with the species obtained in vitro by metal replacement to a canonical Zn 3-βMT structure. The copper-containing βMT clusters formed inside the cells were very stable. In contrast, the nascent β peptide, although it showed cadmium binding ability, produced a highly unstable species, whose stoichiometry depended upon culture conditions. The absence of βMT protein in E.coli proteaseproficient hosts grown in cadmium-supplemented medium pointed to drastic proteolysis of a poorly folded β peptide, somehow enhanced by the presence of cadmium. Possible functional and evolutionary implications of the bioactivity of mammalian βMT in the presence of monovalent and divalent metal ions are discussed.
PLoS ONE, 2012
Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaini... more Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd-thioneins or Cu-thioneins, according to the ecophysiological needs of each kind of organisms.
Journal of Biological Chemistry, 2001
We report the synthesis and characterization of a Homarus americanus MT-cDNA (MTH) through retrot... more We report the synthesis and characterization of a Homarus americanus MT-cDNA (MTH) through retrotranscription of MTH-mRNA from metal-injected lobsters. Heterologous Escherichia coli expression in zinc-and copper-supplemented medium was achieved for MTH, the two domains MTH and ␣MTH and three site-directed mutants, C9H, ␣C37H, and ␣E31C/T34C. The in vivo conformed metal complexes and the in vitro substituted cadmium aggregates were characterized. Major stoichiometries of M II 6-MTH for the entire MTH and M II 3-MTH and M II 3-␣MTH for the independent domains fully validated our expression system. A low affinity binding site for a seventh Zn(II) in the in vivo synthesized MTH was located in the ␣ domain. Additionally, minor M II 4 species were found for each domain. Both single Cys to His mutations exhibited a similar reduction of their in vivo zinc binding ability but differed in their cadmium binding behavior when compared with the wild-type forms. Conversely, the double mutant showed an enhanced zinc and cadmium binding capacity. In vivo synthesis of MTH and of its independent domains in the presence of copper only afforded heterometallic copper-zinc species. These findings allow consideration of MTH as a zinc thionein and question the view of all crustacea MT structures as copper thioneins. Furthermore, a new approach for the evolutionary and functional classification of MT is proposed, based on the stoichiometry of metal-MT species and molecular phylogenetic analysis.
Chemical Communications, 2011
Reaction of ferritin with MTs: Horse spleen ferritin (56 mg/ml) was obtained from Sigma-Aldrich a... more Reaction of ferritin with MTs: Horse spleen ferritin (56 mg/ml) was obtained from Sigma-Aldrich and passed down a Sephadex G-25 column to remove any free iron. Aqueous solutions of proteins were prepared using water purified through the Milli-Q system. Ferritin (0.1 mg/ml, 21 μM in Fe) was incubated at room temperature with each Zn-MTx preparation (1 μM) in the presence of an excess of ferrozine. The experiments were carried out in anaerobic conditions in a glove box and the solutions transferred to screw-cap quartz cuvettes. The development of the iron(II)-ferrozine complex was followed by UV-visible spectroscopy using a Thermospectronic UV300 spectrophotometer against reference containing solutions containing Milli-Q water. Spectra were recorded during 24 h. The concentrations of [Fe(ferrozine) 3 ] 4after 24 h were calculated from the UV-Vis absorbance values at 562 nm using ε 562 = 27.900 mM-1 cm-1. To determine the Zn(II) concentration, the resulting Ft-MTx solutions were filtered and then exhaustively dialyzed at 4 ºC for two days against several changes of Milli-Q water using a dialysis bag with a molecular weight cutoff of 5.000 Da. After 48 h, the water of the dialysis reservoirs (100 ml) were collected and used to determine the Zn(II) concentration by atomic absorption spectrometry. Likewise the Zn(II) concentration of the dialyzed solution was also determined by atomic absorption spectrometry. Two blank experiments were performed at the same conditions either in the absence of ferritin or the Zn-MT complex. In the absence of Zn-MT, the ferritin-ferrozine solution did not show any absorption at 562 nm in the UV-Visible spectrum. Likewise, anaerobic solutions of the Zn-MTx species did not change over time as corroborated by ESI-MS.
BMC Biology, 2011
Background The degree of metal binding specificity in metalloproteins such as metallothioneins (M... more Background The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu+ or Cd2+. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity f...
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Papers by Merce Capdevila