About
90
Publications
9,574
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
858
Citations
Publications
Publications (90)
In this paper we describe the development of a Dictyostelium discoideum strain deficient in frataxin protein (FXN). We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the D. discoideum fxn...
The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved in iron-sulfur cluster assembly. The enzyme catalyzes the desulfurization of the l-Cys substrate, producing a persulfide and l-Ala as products. In this study, we set the measurement of the product l-Ala by NMR in vitro by means of 1H NMR spectra acquisition. T...
SARS-CoV-2 was identified as the pathogenic agent causing the COVID-19 pandemic. Among the proteins codified by this virus, the Spike protein is one of the most-external and -exposed. A fragment of the Spike protein, named the receptor binding domain (RBD), interacts with the ACE2 receptors of human cells, allowing the entrance of the viruses. RBD...
SARS-CoV-2 was identified as the pathogenic agent causing the COVID-19 pandemic. Among the proteins codified by this virus, the Spike is one of the most external and exposed. A fragment of the Spike protein, named the Receptor Binding Protein (RBD) interacts with the ACE2 receptors of human cells, allowing the entrance of the viruses. RBD has been...
In this paper we describe the development of a new model system for Friedreich’s Atax- ia (FA) using Dictyostelium discoideum . We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the fxn lo...
In this work, we have designed and generated a Fe(III)-binding protein with thiol oxidoreductase activity. The consensus iron-binding motif EExxED from the frataxin protein family was grafted on a model peptide and on the surface of thioredoxin (TRX) from E. coli. We investigated metal interactions with a family of peptides containing the motif EEx...
Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigate...
Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigate...
The receptor binding domain (RBD) of the Spike protein from SARS-CoV-2 is a promising candidate to develop effective COVID-19 vaccines since it can induce potent neutralizing antibodies. We have previously reported the highly efficient production of RBD in Pichia pastoris , which is structurally similar to the same protein produced in mammalian HEK...
Frataxin (FXN) is a highly conserved mitochondrial protein whose deficiency causes Friedreich’s ataxia, a neurodegenerative disease. The precise physiological function of FXN is still unclear; however, there is experimental evidence that the protein is involved in biosynthetic iron–sulfur cluster machinery, redox imbalance, and iron homeostasis. FX...
In humans, the loss of frataxin results in Friedreich's Ataxia, a neurodegenerative disease, in which a deficit in the iron–sulfur cluster assembly is observed. In this work, we analyzed three frataxin variants in which one tryptophan was replaced by a glycine: W155G, W168G and W173G. As expected, given its localization in the assembly site, W155G...
We covalently coupled the RBD domain of SARS-CoV-2 produced in Pichia pastoris to a decameric carrier to produce a potent immunogen
Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it...
Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich’s ataxia. Frataxin’s actual physiological function has been debated for a long time without reaching a general agreement; however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulphur cluster (ISC) machinery,...
The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell syste...
The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein
production. In this work we compared the conformation of the receptor binding domain (RBD)
from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in
P. pastoris and in the well established HEK-293T mammalian cell syste...
The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cell...
Several biological activities depend on iron-sulfur clusters ([Fe-S]). Even though they are well-known in several organisms their function and metabolic pathway were poorly understood in the majority of the organisms. We propose to use the amoeba Dictyostelium discoideum, as a biological model to study the biosynthesis of [Fe-S] at the molecular, c...
The relationships between conformational dynamics, stability and protein function are not obvious. Frataxin (FXN) is an essential protein that forms part of a supercomplex dedicated to the iron-sulfur (Fe–S) cluster assembly within the mitochondrial matrix. In humans, the loss of FXN expression or a decrease in its functionality results in Friedrei...
Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally...
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally...
In recent years the mammalian mitochondrial protein complex for iron-sulfur cluster assembly has been the focus of major studies. This is partly because of its high relevance in cell metabolism, but also because mutations of the involved proteins are the cause of several human diseases. Cysteine desulfurase NFS1 is the key enzyme of the complex. At...
Protein dynamics, folding, and thermodynamics represent a central aspect of biophysical chemistry. pH, temperature, and denaturant perturbations inform our understanding of diverse contributors to stability and rates. In this work, we performed a thermodynamic analysis using a combined experimental and computational approach to gain insights into t...
Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also...
The neurodegenerative disease Friedreich ataxia results from a deficiency of frataxin, a mitochondrial protein. Most patients have a GAA expansion in the first intron of both alleles of frataxin gene, whereas a minority of them are heterozygous for the expansion and contain a mutation in the other allele. Frataxin has been claimed to participate in...
Local events that affect specific regions of proteins are of utmost relevance for stability and function. The aim of this study is to quantitatively assess the importance of locally-focused dynamics by means of a simple chemical modification procedure. Taking human Frataxin as a working model, we investigated local fluctuations of the C-terminal re...
Mammalian frataxin is a small mitochondrial protein involved in iron sulfur cluster assembly. Frataxin deficiency causes the neurodegenerative disease Friedreich’s Ataxia. Valuable knowledge has been gained on the structural dynamics of frataxin, metal-ion-protein interactions, as well as on the effect of mutations on protein conformation, stabilit...
Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can...
Peroxiredoxins are thiol‐dependent peroxidases that function in peroxide detoxification and H2O2 induced signaling. Among the six isoforms expressed in humans, PRDX1 and PRDX2 share 97% sequence similarity, 77% sequence identity including the active site, subcellular localization (cytosolic) but they hold different biological functions albeit assoc...
Iron-sulfur clusters are essential cofactors in many biochemical processes. ISD11, one of the subunits of the protein complex that carries out the cluster assembly in mitochondria, is necessary for cysteine desulfurase NFS1 stability and function. Several authors have recently provided evidence showing that ISD11 interacts with the acyl carrier pro...
Friedreich's Ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin (FXN). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN81-210 has been collected, not enough...
Human frataxin (FXN) is a highly conserved mitochondrial protein involved in iron homeostasis and activation of the iron-sulfur cluster assembly. FXN deficiency causes the neurodegenerative disease Friedreich's Ataxia. Here, we investigated the effect of alterations in loop-1, a stretch presumably essential for FXN function, on the conformational s...
Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopi...
Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant...
Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding,...
The aim of this study is to investigate the folding reaction of human frataxin, whose deficiency causes the neurodegenerative disease Friedreich’s Ataxia (FRDA). The characterization of different conformational states would provide knowledge about how frataxin can be stabilized without altering its functionality. Wild-type human frataxin and a set...
Peroxiredoxins (Prxs) constitute a ubiquitous family of Cys-dependent peroxidases that play essential roles in reducing hydrogen peroxide, peroxynitrite and organic hydroperoxides in almost all organisms. Members of the Prx subfamilies show differential oxidizing substrate specificities that await explanations at a molecular level. Among them, alky...
Thioredoxin (TRX) catalyzes redox reactions via the reversible oxidation of the conserved active center CGPC and it is involved in multiple biological processes, some of them linked to redox activity while others not. TRX is a globular, thermodynamically stable and monomeric alpha/beta protein with a structure characterized by a central beta-sheet...
Frataxin is an evolutionary conserved protein that participates in iron metabolism.
Deficiency of this small protein in humans causes a severe neurodegenerative disease known as Friedreich’s ataxia. A number of studies indicate that frataxin binds iron and regulates Fe–S cluster biosynthesis. Previous structural studies showed that metal binding o...
Iron–protein interactions are involved in electron transfer reactions. Alterations of these processes are present in a number of human pathologies; among then, in Friedreich’s ataxia, in which a deficiency in functional frataxin, an iron–binding protein, leads to the progressive neuromuscular degenerative disease. The putative iron–binding motif of...
The exposure to UV-A radiation of bovine serum albumin (BSA) in aerated aqueous solution in the presence of pterin (Ptr), results in chemical and conformational modifications of the protein. Ptr belongs to a family of heterocyclic compounds that are well-known type I (electron-transfer) and type II (singlet oxygen) photosensitizers. The evolution o...
Friedreich's Ataxia (FRDA) is linked to a deficiency of frataxin (FXN), a mitochondrial protein involved in iron-sulfur cluster synthesis. FXN is a small protein with an α/β fold followed by the C-terminal region (CTR) with a nonperiodic structure that packs against the protein core. Here, we explore the impact of the alteration of the CTR on the s...
The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42-80) is not conserved throughout evolution and, under defined experimental conditions, behaves as a random-coil. Overexpression of hFXN56-210 in Escherichia coli yields a multimer, whereas the mature form of hFXN (hFXN81-210) is monomeric. Thus, cumulative experimental evidenc...
Supplementary materials for "The role of the N-terminal tail for the oligomerization, folding and stability of human frataxin".
Adaptation of life to low temperatures influences both protein stability and flexibility. Thus, proteins from psychrophilic organisms are excellent models to study relations between these properties. Here we focused on frataxin from Psychromonas ingrahamii (pFXN), an extreme psychrophilic sea ice bacterium that can grow at temperatures as low as -1...
Frataxin (FXN) is an α/β protein that plays an essential role in iron homeostasis. Apparently, the function of human FXN (hFXN) depends on the cooperative formation of crucial interactions between helix α1, helix α2, and the C-terminal region (CTR) of the protein. In this work we quantitatively explore these relationships using a purified recombina...
Size distribution of hFXN variants as determined by DLS. Experiments were performed in batch mode at 25°C. Samples of 50–100 µL were filtered by 0.22 µm and centrifuged for 20 min at 10000 rpm at 4.0°C. Size distribution by mass was determined using isotropic spheres as the model. Distributions for hFXN90–210 and hFXN90–195 are shown in black and r...
Effect of sodium sulfate on the hFXN90–195 conformation. Far-UV CD spectra of hFXN90–210 (gray) and hFXN90–195 (orange) were acquired in the presence of 200 mM Na2SO4 or in the absence of the osmolyte (FXN90–210 in black, and hFXN90–195 in red). In addition, spectra of the unfolded states of both proteins were acquired in the presence of 5.0 M GdmC...
The application of COREX/BEST (green) and FOLDX (blue) to calculate the the unfolding probability per hFXN residue and the contribution per residue to the protein stability, respectively. In both cases the hFXN structure input used was PDBID = 1EKG. More importantly, the algorithm COREX/BEST identified the loop 1 as the section of hFXN with the hig...
Frustratograph on hFXN: local frustration calculated for pdb code 1EKG [40]. The protein backbone is displayed as gray ribbons for residues 90–195 and blue for residues 96–210. The direct inter-residue interactions with solid lines and the water-mediated interactions with dashed lines. Minimally frustrated interactions are shown in green, highly fr...
Contact matrix of the native and transition state ensembles sampled through the structure based model simulations. Each dot represents a native contact between the amino acid residue in x-axis and the amino acid residue in y-axis. The color range in the right represents the probability of formation of each contact. (A) Contact matrix for the native...
Concentration dependence analysis of sedimentation, at 42000 rpm and 20°C, of hFXN 90–195 and hFXN90–210, in 10 mM Tris-HCl, 100 mM NaCl, pH 7.0. The SV profiles of both variants at the three concentrations were analyzed in terms of one non-interacting species. The species is characterized by the absorbance signal at 280 nm (which is proportional t...
Reversibility of the temperature unfolding reactions for hFXN variants. (A) Transitions were followed by far-UV CD from 4 to 60°C and 4 to 80°C for hFXN90–195 and hFXN90–210, respectively. When proteins reach the highest temperature they were cooled to 4°C in a fast way, without control of the cooling rate. The signal recovery was 96%, and 99% for...
Folding transition for the CTR. (A) Energy profile for the CTR and the hFXN90–210 protein as a function of the native contacts formed, calculated at the melting temperature of the protein. (B) Energy profile for the CTR and hFXN90–210 as a function of the native contacts formed, calculated at the melting temperature of the CTR. (C) Calorific capaci...
Unfolding kinetics of hFXN90–210 and hFXN90–195 followed by tryptophan fluorescence emission intensity. The unfolding reactions were performed at 20°C in buffer 20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.0. GdmCl was added as the chaotropic agent. The concentrations of GdmCl in the experiment were set to establish conditions where the difference...
Analytical ultracentrifugation. Sedimentation velocity of hFXN90–195 (left panels) and hFXN90–210 (right panels) at 42000 rpm and 20°C, in 10 mM Tris-HCl, 100 mM NaCl, pH 7.0. (A) Selection of raw data for both proteins at 1 mg/mL. (B) Superposition of experimental (dots) and fitted (continuous line) profiles corrected for all systematic noise for...
Reversibility of the chemical unfolding reactions for hFXN variants. Reversibility of hFXN90–210 (A, B, C and D) and hFXN90–195 (E, F, G and H) was verified by dialysis. Proteins were incubated for three hours with urea or GdmCl at different concentrations, at room temperature, to ensure equilibrium conditions and to minimize chemical modifications...
Iron induced aggregation of hFXN90–195 (red line) and hFXN90–210 (black line) followed by light scattering. The assay was performed at 20°C in buffer 50 mM HEPES, pH 7.0. Iron (Fe3+) and protein concentration were 50 and 250 µM, respectively. At the 5 min mark, FeCl3 solution was added from a 25 mM stock solution prepared in 0.1 N HCl (arrow) and t...
Proteolytic sites observed between 20 s and 5 min in hFXN90–195 (red) and hFXN90–210 (blue), respectively. The other aromatic residues, potentially sites of chymotrypsin, are highlighted in black through hFXN90–210 amino acid sequence. hFXN variants were incubated at 25°C with chymotrypsin at mass ratios of 1∶200 (protein: protease), in buffer 20 m...
Native contacts between the CTR and the rest of the protein as studied by structure-based model simulations.
(DOC)