Publications by authors named "Adrián Velázquez-Campoy"

174 Publications

The nuclear localization sequence of the epigenetic factor RYBP binds to human importin α3.

Biochim Biophys Acta Proteins Proteom 2021 May 1;1869(8):140670. Epub 2021 May 1.

Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, GBsC-CSIC-BIFI, Universidad de Zaragoza, 50018 Zaragoza, Spain; Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009 Zaragoza, Spain; Fundación ARAID, Government of Aragón, 50018 Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), 28006 Madrid, Spain.

RYBP (Ring1 and YY1 binding protein, UniProt ID: Q8N488) is an epigenetic factor with a key role during embryonic development; it does also show an apoptotic function and an ubiquitin binding activity. RYBP is an intrinsically disordered protein (IDP), with a Zn-finger domain at its N-terminal region, which folds upon binding to DNA. It is predicted that RYBP has a nuclear localization sequence (NLS), comprising residues Asn58 to Lys83, to allow for nuclear translocation. We studied in this work the ability of intact RYBP to bind Impα3 and its truncated species, ΔImpα3, without the importin binding domain (IBB), by using fluorescence and circular dichroism (CD). Furthermore, the binding of the peptide matching the isolated NLS region of RYBP (NLS-RYBP) was also studied using the same methods and isothermal titration calorimetry (ITC), and in silico molecular docking. Moreover, we carried out experiments with NLS-RYBP in the absence or in the presence of NaCl (140 mM). Our results show that RYBP interacted with Impα3 and ΔImpα3, causing protein precipitation. The NLS-RYBP also interacted with both importin species (dissociation constant in the low micromolar range), at low or high ionic strength, as shown by intrinsic fluorescence and ITC. These findings indicate that the NLS region, which was mainly unfolded in isolation in solution, was essentially responsible for the binding of RYBP to each of the importin species. Furthermore, the molecular simulations predict that the anchoring of NLS-RYBP takes place in the major binding site for the NLS of cargo proteins bound to Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in RYBP and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbapap.2021.140670DOI Listing
May 2021

Thermal Liquid Biopsy (TLB) of Blood Plasma as a Potential Tool to Help in the Early Diagnosis of Multiple Sclerosis.

J Pers Med 2021 Apr 13;11(4). Epub 2021 Apr 13.

CNR-NANOTEC, Licryl-UOS Cosenza and CEMIF.Cal, Department of Physics, University of Calabria, 87036 Rende, Italy.

Background: Multiple sclerosis (MS) is frequently characterized by a variety of clinical signs, often exhibiting little specificity. The diagnosis requires a combination of medical observations and instrumental tests, and any support for its objective assessment is helpful.

Objective: Herein, we describe the application of thermal liquid biopsy (TLB) of blood plasma samples, a methodology for predicting the occurrence of MS with a noninvasive, quick blood test.

Methods: TLB allows one to define an index (TLB score), which provides information about overall real-time alterations in plasma proteome that may be indicative of MS.

Results: This pilot study, based on 85 subjects (45 MS patients and 40 controls), showed good performance indexes (sensitivity and specificity both around 70%). The diagnostic methods better discriminate between early stage and low-burden MS patients, and it is not influenced by gender, age, or assumption of therapeutic drugs. TLB is more accurate for patients having low disability level (≤ 3.0, measured by the expanded disability status scale, EDSS) and a relapsing-remitting diagnosis.

Conclusion: Our results suggest that TLB can be applied to MS, especially in an initial phase of the disease when diagnosis is difficult and yet more important (in such cases, accuracy of prediction is close to 80%), as well as in personalized patient periodic monitoring. The next step will be determining its utility in differentiating between MS and other disorders, in particular in inflammatory diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/jpm11040295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069382PMC
April 2021

Rutin Is a Low Micromolar Inhibitor of SARS-CoV-2 Main Protease 3CLpro: Implications for Drug Design of Quercetin Analogs.

Biomedicines 2021 Apr 2;9(4). Epub 2021 Apr 2.

Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, University of Zaragoza, 50018 Zaragoza, Spain.

The pandemic, due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has stimulated the search for antivirals to tackle COVID-19 infection. Molecules with known pharmacokinetics and already approved for human use have been demonstrated or predicted to be suitable to be used either directly or as a base for a scaffold-based drug design. Among these substances, quercetin is known to be a potent in vitro inhibitor of 3CLpro, the SARS-CoV-2 main protease. However, its low in vivo bioavailability calls for modifications to its molecular structure. In this work, this issue is addressed by using rutin, a natural flavonoid that is the most common glycosylated conjugate of quercetin, as a model. Combining experimental (spectroscopy and calorimetry) and simulation techniques (docking and molecular dynamics simulations), we demonstrate that the sugar adduct does not hamper rutin binding to 3CLpro, and the conjugated compound preserves a high potency (inhibition constant in the low micromolar range, = 11 μM). Although showing a disruption of the pseudo-symmetry in the chemical structure, a larger steric volume and molecular weight, and a higher solubility compared to quercetin, rutin is able to associate in the active site of 3CLpro, interacting with the catalytic dyad (His41/Cys145). The overall results have implications in the drug-design of quercetin analogs, and possibly other antivirals, to target the catalytic site of the SARS-CoV-2 3CLpro.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biomedicines9040375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066963PMC
April 2021

Inhibition of the PP2A activity by the histone chaperone ANP32B is long-range allosterically regulated by respiratory cytochrome c.

Redox Biol 2021 Apr 18;43:101967. Epub 2021 Apr 18.

Institute for Chemical Research (IIQ), Scientific Research Centre "Isla de La Cartuja" (cicCartuja), University of Seville, CSIC, Avda. Américo Vespucio 49, Seville, 41092, Spain. Electronic address:

Repair of injured DNA relies on nucleosome dismantling by histone chaperones and de-phosphorylation events carried out by Protein Phosphatase 2A (PP2A). Typical histone chaperones are the Acidic leucine-rich Nuclear Phosphoprotein 32 family (ANP32) members, e.g. ANP32A, which is also a well-known PP2A inhibitor (a.k.a. IPP2A). Here we report the novel interaction between the endogenous family member B-so-called ANP32B-and endogenous cytochrome c in cells undergoing camptothecin-induced DNA damage. Soon after DNA lesions but prior to caspase cascade activation, the hemeprotein translocates to the nucleus to target the Low Complexity Acidic Region (LCAR) of ANP32B; in a similar way, our group recently reported that the hemeprotein targets the acidic domain of SET/Template Activating Factor-Iβ (SET/TAF-Iβ), which is another histone chaperone and PP2A inhibitor (a.k.a. IPP2A). The nucleosome assembly activity of ANP32B is indeed unaffected by cytochrome c binding. Like ANP32A, ANP32B inhibits PP2A activity and is thus herein referred to as IPP2A. Our data demonstrates that ANP32B-dependent inhibition of PP2A is regulated by respiratory cytochrome c, which induces long-distance allosteric changes in the structured N-terminal domain of ANP32B upon binding to the C-terminal LCAR. In agreement with the reported role of PP2A in the DNA damage response, we propose a model wherein cytochrome c is translocated from the mitochondria into the nucleus upon DNA damage to modulate PP2A activity via its interaction with ANP32B.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2021.101967DOI Listing
April 2021

The armadillo-repeat domain of plakophilin 1 binds the C-terminal sterile alpha motif (SAM) of p73.

Biochim Biophys Acta Gen Subj 2021 Apr 17;1865(7):129914. Epub 2021 Apr 17.

Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Joint Units IQFR-CSIC-BIFI, GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain; Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009 Zaragoza, Spain; Fundacion ARAID, Government of Aragon, 50009 Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain.

Background: Plakophilin 1 (PKP1) is a component of desmosomes, which are key structural components for cell-cell adhesion, and can also be found in other cell locations. The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing crucial roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); such domain, SAMp73, is involved in the interaction with other macromolecules.

Methods: We studied the binding of SAMp73 with the armadillo domain of PKP1 (ARM-PKP1) in the absence and the presence of 100 mM NaCl, by using several biophysical techniques, namely fluorescence, far-ultraviolet circular dichroism (CD), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), and molecular docking and simulations.

Results: Association was observed between the two proteins, with a dissociation constant of ~5 μM measured by ITC and fluorescence in the absence of NaCl. The binding region of SAMp73 involved residues of the so-called "middle-loop-end-helix" binding region (i.e., comprising the third helix, together with the C terminus of the second one, and the N-cap of the fourth), as shown by N, H- HSQC-NMR spectra. Molecular modelling provided additional information on the possible structure of the binding complex.

Conclusions: This newly-observed interaction could have potential therapeutic relevance in the tumour pathways where PKP1 is involved, and under conditions when there is a possible inactivation of p53.

General Significance: The discovery of the binding between SAMp73 and ARM-PKP1 suggests a functional role for their interaction, including the possibility that SAMp73 could assist PKP1 in signalling pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2021.129914DOI Listing
April 2021

A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca and Mg binding to EDTA.

Eur Biophys J 2021 Apr 16. Epub 2021 Apr 16.

CIQUP, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto, Portugal.

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00249-021-01523-7DOI Listing
April 2021

Fluid interface calorimetry.

J Colloid Interface Sci 2021 Aug 22;596:119-129. Epub 2021 Mar 22.

Departamento de Fisica de Aplicada, Facultade de Fisica, Universidade de Santiago de Compostela, E-15782 Santiago de Compostela, Spain. Electronic address:

Hypothesis: Amphiphilic molecules spontaneously adsorb to fluid polar-nonpolar interfaces. The timescale of such adsorption depends on the molecular size and structure of the solute. This process should be accompanied by a power heat exchange that could be detected by commercial isothermal calorimeters.

Experiments: Air is injected in the bulk of different aqueous solutions contained in the sample cell of an isothermal titration calorimeter. The formation of the resulting bubbles leads to a liquid/air interface to which the solute molecules spontaneously adsorb. Continuous injection experiments to produce multiple bubbles as well as experiments with static bubbles stand from the capillary tip, aiming to observe slow adsorption processes, were performed.

Findings: The power associated with the formation, growth and release of air bubbles in different liquids was measured. Different independent contributions that can be associated to the pressure change in the gas phase, the evaporation-condensation of the solvent, the increase of interfacial area, the change in the heat capacity of the sample cell content, and the release of the bubble were observed. The periodic pattern produced by the continuous injection of air at a constant rate is used to determine the surface tension of different liquids, including solutions of different molecules and (bio)macromolecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcis.2021.03.098DOI Listing
August 2021

Uncertainty in protein-ligand binding constants: asymmetric confidence intervals versus standard errors.

Eur Biophys J 2021 Apr 10. Epub 2021 Apr 10.

Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, 10257, Vilnius, Lithuania.

Equilibrium binding constants (K) between chemical compounds and target proteins or between interacting proteins provide a quantitative understanding of biological interaction mechanisms. Reported uncertainties of measured experimental parameters are critical for decision-making in many scientific areas, e.g., in lead compound discovery processes and in comparing computational predictions with experimental results. Uncertainties in measured K values are commonly represented by a symmetric normal distribution, often quoted in terms of the experimental value plus-minus the standard deviation. However, in general, the distributions of measured K (and equivalent K) values and the corresponding free energy change ΔG are all asymmetric to varying degree. Here, using a simulation approach, we illustrate the effect of asymmetric K distributions within the realm of isothermal titration calorimetry (ITC) experiments. Further we illustrate the known, but perhaps not widely appreciated, fact that when distributions of any of K, K and ΔG are transformed into each other, their degree of asymmetry is changed. Consequently, we recommend that a more accurate way of expressing the uncertainties of K, K, and ΔG values is to consistently report 95% confidence intervals, in line with other authors' suggestions. The ways to obtain such error ranges are discussed in detail and exemplified for a binding reaction obtained by ITC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00249-021-01518-4DOI Listing
April 2021

Macromolecular interactions in vitro, comparing classical and novel approaches.

Eur Biophys J 2021 Apr 1. Epub 2021 Apr 1.

Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.

Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( https://mosbio.sciencesconf.org/ ). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00249-021-01517-5DOI Listing
April 2021

Isothermal titration calorimetry (ITC): a standard operating procedure (SOP).

Eur Biophys J 2021 Mar 4. Epub 2021 Mar 4.

Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, and Department of Biochemistry and Molecular and Cell Biology, Universidad de Zaragoza, Zaragoza, Spain.

Isothermal titration calorimetry (ITC) is currently widely used in many applied areas of research, spanning protein-ligand binding, metal-ligand interactions, DNA/DNA or protein/DNA interactions, partition to membranes, and polymer surfactant interactions, to mention just a few. This is due to the availability of commercial instruments, and thus the production and spread of an accepted and widely followed SOP is felt by most users, in an effort to produce results that are scientifically correct and comparable. Therefore, within the efforts of Working Group 4 of the ARBRE-MOBIEU COST Action (CA15126), this ITC SOP was generated, alongside SOPs for several other biophysical techniques. Here, we discuss the factors that are fundamental for good experimental design and that need to be carefully considered, as well as machine calibration, in particular chemical calibration, linked to another outcome of Working Group 4 on ITC benchmarking, to be also published in this Special Issue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00249-021-01509-5DOI Listing
March 2021

Unexpected diversity of ferredoxin-dependent thioredoxin reductases in cyanobacteria.

Plant Physiol 2021 Feb 18. Epub 2021 Feb 18.

Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Salamanca 37008, Spain.

Thioredoxin reductases control the redox state of thioredoxins (Trxs)-ubiquitous proteins that regulate a spectrum of enzymes by dithiol-disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/plphys/kiab072DOI Listing
February 2021

Mechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase.

Nat Commun 2021 02 11;12(1):947. Epub 2021 Feb 11.

Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, 46010, Valencia, Spain.

Aspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21165-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878868PMC
February 2021

Influence of the disordered domain structure of MeCP2 on its structural stability and dsDNA interaction.

Int J Biol Macromol 2021 Apr 3;175:58-66. Epub 2021 Feb 3.

Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, Zaragoza 50018, Spain; Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009 Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), 28029 Madrid, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain; Fundación ARAID, Gobierno de Aragón, 50018 Zaragoza, Spain. Electronic address:

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-associated structural protein. MeCP2 deregulation results in two neurodevelopmental disorders: MeCP2 dysfunction is associated with Rett syndrome, while excess of activity is associated with MeCP2 duplication syndrome. MeCP2 is an intrinsically disordered protein (IDP) constituted by six structural domains with variable, small percentage of well-defined secondary structure. Two domains, methyl-CpG binding domain (MBD) and transcription repressor domain (TRD), are the elements responsible for dsDNA binding ability and recruitment of the gene transcription/silencing machinery, respectively. Previously we studied the influence of the completely disordered, MBD-flanking domains (N-terminal domain, NTD, and intervening domain, ID) on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41,635). Here we report the biophysical study of the influence of the remaining domains (transcriptional repressor domain, TRD, and C-terminal domains, CTDα and CTDβ) on the structural stability of MBD and the dsDNA binding capabilities of MBD and ID. The influence of distant disordered domains on MBD properties makes it necessary to consider the NTD-MBD-ID variant as the minimal protein construct for studying dsDNA/chromatin binding properties, while the full-length protein should be considered for transcriptional regulation studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijbiomac.2021.01.206DOI Listing
April 2021

Interplay between non-covalent interactions in 1D supramolecular polymers based on 1,4-bis(iodoethynyl)benzene.

Phys Chem Chem Phys 2021 Feb 29;23(5):3531-3542. Epub 2021 Jan 29.

Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC-Universidad de Zaragoza, Zaragoza, Spain.

A study of the solution-phase, solid-state structures of halogen-bonded co-crystals of 1,4-bis(iodoethynyl)benzene (p-BIB) with three salts, namely, decyltrimethylammonium bromide (DTMABr), tetrapropylammonium bromide (TPABr), and tetrabutylammonium bromide (TBABr), has been carried out, along with theoretical calculations. Isothermal titration calorimetry (ITC) showed that the binding constant of bromide with p-BIB in THF is not strongly dependent on the cation, and that the entropic term clearly dominates the enthalpic one in the free energy of binding. In the three crystal structures, the bromide anion acts as a doubly connected node for halogen bonding interactions, which results in linear or angular open chains. The intrachain angles (IBrI) of the 1D supramolecular polymers based on p-BIB depend on the geometry and size of the cation and vary from 180° for DMTABr to 75° for TBABr. Non-covalent interaction (NCI) analysis of selected motifs and optimized crystals demonstrates that the balance between halogen bonds, hydrogen bonds, and van der Waals interactions, especially type-I halogenhalogen contacts, determines the crystal structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0cp05917jDOI Listing
February 2021

W196 and the -Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing Factor.

Oxid Med Cell Longev 2021 15;2021:6673661. Epub 2021 Jan 15.

Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain.

The human apoptosis-inducing factor (hAIF) is a moonlight flavoprotein involved in mitochondrial respiratory complex assembly and caspase-independent programmed cell death. These functions might be modulated by its redox-linked structural transition that enables hAIF to act as a NAD(H/) redox sensor. Upon reduction with NADH, hAIF undergoes a conformational reorganization in two specific insertions-the flexible regulatory C-loop and the 190-202 -harpin-promoting protein dimerization and the stabilization of a long-life charge transfer complex (CTC) that modulates its monomer-dimer equilibrium and its protein interaction network in healthy mitochondria. In this regard, here, we investigated the precise function of the -hairpin in the AIF conformation landscape related to its redox mechanism, by analyzing the role played by W196, a key residue in the interaction of this motif with the regulatory C-loop. Mutations at W196 decrease the compactness and stability of the oxidized hAIF, indicating that the -hairpin and C-loop coupling contribute to protein stability. Kinetic studies complemented with computational simulations reveal that W196 and the -hairpin conformation modulate the low efficiency of hAIF as NADH oxidoreductase, contributing to configure its active site in a noncompetent geometry for hydride transfer and to stabilize the CTC state by enhancing the affinity for NAD. Finally, the -hairpin motif contributes to define the conformation of AIF's interaction surfaces with its physiological partners. These findings improve our understanding on the molecular basis of hAIF's cellular activities, a crucial aspect for clarifying its associated pathological mechanisms and developing new molecular therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2021/6673661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822688PMC
January 2021

The isolated GTPase-activating-protein-related domain of neurofibromin-1 has a low conformational stability in solution.

Arch Biochem Biophys 2021 03 19;700:108767. Epub 2021 Jan 19.

Instituto de Biocomputación y Física de Sistemas Complejos, Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009, Zaragoza, Spain; Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain; Fundación ARAID, Government of Aragón, 50009, Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009, Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), 28006, Madrid, Spain.

Neurofibromin-1 (NF1) is a large, multidomain tumour suppressor encoded by the NF1 gene. The gene is mutated in neurofibromatosis type I, a disease characterized by malignant tumours of the nervous system and benign neurofibromas. The best-known activity of NF1 is the down-regulation of the mitogen-activated protein kinase pathway via its three-hundred-residue-long GTPase-activating protein (GAP) domain (the so-called GAP-related domain (NF1-GRD)). The NF1-GRD stimulates Ras GTPase activity in turning off signalling. Despite this activity, NF1-GRD has been demonstrated to bind to other different proteins, such as SPRED1 or MC1R. We have embarked on the biophysical and conformational characterization of NF1-GRD in solution by using several spectroscopic (namely fluorescence and circular dichroism (CD)) and biophysical techniques (namely size exclusion chromatography (SEC) and differential scanning calorimetry (DSC)). This biophysical characterization is crucial in deciphering NF1-GRD interactome and in finding biochemical features, modulating possible protein interactions. The native-like structure of NF1-GRD (as monitored by intrinsic fluorescence and far-UV CD) was strongly pH-dependent showing a pH-titration causing a substantial increase in its helicity. NF1-GRD had a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by intrinsic and ANS fluorescence, and CD. Chemical denaturations showed that NF1-GRD unfolded through an intermediate which has a substantial amount of solvent-exposed hydrophobic patches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.abb.2021.108767DOI Listing
March 2021

Enzyme/Nanocopper Hybrid Nanozymes: Modulating Enzyme-like Activity by the Protein Structure for Biosensing and Tumor Catalytic Therapy.

ACS Appl Mater Interfaces 2021 Feb 20;13(4):5111-5124. Epub 2021 Jan 20.

Department of Biocatalysis, Institute of Catalysis (CSIC), c/Marie curie 2, Cantoblanco Campus UAM, 28049 Madrid, Spain.

Artificial enzymes with modulated enzyme-mimicking activities of natural systems represent a challenge in catalytic applications. Here, we show the creation of artificial Cu metalloenzymes based on the generation of Cu nanoparticles in an enzyme matrix. Different enzymes were used, and the structural differences between the enzymes especially influenced the controlled the size of the nanoparticles and the environment that surrounds them. Herein, we demonstrated that the oxidase-like catalytic activity of these copper nanozymes was rationally modulated by enzyme used as a scaffold, with a special role in the nanoparticle size and their environment. In this sense, these nanocopper hybrids have confirmed the ability to mimic a unique enzymatic activity completely different from the natural activity of the enzyme used as a scaffold, such as tyrosinase-like activity or as Fenton catalyst, which has extremely higher stability than natural mushroom tyrosinase. More interestingly, the oxidoreductase-like activity of nanocopper hybrids was cooperatively modulated with the synergistic effect between the enzyme and the nanoparticles improving the catalase activity (no peroxidase activity). Additionally, a novel dual (metallic and enzymatic activity) of the nanozyme made the highly improved catechol-like activity interesting for the design of 3,4-dihydroxy-l-phenylalanine (l-DOPA) biosensor for detection of tyrosinase. These hybrids also showed cytotoxic activity against different tumor cells, interesting in biocatalytic tumor therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsami.0c20501DOI Listing
February 2021

Thermal Liquid Biopsy (TLB) Focused on Benign and Premalignant Pancreatic Cyst Diagnosis.

J Pers Med 2020 Dec 31;11(1). Epub 2020 Dec 31.

Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009 Zaragoza, Spain.

Background: Current efforts in the identification of new biomarkers are directed towards an accurate differentiation between benign and premalignant cysts. Thermal Liquid Biopsy (TLB) has been previously applied to inflammatory and tumor diseases and could offer an interesting point of view in this type of pathology.

Methods: In this work, twenty patients (12 males and 8 females, average ages 62) diagnosed with a pancreatic cyst benign (10) and premalignant (10) cyst lesions were recruited, and biological samples were obtained during the endoscopic ultrasonography procedure.

Results: Proteomic content of cyst liquid samples was studied and several common proteins in the different groups were identified. TLB cyst liquid profiles reflected protein content. Also, TLB serum score was able to discriminate between healthy and cysts patients (71% sensitivity and 98% specificity) and between benign and premalignant cysts (75% sensitivity and 67% specificity).

Conclusions: TLB analysis of plasmatic serum sample, a quick, simple and non-invasive technique that can be easily implemented, reports valuable information on the observed pancreatic lesion. These preliminary results set the basis for a larger study to refine TLB serum score and move closer to the clinical application of TLB providing useful information to the gastroenterologist during patient diagnosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/jpm11010025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823923PMC
December 2020

Proposed mechanism for regulation of H O -induced programmed cell death in plants by binding of cytochrome c to 14-3-3 proteins.

Plant J 2021 Apr 28;106(1):74-85. Epub 2021 Jan 28.

Instituto de Investigaciones Químicas (IIQ) e Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), Centro de Investigaciones Científicas Isla de la Cartuja (cicCartuja), Universidad de Sevilla - Consejo Superior de Investigaciones Científicas (CSIC), Avda. Americo Vespucio 49, Sevilla, 41092, Spain.

Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra-mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14-3-3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase-like activity. This finding establishes the 14-3-3ι protein as a relevant factor at the onset of plant H O -induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14-3-3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14-3-3ι and human 14-3-3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14-3-3ι so as to inhibit caspase-like proteases, which in turn promote cell death upon H O treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tpj.15146DOI Listing
April 2021

Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity.

J Biol Chem 2020 Dec 17. Epub 2020 Dec 17.

Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Spain.

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry (ITC), saturation-transfer difference nuclear magnetic resonance (STD-NMR) and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. Additionally, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemically reacting with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.015103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948413PMC
December 2020

Molecular Context-Dependent Effects Induced by Rett Syndrome-Associated Mutations in MeCP2.

Biomolecules 2020 11 10;10(11). Epub 2020 Nov 10.

Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50018 Zaragoza, Spain.

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. , , 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom10111533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696773PMC
November 2020

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3.

Int J Mol Sci 2020 Oct 8;21(19). Epub 2020 Oct 8.

Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13288 Marseille, France.

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, with several human isoforms; among them, importin α3 (Impα3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Impα3 and its depleted species, ∆Impα3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp α3 with a low micromolar affinity (~5 μM). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to ∆Impα3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Impα3, confirming that the IBB could act as an auto-inhibition region of Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21197428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583046PMC
October 2020

A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin.

Biomolecules 2020 09 11;10(9). Epub 2020 Sep 11.

Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, 163 Avenue de Luminy, 13288 Marseille, France.

Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, of which there are several human isoforms; among them, importin α3 (Impα3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54-74) of NUPR1 towards Impα3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impα3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impα3 with low micromolar affinity (1.7-27 μM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(, + 1) nuclear Overhauser effect (NOE) in the 2D-H-NMR (two-dimensional-proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impα3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom10091313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565984PMC
September 2020

ZZW-115-dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents.

JCI Insight 2020 09 17;5(18). Epub 2020 Sep 17.

Centre de Recherche en Cancérologie de Marseille (CRCM), INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Equipe Labélisée Ligue Nationale Contre le Cancer, Marseille, France.

Establishing the interactome of the cancer-associated stress protein Nuclear Protein 1 (NUPR1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair, and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1, thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further report that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system, indicating that NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation-dependent functions of key proteins involved in the DDR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.138117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526551PMC
September 2020

Structural stability of SARS-CoV-2 3CLpro and identification of quercetin as an inhibitor by experimental screening.

Int J Biol Macromol 2020 Dec 1;164:1693-1703. Epub 2020 Aug 1.

Instituto de Investigación Sanitaria de Aragón (IIS Aragon), 50009 Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), 28029 Madrid, Spain; Institute for Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50009 Zaragoza, Spain; Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain; Fundación ARAID, Gobierno de Aragón, 50018 Zaragoza, Spain. Electronic address:

The global health emergency generated by coronavirus disease 2019 (COVID-19) has prompted the search for preventive and therapeutic treatments for its pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are many potential targets for drug discovery and development to tackle this disease. One of these targets is the main protease, Mpro or 3CLpro, which is highly conserved among coronaviruses. 3CLpro is an essential player in the viral replication cycle, processing the large viral polyproteins and rendering the individual proteins functional. We report a biophysical characterization of the structural stability and the catalytic activity of 3CLpro from SARS-CoV-2, from which a suitable experimental in vitro molecular screening procedure has been designed. By screening of a small chemical library consisting of about 150 compounds, the natural product quercetin was identified as reasonably potent inhibitor of SARS-CoV-2 3CLpro (K ~ 7 μM). Quercetin could be shown to interact with 3CLpro using biophysical techniques and bind to the active site in molecular simulations. Quercetin, with well-known pharmacokinetic and ADMET properties, can be considered as a good candidate for further optimization and development, or repositioned for COVID-19 therapeutic treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijbiomac.2020.07.235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395220PMC
December 2020

Human riboflavin kinase: Species-specific traits in the biosynthesis of the FMN cofactor.

FASEB J 2020 08 10;34(8):10871-10886. Epub 2020 Jul 10.

Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain.

Human riboflavin kinase (HsRFK) catalyzes vitamin B (riboflavin) phosphorylation to flavin mononucleotide (FMN), obligatory step in flavin cofactor synthesis. HsRFK expression is related to protection from oxidative stress, amyloid-β toxicity, and some malignant cancers progression. Its downregulation alters expression profiles of clock-controlled metabolic-genes and destroys flavins protection on stroke treatments, while its activity reduction links to protein-energy malnutrition and thyroid hormones decrease. We explored specific features of the mechanisms underlying the regulation of HsRFK activity, showing that both reaction products regulate it through competitive inhibition. Fast-kinetic studies show that despite HsRFK binds faster and preferably the reaction substrates, the complex holding both products is kinetically most stable. An intricate ligand binding landscape with all combinations of substrates/products competing with the catalytic complex and exhibiting moderate cooperativity is also presented. These data might contribute to better understanding the molecular bases of pathologies coursing with aberrant HsRFK availability, and envisage that interaction with its client-apoproteins might favor FMN release. Finally, HsRFK parameters differ from those of the so far evaluated bacterial counterparts, reinforcing the idea of species-specific mechanisms in RFK catalysis. These observations support HsRFK as potential therapeutic target because of its key functions, while also envisage bacterial RFK modules as potential antimicrobial targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.202000566RDOI Listing
August 2020

Tolcapone, a potent aggregation inhibitor for the treatment of familial leptomeningeal amyloidosis.

FEBS J 2021 Jan 11;288(1):310-324. Epub 2020 May 11.

Departament de Bioquímica i Biologia Molecular, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Spain.

Hereditary transthyretin amyloidosis (ATTR) is a disease characterized by the extracellular deposition of transthyretin (TTR) amyloid fibrils. Highly destabilizing TTR mutations cause leptomeningeal amyloidosis, a rare, but fatal, disorder in which TTR aggregates in the brain. The disease remains intractable, since liver transplantation, the reference therapy for systemic ATTR, does not stop mutant TTR production in the brain. In addition, despite current pharmacological strategies have shown to be effective against in vivo TTR aggregation by stabilizing the tetramer native structure and precluding its dissociation, they display low brain permeability. Recently, we have repurposed tolcapone as a molecule to treat systemic ATTR. Crystal structures and biophysical analysis converge to demonstrate that tolcapone binds with high affinity and specificity to three unstable leptomeningeal TTR variants, stabilizing them and, consequently, inhibiting their aggregation. Because tolcapone is an FDA-approved drug that crosses the blood-brain barrier, our results suggest that it can translate into a first disease-modifying therapy for leptomeningeal amyloidosis. DATABASES: PDB codes for A25T-TTR, V30G-TTR, and Y114C-TTR bound to tolcapone are 6TXV, 6TXW, and 6XTK, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.15339DOI Listing
January 2021

Small Molecule Inhibitors of the Response Regulator ArsR Exhibit Bactericidal Activity against .

Microorganisms 2020 Apr 1;8(4). Epub 2020 Apr 1.

Aragon Institute for Health Research (IIS Aragón), San Juan Bosco 13, 50009 Zaragoza, Spain.

is considered the most prevalent bacterial pathogen in humans. The increasing antibiotic resistance evolved by this microorganism has raised alarm bells worldwide due to the significant reduction in the eradication rates of traditional standard therapies. A major challenge in this antibiotic resistance crisis is the identification of novel microbial targets whose inhibitors can overcome the currently circulating resistome. In the present study, we have validated the use of the essential response regulator ArsR as a novel and promising therapeutic target against infections. A high-throughput screening of a repurposing chemical library using a fluorescence-based thermal shift assay identified several ArsR binders. At least four of these low-molecular weight compounds noticeably inhibited the DNA binding activity of ArsR and showed bactericidal effects against antibiotic-resistant strains of . Among the ArsR inhibitors, a human secondary bile acid, lithocholic acid, quickly destroyed cells and exhibited partial synergistic action in combination with clarithromycin or levofloxacin, while the antimicrobial effect of this compound against representative members of the normal human microbiota such as and appeared irrelevant. Our results enhance the battery of novel therapeutic tools against refractory infections caused by multidrug-resistant strains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/microorganisms8040503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232201PMC
April 2020

Repurposing Dihydropyridines for Treatment of Infection.

Pharmaceutics 2019 Dec 15;11(12). Epub 2019 Dec 15.

Aragon Institute for Health Research (IIS Aragón), San Juan Bosco 13, 50009 Zaragoza, Spain.

Antibiotic resistance is a major cause of the increasing failures in the current eradication therapies against . In this scenario, repurposing drugs could be a valuable strategy to fast-track novel antimicrobial agents. In the present study, we analyzed the inhibitory capability of 1,4-dihydropyridine (DHP) antihypertensive drugs on the essential function of the response regulator HsrA and investigated both the in vitro antimicrobial activities and the in vivo efficacy of DHP treatments against . Six different commercially available and highly prescribed DHP drugs-namely, Nifedipine, Nicardipine, Nisoldipine, Nimodipine, Nitrendipine, and Lercanidipine-noticeably inhibited the DNA binding activity of HsrA and exhibited potent bactericidal activities against both metronidazole- and clarithromycin-resistant strains of , with minimal inhibitory concentration (MIC) values in the range of 4 to 32 mg/L. The dynamics of the decline in the bacterial counts at 2 × MIC appeared to be correlated with the lipophilicity of the drugs, suggesting different translocation efficiencies of DHPs across the bacterial membrane. Oral treatments with 100 mg/kg/day of marketed formulations of Nimodipine or Nitrendipine in combination with omeprazole significantly reduced the gastric colonization in mice. The results presented here support a novel therapeutic solution for treatment of antibiotic-resistant infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/pharmaceutics11120681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969910PMC
December 2019