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1.
Anal Chem ; 96(2): 615-619, 2024 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-38165272

RESUMEN

STD NMR spectroscopy is a powerful ligand-observed NMR tool for screening and characterizing the interactions of small molecules and low molecular weight fragments with a given macromolecule, identifying the main intermolecular contacts in the bound state. It is also a powerful analytical technique for the accurate determination of protein-ligand dissociation constants (KD) of medium-to-weak affinity, of interest in the pharmaceutical industry. However, accurate KD determination and epitope mapping requires a long series of experiments at increasing saturation times to carry out a full analysis using the so-called STD NMR build-up curve approach and apply the "initial slopes approximation". Here, we have developed a new protocol to bypass this important limitation, which allows us to obtain initial slopes by using just two saturation times and, hence, to very quickly determine precise protein-ligand dissociation constants by STD NMR.


Asunto(s)
Imagen por Resonancia Magnética , Proteínas , Ligandos , Proteínas/química , Espectroscopía de Resonancia Magnética/métodos , Mapeo Epitopo , Unión Proteica
2.
J Biomol NMR ; 74(8-9): 421-429, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32683519

RESUMEN

A major driving force for protein-nucleic acid association is electrostatic interactions via ion pairs of the positively charged basic side chains and negatively charged phosphates. For a better understanding of how proteins scan DNA and recognize particular signatures, it is important to gain atomic-level insight into the behavior of basic side chains at the protein-DNA interfaces. NMR spectroscopy is a powerful tool for investigating the structural, dynamic, and kinetic aspects of protein-DNA interactions. However, resonance assignment of basic side-chain cationic moieties at the molecular interfaces remains to be a major challenge. Here, we propose a fast, robust, and inexpensive approach that greatly facilitates resonance assignment of interfacial moieties and also allows for kinetic measurements of protein translocation between two DNA duplexes. This approach utilizes site-specific incorporation of racemic phosphorothioate at the position of a phosphate that interacts with a protein side chain. This modification retains the electric charge of phosphate and therefore is mild, but causes significant chemical shift perturbations for the proximal protein side chains, which facilitates resonance assignment. Due to the racemic nature of the modification, two different chemical shifts are observed for the species with different diastereomers RP and SP of the incorporated phosphorothioate group. Kinetic information on the exchange of the protein molecule between RP and SP DNA duplexes can be obtained by 15Nz exchange spectroscopy. We demonstrate the applications of this approach to the Antennapedia homeodomain-DNA complex and the CREB1 basic leucine-zipper (bZIP)-DNA complex.


Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Sustancias Macromoleculares/química , Resonancia Magnética Nuclear Biomolecular , Fosfatos/química , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Dominios y Motivos de Interacción de Proteínas
3.
Chemistry ; 26(44): 10024-10034, 2020 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-32449563

RESUMEN

Ligand-based NMR techniques to study protein-ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi-frequency STD NMR approach, differential epitope mapping (DEEP)-STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so-called DEEP-STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP-STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi-subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.


Asunto(s)
Toxina del Cólera/química , Toxina del Cólera/metabolismo , Gangliósido G(M1)/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Simulación de Dinámica Molecular , Sitios de Unión , Ligandos , Unión Proteica
4.
Angew Chem Int Ed Engl ; 59(4): 1465-1468, 2020 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-31743557

RESUMEN

Due to a high density of negative charges on its surface, DNA condenses cations as counterions, forming the so-called "ion atmosphere". Although the release of counterions upon DNA-protein association has been postulated to have a major contribution to the binding thermodynamics, this release remains to be confirmed through a direct observation of the ions. Herein, we report the characterization of the ion atmosphere around DNA using NMR spectroscopy and directly detect the release of counterions upon DNA-protein association. NMR-based diffusion data reveal the highly dynamic nature of counterions within the ion atmosphere around DNA. Counterion release is observed as an increase in the apparent ionic diffusion coefficient, which directly provides the number of counterions released upon DNA-protein association.


Asunto(s)
ADN/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas/química , Humanos
5.
Glycobiology ; 29(1): 45-58, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30371779

RESUMEN

Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed serine-rich repeat protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC-MS and GC-MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP100-23 and SRRP53608 with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP53608 variants with α-GlcNAc and GlcNAcß(1→6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications.


Asunto(s)
Adhesinas Bacterianas/química , Limosilactobacillus reuteri/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Glicosilación , Limosilactobacillus reuteri/genética , Limosilactobacillus reuteri/metabolismo , Mutación , Resonancia Magnética Nuclear Biomolecular , Secuencias Repetitivas de Aminoácido
6.
Chembiochem ; 20(3): 340-344, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30379391

RESUMEN

Differential epitope mapping saturation transfer difference (DEEP-STD) NMR spectroscopy is a recently developed powerful approach for elucidating the structure and pharmacophore of weak protein-ligand interactions, as it reports key information on the orientation of the ligand and the architecture of the binding pocket. The method relies on selective saturation of protein residues in the binding site and the generation of a differential epitope map by observing the ligand, which depicts the nature of the protein residues making contact with the ligand in the bound state. Selective saturation requires knowledge of the chemical-shift assignment of the protein residues, which can be obtained either experimentally by NMR spectroscopy or predicted from 3D structures. Herein, we propose a simple experimental procedure to expand the DEEP-STD NMR methodology to protein-ligand cases in which the spectral assignment of the protein is not available. This is achieved by experimentally identifying the chemical shifts of the residues present in binding hot-spots on the surface of the receptor protein by using 2D NMR experiments combined with a paramagnetic probe.


Asunto(s)
Óxidos N-Cíclicos/química , Mapeo Epitopo , Epítopos/química , Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Sitios de Unión , Ligandos , Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Marcadores de Spin
7.
Int J Mol Sci ; 20(5)2019 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-30857167

RESUMEN

The newly identified CUBAN (Cullin binding domain associating with NEDD8) domain recognizes both ubiquitin and the ubiquitin-like NEDD8. Despite the high similarity between the two molecules, CUBAN shows a clear preference for NEDD8, free and conjugated to cullins. We previously characterized the domain structure, both alone and in complex with NEDD8. The results here reported are addressed to investigate the determinants that drive the selective binding of CUBAN towards NEDD8 and ubiquitin. The 15N HSQC NMR perturbation pattern of the labeled CUBAN domain, when combined with either NEDD8 or ubiquitin, shows a clear involvement of hydrophobic residues that characterize the early stages of these interactions. After a slow conformational selection step, hydrophobic and then neutral and polar interactions take place, which drive the correct orientation of the CUBAN domain, leading to differences in the recognition scheme of NEDD8 and ubiquitin. As a result, a cascade of induced fit steps seems to determine the structural preference shown for NEDD8 and therefore the basis of the selectivity of the CUBAN domain. Finally, molecular dynamics analysis was performed to determine by fluctuations the internal flexibility of the CUBAN/NEDD8 complex. We consider that our results, based on a structural investigation mainly focused on the early stages of the recognition, provide a fruitful opportunity to report the different behavior of the same protein with two highly similar binding partners.


Asunto(s)
Proteína NEDD8/metabolismo , Ubiquitina/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteína NEDD8/química , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Ubiquitina/química , Ubiquitinación
8.
Molecules ; 22(11)2017 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-29077041

RESUMEN

Thymosin α1 (Tα1), is a peptidic hormone, whose immune regulatory properties have been demonstrated both in vitro and in vivo and approved in different countries for treatment of several viral infections and cancers. Tα1 assumes a conformation in negative membranes upon insertion into the phosphatidylserine exposure as found in several pathologies and in apoptosis. These findings are in agreement with the pleiotropy of Tα1, which targets both normal and tumor cells, interacting with multiple cellular components, and have generated renewed interest in the topic. Hyaluronan (HA) occurs ubiquitously in the extracellular matrix and on cell surfaces and has been related to a variety of diseases, and developmental and physiological processes. Proteins binding HA, among them CD44 and the Receptor for HA-mediated motility (RHAMM) receptors, mediate its biological effects. NMR spectroscopy indicated preliminarily that an interaction of Tα1 with HA occurs specifically around lysine residues of the sequence LKEKK of Tα1 and is suggestive of a possible interference of Tα1 in the binding of HA with CD44 and RHAMM. Further studies are needed to deepen these observations because Tα1 is known to potentiate the T-cell immunity and anti-tumor effect. The binding inhibitory activity of Tα1 on HA-CD44 or HA-RHAMM interactions can suppress both T-cell reactivity and tumor progression.


Asunto(s)
Secuencia de Aminoácidos , Ácido Hialurónico/química , Dominios y Motivos de Interacción de Proteínas , Electricidad Estática , Timosina/análogos & derivados , Espectroscopía de Resonancia Magnética , Unión Proteica , Timalfasina , Timosina/química
9.
Biochemistry ; 55(10): 1462-72, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26909491

RESUMEN

Thymosin α1 is a peptidic hormone with pleiotropic activity and is used in the therapy of several diseases. It is unstructured in water solution and interacts with negative regions of vesicles by assuming two tracts of helical conformation with a structural break between them. This study reports on Thymosin α1's interaction with mixed phospholipids phosphatidylcholine and phosphatidylserine, the negative component of the membranes, by ¹H and natural abundance ¹5N nuclear magnetic resonance (NMR). The results indicate that interaction occurs when the membrane is negatively charged by exposing phosphatidylserine. Moreover, the direct interaction of Thymosin α1 with K562 cells with an overexposure of phosphatidylserine as a consequence of resveratrol-induced apoptosis was conducted. Thymosin α1's interaction with human serum albumin was also investigated by NMR spectroscopy. Steady-state saturation transfer, transfer nuclear Overhauser effect spectroscopy, and diffusion-ordered spectroscopy methodologies all reveal that the C-terminal region of Thymosin α1 is involved in the interaction with serum albumin. These results may shed more light on Thymosin α1's mechanism of action by its insertion in negative regions of membranes due to the exposure of phosphatidylserine. Once Thymosin α1's N-terminus has been inserted into the membrane, the rest may interact with nearby proteins and/or receptors acting as effectors and causing a biological signaling cascade, thus exerting thymosin α1's pleiotropy.


Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Fosfatidilserinas/metabolismo , Albúmina Sérica/metabolismo , Timosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Bovinos , Humanos , Células K562 , Datos de Secuencia Molecular , Fosfatidilserinas/química , Fosfatidilserinas/genética , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Albúmina Sérica/química , Albúmina Sérica/genética , Timalfasina , Timosina/química , Timosina/genética , Timosina/metabolismo
10.
Amino Acids ; 48(5): 1231-9, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26801937

RESUMEN

Thymosin α1 is a peptidic hormone already used in the therapy of several diseases. Until now, the description of the precise receptor and mechanism for its action still remains elusive. The interaction of Thymosin α1, which is unstructured in water solution, has been recently studied in sodium dodecylsulphate micellar systems and it was reported that Thymosin α1 inserts in micelle assuming a conformation with two tracts of helix with a structural break in between. An investigation of its interaction both with micelles of dodecylphosphocholine alone and with mixed dodecylphosphocholine-sodium dodecylsulphate micelles is here reported. In these environments the results indicate that Thymosin α1 in phospholipidic membrane exposing choline polar heads interacts by aspecific modality and, oppositely, in the mixed dodecylphosphocholine-sodium dodecylsulphate micelles an insertion in the micellar hydrophobic region conformationally similar to that found in sodium dodecylsulphate micelles occurs. In presence of mixed micelles the insertion and structuration occur in preferred regions when the membrane models are negatively charged. From the point of view of the mechanism of action, insertion its N terminus in negative regions of membrane led to hypothesize that this process would be similar to a binding to phosphatidylserine exposed like in apoptotic cells. Thymosin α1 when inserted may interact with nearby proteins and/or receptors acting as effector and causing a biological signaling cascade. The recent attention to the phosphatidylserine exposure in cells may enforce the interest for these findings.


Asunto(s)
Membrana Celular/metabolismo , Timosina/análogos & derivados , Membrana Celular/química , Interacciones Hidrofóbicas e Hidrofílicas , Micelas , Modelos Biológicos , Conformación Molecular , Unión Proteica , Timalfasina , Timosina/química , Timosina/metabolismo
11.
Amino Acids ; 46(12): 2841-53, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25261131

RESUMEN

The structural characterization of [W8S]contryphan Vn, an analogue of Contryphan Vn with tryptophan 8 substituted with a serine residue (W8S), was performed by NMR spectroscopy, molecular dynamics simulations and fluorescence spectroscopy. Contryphan Vn, a bioactive cyclic peptide from the venom of the cone snail Conus ventricosus, contains an S-S bridge between two cysteines and a D-tryptophan. Like other Contryphans, [W8S]contryphan Vn has proline 7 isomerized trans, while the proline 4 has nearly equivalent populations of cis and trans configurations. The thermodynamic and kinetic parameters of the trans-cis isomerization of proline 4 were measured. The isomers of [W8S]contryphan Vn with proline 4 in cis and trans show structural differences. The absence of the salt bridge between the same Asp2 and Lys6, present in Contryphan Vn, may be attributed to the lack of the hydrophobic side chain of Trp8 where it likely protects the electrostatic interactions. These results may contribute to identifying, in these cyclic peptides, the structural determinants of the mechanism of proline trans-cis isomerization, this being also an important step in protein folding.


Asunto(s)
Péptidos Cíclicos/química , Prolina/química , Serina/química , Triptófano/química , Secuencia de Aminoácidos , Isomerismo , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos Cíclicos/síntesis química
12.
J Med Chem ; 67(12): 10025-10034, 2024 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-38848103

RESUMEN

Low-affinity protein-ligand interactions are important for many biological processes, including cell communication, signal transduction, and immune responses. Structural characterization of these complexes is also critical for the development of new drugs through fragment-based drug discovery (FBDD), but it is challenging due to the low affinity of fragments for the binding site. Saturation transfer difference (STD) NMR spectroscopy has revolutionized the study of low-affinity receptor-ligand interactions enabling binding detection and structural characterization. Comparison of relaxation and exchange matrix calculations with 1H STD NMR experimental data is essential for the validation of 3D structures of protein-ligand complexes. In this work, we present a new approach based on the calculation of a reduced relaxation matrix, in combination with funnel metadynamics MD simulations, that allows a very fast generation of experimentally STD-NMR-validated 3D structures of low-affinity protein-ligand complexes.


Asunto(s)
Proteínas , Ligandos , Proteínas/química , Proteínas/metabolismo , Simulación de Dinámica Molecular , Modelos Moleculares , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Humanos , Unión Proteica , Sitios de Unión , Descubrimiento de Drogas
13.
Int Immunopharmacol ; 118: 110113, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-37028279

RESUMEN

The study of mechanism of action of Thymosin alpha 1 (Tα1) and the basis of the pleiotropic effect in health and disease, is one of the main focus of our ongoing research. Tα1 is a thymic peptide that demonstrates a peculiar ability to restore homeostasis in different physiological and pathological conditions (i.e., infections, cancer, immunodeficiency, vaccination, and aging) acting as multitasking protein depending on the host state of inflammation or immune dysfunction. However, few are the information about mechanisms of action mediated by specific Tα1-target protein interaction that could explain its pleiotropic effect. We investigated the interaction of Tα1 with Galectin-1 (Gal-1), a protein belonging to an oligosaccharide binding protein family involved in a variety of biological and pathological processes, including immunoregulation, infections, cancer progression and aggressiveness. Using molecular and cellular methodological approaches, we demonstrated the interaction between these two proteins. Tα1 specifically inhibited the hemagglutination activity of Gal-1, the Gal-1 dependent in vitro formation of endothelial cell tubular structures, and the migration of cancer cells in wound healing assay. Physico-chemical methods revealed the details of the molecular interaction of Tα1 with Gal-1. Hence, the study allowed the identification of the not known until now specific interaction between Tα1 and Gal-1, and unraveled a novel mechanism of action of Tα1 that could support understanding of its pleiotropic activity.


Asunto(s)
Neoplasias , Timosina , Humanos , Timalfasina , Galectina 1
14.
Microb Pathog ; 53(3-4): 135-46, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22771837

RESUMEN

Mycobacterium tuberculosis (MTB) colony morphology was associated to the pathogen's virulence. We isolated a new MTB H37Rv smooth colony, which only appeared following human macrophages (MDM) infection. The new phenotype was Alcohol-Acid resistant, but devoid of a covering capsule and biofilm defective. We ascertained that there were no deletions in the Rv0096-Rv0101 PDIM Operon, but that its expression was repressed as compared to MTB wild type (wt). Its lipid composition displayed lower PDIM components and higher TAG as compared to wt. In MDM it induced the sigma factors sigB, sigI and sigL expression vs. synthetic medium culture, while it repressed other six sigma factors. It also induced, significantly more than wt, mprA, a mycobacterial persistence regulator. It was phagocytosed more than wt by MDM, where it grew significantly less, but persisted therein till 14 days infection. It induced significantly less IFN-γ, IL-12 and IL-27 transcription than wt in infected MDM, while it increased the transcription of inducible NOS. It resided in mature, LAMP-3 positive phagolysosomes, where it never formed cords. This apparently "weaker" colony might represent an adaptive intracellular phenotype, whose infection may be less productive, but probably better equipped for a long lasting persistence in mildly activated host cells.


Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Macrófagos/microbiología , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/genética , Operón , Tuberculosis/microbiología , Proteínas Bacterianas/metabolismo , Células Cultivadas , Regulación hacia Abajo , Humanos , Interferones/genética , Interferones/inmunología , Interleucina-12/genética , Interleucina-12/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/metabolismo , Tuberculosis/genética , Tuberculosis/inmunología
15.
Front Mol Biosci ; 8: 727980, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34604306

RESUMEN

Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms.

16.
J Colloid Interface Sci ; 594: 217-227, 2021 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-33756365

RESUMEN

HYPOTHESIS: The classical STD NMR protocol to monitor solvent interactions in gels is strongly dependent on gelator and solvent concentrations and does not report on the degree of structuration of the solvent at the particle/solvent interface. We hypothesised that, for suspensions of large gelator particles, solvent structuration could be characterised by STD NMR when taking into account the particle-to-solvent 1H-1H spin diffusion transfer using the 1D diffusion equation. EXPERIMENTS: We have carried out a systematic study on effect of gelator and solvent concentrations, and gelator surface charge, affecting the behaviour of the classical STD NMR build-up curves. To do so, we have characterised solvent interactions in dispersions of starch and cellulose-like particles prepared in deuterated water and alcohol/D2O mixtures. FINDINGS: The Spin Diffusion Transfer Difference (SDTD) NMR protocol is independent of the gelator and solvent concentrations, hence allowing the estimation of the degree of solvent structuration within different particle networks. In addition, the simulation of SDTD build-up curves using the general one-dimensional diffusion equation allows the determination of minimum distances (r) and spin diffusion rates (D) at the particle/solvent interface. This novel NMR protocol can be readily extended to characterise the solvent(s) organisation in any type of colloidal systems constituted by large particles.

17.
Biochemistry ; 49(2): 252-8, 2010 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-20000484

RESUMEN

RTN1-C protein is a membrane protein localized in the ER and expressed in the nervous system, and its biological role is not completely clarified. Our previous studies have shown that the C-terminal region of RTN1-C, corresponding to the fragment from residues 186 to 208, was able to bind the nucleic acids and to interact with histone deacetylase (HDAC) enzymes. In the present work the properties of the synthetic RTN1-C(CT) peptide corresponding to this region were studied with relation to its ability to bind the metal ions in its N-terminal region. RTN1-C(CT) peptide is characterized by the presence of high-affinity copper and nickel ion sites. The nuclease activity of the metal-peptide complex was observed due to the presence of an ATCUN-binding motif. Moreover, the effect of the Cu/Ni-RTN1-C(CT) complexes on the HDAC activity was investigated. The histone deacetylase inhibitors are a new class of antineoplastic agents currently being evaluated in clinical trials. Our data show that the acetylated form of the metal-peptide complex is able to inhibit the HDAC activity at micromolar concentrations. These results allow to propose the Cu/Ni-RTN1-C(CT) complexes as models for the design of antitumor agents.


Asunto(s)
Histona Desacetilasas/metabolismo , Proteínas del Tejido Nervioso/química , Acetilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dicroismo Circular , Secuencia de Consenso , ADN/metabolismo , Retículo Endoplásmico/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/química , Humanos , Metales/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/farmacología , Péptidos/síntesis química , Péptidos/química , Plásmidos , Ratas , Espectrometría de Fluorescencia
18.
J Magn Reson ; 312: 106687, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31982802

RESUMEN

Hydrogen exchange between solute and water molecules occurs across a wide range of timescales. Rapid hydrogen-exchange processes can effectively diminish 1H-15N scalar couplings. We demonstrate that the self-decoupling of 15N nuclear magnetic resonance can allow quantitative investigations of hydrogen exchange on a micro- to millisecond timescale, which is relatively difficult to analyze with other methods. Using a Liouvillian matrix incorporating hydrogen exchange as a mechanism for scalar relaxation, the hydrogen exchange rate can be determined from 15N NMR line shapes recorded with and without 1H decoupling. Self-decoupling offers a simple approach to analyze the kinetics of hydrogen exchange in a wide range of timescale.


Asunto(s)
Compuestos de Amonio/química , Hidrógeno/química , Espectroscopía de Resonancia Magnética/métodos , Isótopos de Nitrógeno/química , Concentración de Iones de Hidrógeno , Cinética
19.
Biomolecules ; 10(11)2020 10 24.
Artículo en Inglés | MEDLINE | ID: mdl-33114342

RESUMEN

The stability and the degradation of polymers in physiological conditions are very important issues in biomedical applications. The copolymer of hyaluronic acid and poly-D,L-lactic acid (made available in a product called DAC®) produces a hydrogel which retains the hydrophobic character of the poly-D,L-lactide sidechains and the hydrophilic character of a hyaluronic acid backbone. This hydrogel is a suitable device for the coating of orthopedic implants with structured surfaces. In fact, this gel creates a temporary barrier to bacterial adhesion by inhibiting colonization, thus preventing the formation of the biofilm and the onset of an infection. Reabsorbed in about 72 h after the implant, this hydrogel does not hinder bone growth processes. In the need to assess stability and degradation of both the hyaluronan backbone and of the polylactic chains along time and temperature, we identified NMR spectroscopy as a privileged technique for the characterization of the released species, and we applied diffusion-ordered NMR spectroscopy (DOSY-NMR) for the investigation of molecular weight dispersion. Our diffusion studies of DAC® in physiological conditions provided a full understanding of the product degradation by overcoming the limitations observed in applying classical chromatography approaches by gel permeation UV.


Asunto(s)
Materiales Biocompatibles/química , Ácido Hialurónico/química , Hidrogeles/química , Poliésteres/química , Estabilidad de Medicamentos , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Peso Molecular
20.
Colloids Surf B Biointerfaces ; 195: 111266, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32739771

RESUMEN

The development of new therapeutic strategies against multidrug resistant Gram-negative bacteria is a major challenge for pharmaceutical research. In this respect, it is increasingly recognized that an efficient treatment for resistant bacterial infections should combine antimicrobial and anti-inflammatory effects. Here, we explore the multifunctional therapeutic potential of nanostructured self-assemblies from a cationic bolaamphiphile, which target bacterial lipopolysaccharides (LPSs) and associates with an anti-bacterial nucleic acid to form nanoplexes with therapeutic efficacy against Gram-negative bacteria. To understand the mechanistic details of these multifunctional antimicrobial-anti-inflammatory properties, we performed a fundamental study, comparing the interaction of these nanostructured therapeutics with synthetic biomimetic bacterial membranes and live bacterial cells. Combining a wide range of experimental techniques (Confocal Microscopy, Fluorescence Correlation Spectroscopy, Microfluidics, NMR, LPS binding assays), we demonstrate that the LPS targeting capacity of the bolaamphiphile self-assemblies, comparable to that exerted by Polymixin B, is a key feature of these nanoplexes and one that permits entry of therapeutic nucleic acids in Gram-negative bacteria. These findings enable a new approach to the design of efficient multifunctional therapeutics with combined antimicrobial and anti-inflammatory effects and have therefore the potential to broadly impact fundamental and applied research on self-assembled nano-sized antibacterials for antibiotic resistant infections.


Asunto(s)
Antiinfecciosos , Lipopolisacáridos , Antibacterianos/farmacología , Antiinfecciosos/farmacología , ADN , Bacterias Gramnegativas , Pruebas de Sensibilidad Microbiana
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