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Introduction: Aryl-alcohol oxidase (AAO) shows a pronounced duality as oxidase and dehydrogenase similar to that described for other glucose-methanol-choline (GMC) oxidase/dehydrogenase superfamily proteins involved in lignocellulose decomposition. In this work, we detail the overall mechanism of AAOs from Pleurotus eryngii and Bjerkandera adusta for catalyzing the oxidation of natural aryl-alcohol substrates using either oxygen or quinones as electron acceptors and describe the crystallographic structure of AAO from B. adusta in complex with a product analogue. Methods: Kinetic studies with 4-methoxybenzyl and 3-chloro-4- methoxybenzyl alcohols, including both transient-state and steady-state analyses, along with interaction studies, provide insight into the oxidase and dehydrogenase mechanisms of these enzymes. Moreover, the resolution of the crystal structure of AAO from B. adusta allowed us to compare their overall folding and the structure of the active sites of both AAOs in relation to their activities. Results and Discussion: Although both enzymes show similar mechanistic properties, notable differences are highlighted in this study. In B. adusta, the AAO oxidase activity is limited by the reoxidation of the flavin, while in P. eryngii the slower step takes place during the reductive half-reaction, which determines the overall reaction rate. By contrast, dehydrogenase activity in both enzymes, irrespective of the alcohol participating in the reaction, is limited by the hydroquinone release from the active site. Despite these differences, both AAOs are more efficient as dehydrogenases, supporting the physiological role of this activity in lignocellulosic decay. This dual activity would allow these enzymes to adapt to different environments based on the available electron acceptors.
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Compartmentalization of proteins by liquid-liquid phase separation (LLPS) is used by cells to control biochemical reactions spatially and temporally. Among them, the recruitment of proteins to DNA foci and nucleolar trafficking occur by biomolecular condensation. Within this frame, the oncoprotein SET/TAF-Iß plays a key role in both chromatin remodeling and DNA damage response, as does nucleophosmin (NPM1) which indeed participates in nucleolar ribosome synthesis. Whereas phase separation by NPM1 is widely characterized, little is known about that undergone by SET/TAF-Iß. Here, we show that SET/TAF-Iß experiences phase separation together with respiratory cytochrome c (Cc), which translocates to the nucleus upon DNA damage. Here we report the molecular mechanisms governing Cc-induced phase separation of SET/TAF-Iß and NPM1, where two lysine-rich clusters of Cc are essential to recognize molecular surfaces on both partners in a specific and coordinated manner. Cc thus emerges as a small, globular protein with sequence-encoded heterotypic phase-separation properties.
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Human antigen R (HuR) is an RNA binding protein mainly involved in maintaining the stability and controlling the translation of mRNAs, critical for immune response, cell survival, proliferation and apoptosis. Although HuR is a nuclear protein, its mRNA translational-related function occurs at the cytoplasm, where the oligomeric form of HuR is more abundant. However, the regulation of nucleo-cytoplasmic transport of HuR and its connection with protein oligomerization remain unclear. In this work, we describe the phosphorylation of Tyr5 as a new hallmark for HuR activation. Our biophysical, structural and computational assays using phosphorylated and phosphomimetic HuR proteins demonstrate that phosphorylation of Tyr5 at the disordered N-end stretch induces global changes on HuR dynamics and conformation, modifying the solvent accessible surface of the HuR nucleo-cytoplasmic shuttling (HNS) sequence and releasing regions implicated in HuR dimerization. These findings explain the preferential cytoplasmic accumulation of phosphorylated HuR in HeLa cells, aiding to comprehend the mechanisms underlying HuR nucleus-cytoplasm shuttling and its later dimerization, both of which are relevant in HuR-related pathogenesis.
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Citoplasma , Proteína 1 Similar a ELAV , Multimerización de Proteína , Humanos , Citoplasma/metabolismo , Fosforilación , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Células HeLa , Núcleo Celular/metabolismoRESUMEN
PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions - i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.
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Citrulinación , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Arginina Deiminasa Proteína-Tipo 4/química , Humanos , Desiminasas de la Arginina Proteica/metabolismo , Desiminasas de la Arginina Proteica/química , Conformación Proteica , Péptidos/química , Péptidos/metabolismo , Citrulina/química , Citrulina/metabolismo , Unión Proteica , Secuencia de AminoácidosRESUMEN
The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.
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Antivirales , Proteasas 3C de Coronavirus , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Inhibidores de Proteasas , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/metabolismo , Antivirales/farmacología , Antivirales/química , Humanos , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , COVID-19/virología , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Evaluación Preclínica de Medicamentos/métodos , Unión Proteica , LigandosRESUMEN
Polycomb groups (PcGs) are transcriptional repressors, formed by a complex of several proteins, involved in multicellular development and cancer epigenetics. One of these proteins is the E3 ubiquitin-protein ligase RING1 (or RING1B), associated with the regulation of transcriptional repression and responsible for monoubiquitylation of the histone H2A. On the other hand, PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline, and it is also involved in the development of glioblastoma, among other types of cancers. In this work, we showed the association of PADI4 and RING1B in the nucleus and cytosol in several cancer cell lines by using immunofluorescence and proximity ligation assays. Furthermore, we demonstrated that binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that RING1B could bind to the active site of PADI4, as confirmed by protein-protein docking simulations. In vitro and in silico findings showed that binding to PADI4 occurred for the isolated fragments corresponding to both the N-terminal (residues 1-221) and C-terminal (residues 228-336) regions of RING1B. Binding to PADI4 was also hampered by GSK484, as shown by isothermal titration calorimetry (ITC) experiments for the sole N-terminal region, and by both NMR and ITC for the C-terminal one. The dissociation constants between PADI4 and any of the two isolated RING1B fragments were in the low micromolar range (~2-10 µM), as measured by fluorescence and ITC. The interaction between RING1B and PADI4 might imply citrullination of the former, leading to several biological consequences, as well as being of potential therapeutic relevance for improving cancer treatment with the generation of new antigens.
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Unión Proteica , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Humanos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citrulinación , Simulación del Acoplamiento Molecular , Neoplasias/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/química , Complejo Represivo Polycomb 1/genética , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Desiminasas de la Arginina Proteica/metabolismo , Desiminasas de la Arginina Proteica/genéticaRESUMEN
Objectives: The rise of antibiotic-resistant Streptococcus pneumoniae (Sp) poses a significant global health threat, urging the quest for novel antimicrobial solutions. We have discovered that the human hormone l-thyroxine has antibacterial properties. In order to explore its drugability we perform here the characterization of a series of l-thyroxine analogues and describe the structural determinants influencing their antibacterial efficacy. Method: We performed a high-throughput screening of a library of compounds approved for use in humans, complemented with ITC assays on purified Sp-flavodoxin, to pinpoint molecules binding to this protein. Antimicrobial in vitro susceptibility assays of the hit compound (l-thyroxine) as well as of 13 l-thyroxine analogues were done against a panel of Gram-positive and Gram-negative bacteria. Toxicity of compounds on HepG2 cells was also assessed. A combined structure-activity and computational docking analysis was carried out to uncover functional groups crucial for the antimicrobial potency of these compounds. Results: Human l-thyroxine binds to Sp-flavodoxin, forming a 1:1 complex of low micromolar Kd. While l-thyroxine specifically inhibited Sp growth, some derivatives displayed activity against other Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, while remaining inactive against Gram-negative pathogens. Neither l-thyroxine nor some selected derivatives exhibited toxicity to HepG2 cells. Conclusions: l-thyroxine derivatives targeting bacterial flavodoxins represent a new and promising class of antimicrobials.
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Plakophilin 1 (PKP1), a member of the p120ctn subfamily of the armadillo (ARM)-repeat-containing proteins, is an important structural component of cell-cell adhesion scaffolds although it can also be ubiquitously found in the cytoplasm and the nucleus. RYBP (RING 1A and YY1 binding protein) is a multifunctional intrinsically disordered protein (IDP) best described as a transcriptional regulator. Both proteins are involved in the development and metastasis of several types of tumors. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with RYBP by using in cellulo methods, namely immunofluorescence (IF) and proximity ligation assay (PLA), and in vitro biophysical techniques, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), and isothermal titration calorimetry (ITC). We also characterized the binding of the two proteins by using in silico experiments. Our results showed that there was binding in tumor and non-tumoral cell lines. Binding in vitro between the two proteins was also monitored and found to occur with a dissociation constant in the low micromolar range (~10 µM). Finally, in silico experiments provided additional information on the possible structure of the binding complex, especially on the binding ARM-PKP1 hot-spot. Our findings suggest that RYBP might be a rescuer of the high expression of PKP1 in tumors, where it could decrease the epithelial-mesenchymal transition in some cancer cells.
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Proteínas Intrínsecamente Desordenadas , Placofilinas , Unión Proteica , Humanos , Placofilinas/metabolismo , Placofilinas/genética , Placofilinas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas del Dominio Armadillo/metabolismo , Proteínas del Dominio Armadillo/química , Proteínas del Dominio Armadillo/genética , Dominios Proteicos , Dicroismo CircularRESUMEN
MeCP2 is a general regulator of transcription involved in the repression/activation of genes depending on the local epigenetic context. It acts as a chromatin regulator and binds with exquisite specificity to gene promoters. The set of epigenetic marks recognized by MeCP2 has been already established (mainly, cytosine modifications in CpG and CpA), as well as many of the constituents of its interactome. We unveil a new set of interactions for MeCP2 with the four canonical nucleosomal histones. MeCP2 interacts with high affinity with H2A, H2B, H3 and H4. In addition, Rett syndrome associated mutations in MeCP2 and histone epigenetic marks modulate these interactions. Given the abundance and the structural/functional relevance of histones and their involvement in epigenetic regulation, this new set of interactions and its modulating elements provide a new addition to the 'alphabet' for this epigenetic reader.