RESUMEN
Targeting of epigenetic mechanisms, such as the hydroxymethylation of DNA, has been intensively studied, with respect to the treatment of many serious pathologies, including oncological disorders. Recent studies demonstrated that promising therapeutic strategies could potentially be based on the inhibition of the TET1 protein (ten-eleven translocation methylcytosine dioxygenase 1) by specific iron chelators. Therefore, in the present work, we prepared a series of pyrrolopyrrole derivatives with hydrazide (1) or hydrazone (2-6) iron-binding groups. As a result, we determined that the basic pyrrolo[3,2-b]pyrrole derivative 1 was a strong inhibitor of the TET1 protein (IC50 = 1.33 µM), supported by microscale thermophoresis and molecular docking. Pyrrolo[3,2-b]pyrroles 2-6, bearing substituted 2-hydroxybenzylidene moieties, displayed no significant inhibitory activity. In addition, in vitro studies demonstrated that derivative 1 exhibits potent anticancer activity and an exclusive mitochondrial localization, confirmed by Pearson's correlation coefficient of 0.92.
Asunto(s)
Dioxigenasas , Pirroles , ADN , Dioxigenasas/metabolismo , Hidrazonas/química , Hierro , Quelantes del Hierro , Proteínas Mitocondriales , Simulación del Acoplamiento Molecular , Pirroles/química , Pirroles/farmacologíaRESUMEN
COVID-19 is a pandemic respiratory disease caused by the SARS-CoV-2 coronavirus. The worldwide epidemiologic data showed higher mortality in males compared to females, suggesting a hypothesis about the protective effect of estrogens against severe disease progression with the ultimate end being patient's death. This article summarizes the current knowledge regarding the potential effect of estrogens and other modulators of estrogen receptors on COVID-19. While estrogen receptor activation shows complex effects on the patient's organism, such as an influence on the cardiovascular/pulmonary/immune system which includes lower production of cytokines responsible for the cytokine storm, the receptor-independent effects directly inhibits viral replication. Furthermore, it inhibits the interaction of IL-6 with its receptor complex. Interestingly, in addition to natural hormones, phytestrogens and even synthetic molecules are able to interact with the estrogen receptor and exhibit some anti-COVID-19 activity. From this point of view, estrogen receptor modulators have the potential to be included in the anti-COVID-19 therapeutic arsenal.
Asunto(s)
COVID-19/patología , Moduladores de los Receptores de Estrógeno/farmacología , SARS-CoV-2/efectos de los fármacos , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , COVID-19/complicaciones , COVID-19/virología , Moduladores de los Receptores de Estrógeno/metabolismo , Moduladores de los Receptores de Estrógeno/uso terapéutico , Femenino , Humanos , Receptores de Estrógenos/química , Receptores de Estrógenos/metabolismo , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/fisiología , Proteínas de la Matriz Viral/antagonistas & inhibidores , Proteínas de la Matriz Viral/metabolismo , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Interleukin-6 (IL-6) is a cytokine with multifaceted effects playing a remarkable role in the initiation of the immune response. The increased level of this cytokine in the elderly seems to be associated with the chronic inflammatory setting of the microenvironment in aged individuals. IL-6 also represents one of the main signals in communication between cancer cells and their non-malignant neighbours within the tumour niche. IL-6 also participates in the development of a premetastatic niche and in the adjustment of the metabolism in terminal-stage patients suffering from a malignant disease. IL-6 is a fundamental factor of the cytokine storm in patients with severe COVID-19, where it is responsible for the fatal outcome of the disease. A better understanding of the role of IL-6 under physiological as well as pathological conditions and the preparation of new strategies for the therapeutic control of the IL-6 axis may help to manage the problems associated with the elderly, cancer, and serious viral infections.
Asunto(s)
Envejecimiento/metabolismo , Infecciones por Coronavirus/metabolismo , Interleucina-6/metabolismo , Neoplasias/metabolismo , Neumonía Viral/metabolismo , Envejecimiento/patología , Animales , COVID-19 , Infecciones por Coronavirus/patología , Humanos , Interleucina-6/genética , Neoplasias/patología , Pandemias , Neumonía Viral/patología , Transducción de SeñalRESUMEN
The tail of Caudovirales bacteriophages serves as an adsorption device, a host cell wall-perforating machine, and a genome delivery pathway. In Siphoviridae, the assembly of the long and flexible tail is a highly cooperative and regulated process that is initiated from the proteins forming the distal tail tip complex. In Gram-positive-bacterium-infecting siphophages, the distal tail (Dit) protein has been structurally characterized and is proposed to represent a baseplate hub docking structure. It is organized as a hexameric ring that connects the tail tube and the adsorption device. In this study, we report the characterization of pb9, a tail tip protein of Escherichia coli bacteriophage T5. By immunolocalization, we show that pb9 is located in the upper part of the cone of the T5 tail tip, at the end of the tail tube. The crystal structure of pb9 reveals a two-domain protein. Domain A exhibits remarkable structural similarity with the N-terminal domain of known Dit proteins, while domain B adopts an oligosaccharide/oligonucleotide-binding fold (OB-fold) that is not shared by these proteins. We thus propose that pb9 is the Dit protein of T5, making it the first Dit protein described for a Gram-negative-bacterium-infecting siphophage. Multiple sequence alignments suggest that pb9 is a paradigm for a large family of Dit proteins of siphophages infecting mostly Gram-negative hosts. The modular structure of the Dit protein maintains the basic building block that would be conserved among all siphophages, combining it with a more divergent domain that might serve specific host adhesion properties.
Asunto(s)
Bacteriófagos/química , Siphoviridae/química , Proteínas de la Cola de los Virus/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Cristalografía por Rayos X , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Siphoviridae/genética , Siphoviridae/metabolismo , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/metabolismoRESUMEN
Immune checkpoints regulate the immune system response. Recent studies suggest that flavonoids, known as phytoestrogens, may inhibit the PD-1/PD-L1 axis. We explored the potential of estrogens and 17 Selective Estrogen Receptor Modulators (SERMs) as inhibiting ligands for immune checkpoint proteins (CTLA-4, PD-L1, PD-1, and CD80). Our docking studies revealed strong binding energy values for quinestrol, quercetin, and bazedoxifene, indicating their potential to inhibit PD-1 and CTLA-4. Quercetin and bazedoxifene, known to modulate EGFR and IL-6R alongside estrogen receptors, can influence the immune checkpoint functionality. We discuss the impact of SERMs on PD-1 and CTLA-4, suggesting that these SERMs could have therapeutic effects through immune checkpoint inhibition. This study highlights the potential of SERMs as inhibitory ligands for immune checkpoint proteins, emphasizing the importance of considering PD-1 and CTLA-4 inhibition when evaluating SERMs as therapeutic agents. Our findings open new avenues for cancer immunotherapy by exploring the interaction between various SERMs and immune checkpoint pathways.
Asunto(s)
Proteínas de Punto de Control Inmunitario , Neoplasias , Humanos , Antígeno CTLA-4 , Antígeno B7-H1 , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Receptor de Muerte Celular Programada 1 , Moduladores de los Receptores de Estrógeno , Quercetina , Inmunoterapia , Neoplasias/terapiaRESUMEN
Mitochondrial dysregulation plays a significant role in the carcinogenesis. On the other hand, its destabilization strongly represses the viability and metastatic potential of cancer cells. Photodynamic and photothermal therapies (PDT and PTT) target mitochondria effectively, providing innovative and non-invasive anticancer therapeutic modalities. Cyanine dyes, with strong mitochondrial selectivity, show significant potential in enhancing PDT and PTT. The potential and limitations of cyanine dyes for mitochondrial PDT and PTT are discussed, along with their applications in combination therapies, theranostic techniques, and optimal delivery systems. Additionally, novel approaches for sonodynamic therapy using photoactive cyanine dyes are presented, highlighting advances in cancer treatment.
RESUMEN
The mechanisms by which myelodysplastic syndrome (MDS) cells resist the effects of hypomethylating agents (HMA) are currently the subject of intensive research. A better understanding of mechanisms by which the MDS cell becomes to tolerate HMA and progresses to acute myeloid leukemia (AML) requires the development of new cellular models. From MDS/AML cell lines we developed a model of 5-azacytidine (AZA) resistance whose stability was validated by a transplantation approach into immunocompromised mice. When investigating mRNA expression and DNA variants of the AZA resistant phenotype we observed deregulation of several cancer-related pathways including the phosphatidylinosito-3 kinase signaling. We have further shown that these pathways can be modulated by specific inhibitors that, while blocking the proliferation of AZA resistant cells, are unable to increase their sensitivity to AZA. Our data reveal a set of molecular mechanisms that can be targeted to expand therapeutic options during progression on AZA therapy.
Asunto(s)
Azacitidina/farmacología , Resistencia a Antineoplásicos , Modelos Biológicos , Animales , ADN de Neoplasias/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Ratones , Ratones SCID , Anotación de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
Lanthanoid ions exhibit extremely large anomalous X-ray scattering at their L(III) absorption edge. They are thus well suited for anomalous diffraction experiments. A novel class of lanthanoid complexes has been developed that combines the physical properties of lanthanoid atoms with functional chemical groups that allow non-covalent binding to proteins. Two structures of large multimeric proteins have already been determined by using such complexes. Here the use of the luminescent europium tris-dipicolinate complex [Eu(DPA)(3)](3-) to solve the low-resolution structure of a 444 kDa homododecameric aminopeptidase, called PhTET1-12s from the archaea Pyrococcus horikoshii, is reported. Surprisingly, considering the low resolution of the data, the experimental electron density map is very well defined. Experimental phases obtained by using the lanthanoid complex lead to maps displaying particular structural features usually observed in higher-resolution maps. Such complexes open a new way for solving the structure of large molecular assemblies, even with low-resolution data.
Asunto(s)
Aminopeptidasas/química , Proteínas Arqueales/química , Complejos de Coordinación/química , Cristalografía por Rayos X/métodos , Elementos de la Serie de los Lantanoides/química , Cristalización , Sustancias Macromoleculares , Pyrococcus horikoshii/enzimología , Dispersión de Radiación , Difracción de Rayos XRESUMEN
The structure of a 468 kDa peptidase complex from the hyperthermophile Pyrococcus horikoshii has been solved at 1.9 A resolution. The monomer contains the M42 peptidase typical catalytic domain, and a dimerization domain that allows the formation of dimers that assemble as a 12-subunit self-compartmentalized tetrahedron, similar to those described for the TET peptidases. The biochemical analysis shows that the enzyme is cobalt-activated and cleaves peptides by a non-processive mechanism. Consequently, this protein represents the third TET peptidase complex described in P. horikoshii, thereby called PhTET3. It is a lysyl aminopeptidase with a strong preference for basic residues, which are poorly cleaved by PhTET1 and PhTET2. The structural analysis of PhTET3 and its comparison with PhTET1 and PhTET2 unravels common features explaining the general mode of action of the TET molecular machines as well as differences that can be associated with strong substrate discriminations. The question of the stability of the TET assemblies under extreme temperatures has been addressed. PhTET3 displays its maximal activity at 95 degrees C and small-angle neutron scattering experiments at 90 degrees C demonstrate the absence of quaternary structure alterations after extensive incubation times. In conclusion, PhTETs are complementary peptide destruction machines that may play an important role in the metabolism of P. horikoshii.
Asunto(s)
Aminopeptidasas/metabolismo , Proteínas Arqueales/metabolismo , Pyrococcus horikoshii/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/genética , Aminopeptidasas/aislamiento & purificación , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Cobalto/metabolismo , Calor , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Pyrococcus horikoshii/genéticaRESUMEN
The crystal structure of the glycosomal enzyme pyruvate phosphate dikinase from the African protozoan parasite Trypanosoma brucei has been solved to 3.0 A resolution by molecular replacement. The search model was the 2.3 A resolution structure of the Clostridium symbiosum enzyme. Due to different relative orientations of the domains and sub-domains in the two structures, molecular replacement could be achieved only by positioning these elements (four bodies altogether) sequentially in the asymmetric unit of the P2(1)2(1)2 crystal, which contains one pyruvate phosphate dikinase (PPDK) subunit. The refined model, comprising 898 residues and 188 solvent molecules per subunit, has a crystallographic residual index Rf = 0.245 (cross-validation residual index Rfree = 0.291) and displays satisfactory stereochemistry. Eight regions, comprising a total of 69 amino acid residues at the surface of the molecule, are disordered in this crystal form. The PPDK subunits are arranged around the crystallographic 2-fold axis as a dimer, analogous to that observed in the C. symbiosum enzyme. Comparison of the two structures was carried out by superposition of the models. Although the fold of each domain or sub-domain is similar, the relative orientations of these constitutive elements are different in the two structures. The trypanosome enzyme is more "bent" than the bacterial enzyme, with bending increasing from the center of the molecule (close to the molecular 2-fold axis) towards the periphery where the N-terminal domain is located. As a consequence of this increased bending and of the differences in relative positions of subdomains, the nucleotide-binding cleft in the amino-terminal domain is wider in T. brucei PPDK: the N-terminal fragment of the amino-terminal domain is distant from the catalytic, phospho-transfer competent histidine 482 (ca 10 A away). Our observations suggest that the requirements of domain motion during enzyme catalysis might include widening of the nucleotide-binding cleft to allow access and departure of the AMP or ATP ligand.
Asunto(s)
Piruvato Ortofosfato Diquinasa/química , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Piruvato Ortofosfato Diquinasa/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Trypanosoma brucei brucei/químicaRESUMEN
The three-dimensional crystal structure of the (R207S, R292S) mutant of malate dehydrogenase from Haloarcula marismortui was solved at 1.95A resolution in order to determine the role of salt bridges and solvent ions in halophilic adaptation and quaternary structure stability. The mutations, located at the dimer-dimer interface, disrupt two inter-dimeric salt bridge clusters that are essential for wild-type tetramer stabilisation. Previous experiments in solution, performed on the double mutant, had shown a tetrameric structure in 4M NaCl, which dissociated into active dimers in 2M NaCl. In order to establish if the active dimeric form is a product of the mutation, or if it also exists in the wild-type protein, complementary studies were performed on the wild-type enzyme by analytical centrifugation and small angle neutron scattering experiments. They showed the existence of active dimers in NaF, KF, Na(2)SO(4), even in the absence of NADH, and in the presence of NADH at concentrations of NaCl below 0.3M. The crystal structure shows a tetramer that, in the absence of the salt bridge clusters, appears to be stabilized by a network of ordered water molecules and by Cl(-) binding at the dimer-dimer interface. The double mutant and wild-type dimer folds are essentially identical (the r.m.s. deviation between equivalent C(alpha) positions is 0.39A). Chloride ions are also observed at the monomer-monomer interfaces of the mutant, contributing to the stability of each dimer against low salt dissociation. Our results support the hypothesis that extensive binding of water and salt is an important feature of adaptation to a halophilic environment.
Asunto(s)
Haloarcula marismortui/enzimología , Malato Deshidrogenasa/química , Solventes/química , Biopolímeros , Cristalografía por Rayos X , Dimerización , Malato Deshidrogenasa/metabolismo , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The crystal structure of malate dehydrogenase from the hyperthermophilic archaeon Archeoglobus fulgidus, in complex with its cofactor NAD, was solved at 2.9A resolution. The crystal structure shows a compact homodimer with one coenzyme bound per subunit. The substrate binding site is occupied by a sulphate ion. In order to gain insight into adaptation mechanisms, which allow the protein to be stable and active at high temperatures, the 3D structure was compared to those of several thermostable and hyperthermostable homologues, and to halophilic malate dehydrogenase. The hyperthermostable A. fulgidus MalDH protein displays a reduction of the solvent-exposed surface, an optimised compact hydrophobic core, a high number of hydrogen bonds, and includes a large number of ion pairs at the protein surface. These features occur concomitantly with a reduced number of residues in the protein subunit, due to several deletions in loop regions. The loops are further stiffened by ion pair links with secondary structure elements. A. fulgidus malate dehydrogenase is the only dimeric protein known to date that belongs to the [LDH-like] MalDH family. All the other known members of this family are homo-tetramers. The crystal structures revealed that the association of the dimers to form tetramers is prevented by several deletions, taking place at the level of two loops that are known to be essential for the tetramerisation process within the LDH and [LDH-like] MalDH enzymes.
Asunto(s)
Archaeoglobus fulgidus/enzimología , Malato Deshidrogenasa/química , Cristalografía por Rayos X , Dimerización , Estabilidad de Enzimas , Estructura Molecular , NAD/química , Conformación Proteica , Homología Estructural de Proteína , TemperaturaRESUMEN
Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate with concomitant oxidation of NADH during the last step in anaerobic glycolysis. In the present study, we present a comparative biochemical and structural analysis of various LDHs adapted to function over a large temperature range. The enzymes were from Champsocephalus gunnari (an Antarctic fish), Deinococcus radiodurans (a mesophilic bacterium) and Thermus thermophilus (a hyperthermophilic bacterium). The thermodynamic activation parameters of these LDHs indicated that temperature adaptation from hot to cold conditions was due to a decrease in the activation enthalpy and an increase in activation entropy. The crystal structures of these LDHs have been solved. Pairwise comparisons at the structural level, between hyperthermophilic versus mesophilic LDHs and mesophilic versus psychrophilic LDHs, have revealed that temperature adaptation is due to a few amino acid substitutions that are localized in critical regions of the enzyme. These substitutions, each having accumulating effects, play a role in either the conformational stability or the local flexibility or in both. Going from hot- to cold-adapted LDHs, the various substitutions have decreased the number of ion pairs, reduced the size of ionic networks, created unfavorable interactions involving charged residues and induced strong local disorder. The analysis of the LDHs adapted to extreme temperatures shed light on how evolutionary processes shift the subtle balance between overall stability and flexibility of an enzyme.
Asunto(s)
Adaptación Fisiológica , Bacterias Anaerobias Gramnegativas/enzimología , Calor , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Estabilidad de Enzimas , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Propiedades de SuperficieRESUMEN
Intense synchrotron radiation produces specific structural and chemical damage to crystalline proteins even at 100 K. Carboxyl groups of acidic residues (Glu, Asp) losing their definition is one of the major effects observed. Here, the susceptibilities to X-ray damage of acidic residues in tetrameric malate dehydrogenase from Haloarcula marismortui are investigated. The marked excess of acidic residues in this halophilic enzyme makes it an ideal target to determine how specific damage to acidic residues is related to their structural and chemical environment. Four conclusions are drawn. (i) Acidic residues interacting with the side-chains of lysine and arginine residues are less affected by radiation damage than those interacting with serine, threonine and tyrosine side-chains. This suggests that residues with higher pK(a) values are more vulnerable to damage than those with a lower pK(a). However, such a correlation was not found when calculated pK(a) values were inspected. (ii) Acidic side-chains located in the enzymatic active site are the most radiation-sensitive ones. (iii) Acidic residues in the internal cavity formed by the four monomers and those involved in crystal contacts appear to be particularly susceptible. (iv) No correlation was found between radiation susceptibility and solvent accessibility.
Asunto(s)
Aminoácidos/química , Aminoácidos/efectos de la radiación , Cristalografía por Rayos X/métodos , Haloarcula marismortui/química , Malato Deshidrogenasa/química , Malato Deshidrogenasa/efectos de la radiación , Modelos Químicos , Simulación por Computador , Relación Dosis-Respuesta en la Radiación , Concentración de Iones de Hidrógeno , Malato Deshidrogenasa/ultraestructura , Modelos Moleculares , Conformación Proteica/efectos de la radiación , Desnaturalización Proteica/efectos de la radiación , Dosis de Radiación , Soluciones , Solventes/química , Relación Estructura-Actividad , Rayos XRESUMEN
Cellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data. The internal organization of the PhTET1 particles reveals highly self-compartmentalized systems made of networks of access channels extended by vast catalytic chambers. The two edifices display aminopeptidase activity, and their organizations indicate substrate navigation mechanisms different from those described in other large peptidase complexes. Compared with the tetrahedron, the octahedron forms a more expanded hollow structure, representing a new type of giant peptidase complex. PhTET1 assembles into two different quaternary structures because of quasi-equivalent contacts that previously have only been identified in viral capsids.
Asunto(s)
Aminopeptidasas/química , Pyrococcus/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/metabolismo , Cápside/química , Catálisis , Microscopía por Crioelectrón , Cristalografía por Rayos X , Dimerización , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Conformación Proteica , Estructura Cuaternaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The crystal structure of the sulfolactate dehydrogenase from the hyperthermophilic and methanogenic archaeon Methanocaldococcus jannaschii was solved at 2.5 A resolution (PDB id. 1RFM). The asymmetric unit contains a tetramer of tight dimers. This structure, complexed with NADH, does not contain a cofactor-binding domain with 'Rossmann-fold' topology. Instead, the tertiary and quaternary structures indicate a novel fold. The NADH is bound in an extended conformation in each active site, in a manner that explains the pro-S specificity. Cofactor binding involves residues belonging to both subunits within the tight dimers, which are therefore the smallest enzymatically active units. The protein was found to be a homodimer in solution by size-exclusion chromatography, analytical ultracentrifugation and small-angle neutron scattering. Various compounds were tested as putative substrates. The results indicate the existence of a substrate discrimination mechanism, which involves electrostatic interactions. Based on sequence homology and phylogenetic analyses, several other enzymes were classified as belonging to this novel family of homologous (S)-2-hydroxyacid dehydrogenases.