RESUMEN
People generally need more support as they grow older to maintain healthy and active lifestyles. Older people living with chronic conditions are particularly dependent on healthcare services. Yet, in an increasingly digital society, there is a danger that efforts to drive innovations in eHealth will neglect the needs of those who depend on healthcare the most-our ageing population. The SHAPES (Smart and Healthy Ageing through People Engaging in Supportive Systems) Innovation Action aims to create an open European digital platform that facilitates the provision of meaningful, holistic support to older people living independently. A pan-European pilot campaign will evaluate a catalogue of digital solutions hosted on the platform that have been specifically adapted for older people. 'Medicines control and optimisation' is one of seven themes being explored in the campaign and will investigate the impact of digital solutions that aim to optimise medicines use by way of fostering effective self-management, while facilitating timely intervention by clinicians based on remote monitoring and individualised risk assessments powered by artificial intelligence. If successful, the SHAPES Innovation Action will lead to a greater sense of self-sufficiency and empowerment in people living with chronic conditions as they grow older.
RESUMEN
The pseudokinase TRIB1 controls cell function in a range of contexts, by regulating MAP kinase activation and mediating protein degradation via the COP1 ubiquitin ligase. TRIB1 regulates polarization of macrophages and dysregulated Trib1 expression in murine models has been shown to alter atherosclerosis burden and adipose homeostasis. Recently, TRIB1 has also been implicated in the pathogenesis of prostate cancer, where it is often overexpressed, even in the absence of genetic amplification. Well described TRIB1 effectors include MAP kinases and C/EBP transcription factors, both in immune cells and in carcinogenesis. However, the mechanisms that regulate TRIB1 itself remain elusive. Here, we show that the long and conserved 3'untranslated region (3'UTR) of TRIB1 is targeted by miRNAs in macrophage and prostate cancer models. By using a systematic in silico analysis, we identified multiple "high confidence" miRNAs potentially binding to the 3'UTR of TRIB1 and report that miR-101-3p and miR-132-3p are direct regulators of TRIB1 expression and function. Binding of miR-101-3p and miR-132-3p to the 3'UTR of TRIB1 mRNA leads to an increased transcription and secretion of interleukin-8. Our data demonstrate that modulation of TRIB1 by miRNAs alters the inflammatory profile of both human macrophages and prostate cancer cells.
Asunto(s)
Citocinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Macrófagos/metabolismo , MicroARNs/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Regiones no Traducidas 3' , Animales , Sitios de Unión , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Inflamación , Interleucina-8/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Transgénicos , MicroARNs/genética , Fenotipo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The heptapeptide angiotensin (Ang)-(1-7) is part of the beneficial arm of the renin-angiotensin system. Ang-(1-7) has cardiovascular protective effects, stimulates regeneration, and opposes the often detrimental effects of AngII. We recently identified the G protein-coupled receptors Mas and MrgD as receptors for the heptapeptide. Ala1-Ang-(1-7) (Alamandine), a decarboxylated form of Ang-(1-7), has similar vasorelaxant effects, but has been described as only stimulating MrgD. Therefore, this study aimed to characterise the consequences of the lack of the carboxyl group in amino acid 1 on intracellular signalling and to identify the receptor fingerprint for Ala1-Ang-(1-7). In primary endothelial and mesangial cells, Ala1-Ang-(1-7) elevated cAMP concentration. Dose response curves generated with Ang-(1-7) and Ala1-Ang-(1-7) significantly differed from each other, with a much lower EC50 and a bell-shape curve for Ala1-Ang-(1-7). We provided pharmacological proof that both, Mas and MrgD, are functional receptors for Ala1-Ang-(1-7). Consequently, in primary mesangial cells with genetic deficiency in both receptors, the heptapeptide failed to increase cAMP concentration. As we previously described for Ang-(1-7), the Ala1-Ang-(1-7)-mediated cAMP increase in Mas/MrgD-transfected HEK293 cells and primary cells was blocked by the AT2 receptor blocker, PD123319. The very distinct dose-response curves for both heptapeptides could be explained by in silico modelling, electrostatic potential calculations, and an involvement of Galpha i for higher concentrations of Ala1-Ang-(1-7). Our results identify Ala1-Ang-(1-7) as a peptide with specific pharmacodynamic properties and builds the basis for the design of more potent and efficient Ang-(1-7) analogues for therapeutic intervention in a rapidly growing number of diseases.
Asunto(s)
Angiotensina I/metabolismo , Oligopéptidos/farmacología , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Descarboxilación , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Células Mesangiales/efectos de los fármacos , Células Mesangiales/metabolismo , Ratones Noqueados , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Angiotensin (Ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental Ang II within the renin-angiotensin system. Although it is well accepted that the G-protein-coupled receptor Mas is a receptor for the heptapeptide, the lack in knowing initial signaling molecules stimulated by Ang-(1-7) prevented definitive characterization of ligand/receptor pharmacology as well as identification of further hypothesized receptors for the heptapeptide. The study aimed to identify a second messenger stimulated by Ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. Ang-(1-7) elevates cAMP concentration in primary cells, such as endothelial or mesangial cells. Using cAMP as readout in receptor-transfected human embryonic kidney (HEK293) cells, we provided pharmacological proof that Mas is a functional receptor for Ang-(1-7). Moreover, we identified the G-protein-coupled receptor MrgD as a second receptor for Ang-(1-7). Consequently, the heptapeptide failed to increase cAMP concentration in primary mesangial cells with genetic deficiency in both Mas and MrgD Mice deficient in MrgD showed an impaired hemodynamic response after Ang-(1-7) administration. Furthermore, we excluded the Ang II type 2 receptor as a receptor for the heptapeptide but discovered that the Ang II type 2 blocker PD123319 can also block Mas and MrgD receptors. Our results lead to an expansion and partial revision of the renin-angiotensin system, by identifying a second receptor for Ang-(1-7), by excluding Ang II type 2 as a receptor for the heptapeptide, and by enforcing the revisit of such publications which concluded Ang II type 2 function by only using PD123319.
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Adenilil Ciclasas/metabolismo , Angiotensina I/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hipertensión/enzimología , Fragmentos de Péptidos/farmacología , Adenilil Ciclasas/efectos de los fármacos , Animales , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Células HEK293/efectos de los fármacos , Células HEK293/metabolismo , Humanos , Hipertensión/tratamiento farmacológico , Masculino , Células Mesangiales/efectos de los fármacos , Células Mesangiales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfotransferasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Sensibilidad y EspecificidadRESUMEN
Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G(1) phase. Aberrant expression of CDK4 and CDK6 is a hallmark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM (the calcein acetoxymethyl-ester) is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G(1) phase. The metabolic effects of calcein AM on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phosphate pathway was significantly altered. To elucidate whether these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.
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The methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of the isoprenoid universal building blocks (isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)) is present in most of human pathogens and is absent in animals, turning it into a promising therapeutic druggable pathway. Two different strategies, a pharmacophore-directed virtual screening and a protein-protein interaction (PPI)-mimicking cyclic peptide were used to search for compounds that bind to the PPI surface of the 4-(cytidine 5-diphospho)-2C-methyl-D-erythritol kinase (CMK), which catalyzes the fourth step of the MEP pathway. A significant part of the pharmacophore hypothesis used in this study was designed by mimicking water-mediated PPI relevant in the CMK homodimer complex stabilization. After database search and with the aid of docking and molecular dynamics (MD) simulations, a 7H-furo[3,2-g]chromen-7-one derivative and a cyclic peptide were chosen as candidates to be ligands of CMK. Their binding affinities were measured using surface plasmon resonance (SPR) technology.
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Diseño de Fármacos , Proteínas de Escherichia coli/metabolismo , Ligandos , Imitación Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas de Escherichia coli/genética , Conformación Molecular , Simulación de Dinámica Molecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Unión Proteica , Solventes/química , Resonancia por Plasmón de SuperficieRESUMEN
The 2C-methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate, which are the precursors of isoprenoids, is present in plants, in the malaria parasite Plasmodium falciparum and in most eubacteria, including pathogenic agents. However, the MEP pathway is absent from fungi and animals, which have exclusively the mevalonic acid pathway. Given the characteristics of the MEP pathway, its enzymes represent potential targets for the generation of selective antibacterial, antimalarial and herbicidal molecules. We have focussed on the enzyme 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (CMK), which catalyses the fourth reaction step of the MEP pathway. A molecular dynamics simulation was carried out on the CMK dimer complex, and protein-protein interactions analysed, considering also water-mediated interactions between monomers. In order to find small molecules that bind to CMK and disrupt dimer formation, interactions observed in the dynamics trajectory were used to model a pharmacophore used in database searches. Using an intensity-fading matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry approach, one compound was found to interact with CMK. The data presented here indicate that a virtual screening approach can be used to identify candidate molecules that disrupt the CMK-CMK complex. This strategy can contribute to speeding up the discovery of new antimalarial, antibacterial, and herbicidal compounds.
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Proteínas de Escherichia coli/química , Fosfotransferasas/química , Multimerización de Proteína , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Solventes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Electricidad EstáticaRESUMEN
The tumor suppressor gene p16INK4a is commonly found altered in numerous and different types of cancer. The encoded protein arrests cell cycle in G1 phase by binding to CDK4 and CDK6, inhibiting their kinase function. In 1995, a 20-residue peptide, extracted from p16INK4a protein sequence, was discovered that retains the cell cycle inhibition properties of the endogenous tumor suppressor. However, its structure has not been determined yet. In this article, the features of a theoretical structure of the peptide bound to CDK6 are reported. The complex was modeled from CDK6-p16INK4a X-ray crystal structure and through molecular dynamics. Final structure was assessed by comparing computed binding free energy changes, when single-alanine substitutions were brought about on the peptide, to experimental data. Better concordance was obtained when including a high level of solvation effects. Solute-solvent vdW energy and electrostatic energy between solute and first shells of water, computed through a force field and considering explicit waters, were also to be included to achieve reasonably good concordance between theoretical and experimental data.
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Alanina/metabolismo , Quinasa 4 Dependiente de la Ciclina/química , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/química , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/química , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Alanina/química , Sitios de Unión , Simulación por Computador , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Electricidad Estática , Homología Estructural de Proteína , Termodinámica , AguaRESUMEN
Cyclin-dependent kinases 4, 6 and 2 (Cdk4/6/2), are proteins that lead progression through the G1-S transition, a step strictly regulated in the process of cell proliferation. The p16(INK4a) tumor suppressor, whose expression is inhibited in a high number of cancers, binds to Cdk4/6 and inhibits phosphorylation of the retinoblastoma protein, forcing cells to remain in the G1 phase and therefore, arresting cell division. Accordingly, the design of small compounds mimicking the inhibition of p16(INK4a) appears to be a promising way to treat cancer. In order to get some insight into the key interactions governing recognition between different cyclin-dependent kinases and the p16(INK4a) tumor suppressor, the present work reports the results of molecular dynamics simulations of both, the Cdk6-p16(INK4a) complex and the Cdk4-p16(INK4a) complex, respectively at 300 K. Most of the key interactions observed, were already anticipated in the analysis of the crystal structure of Cdk6-p16(INK4a). However, a few different features found out from the analysis of these calculations provide a better understanding of the role of the T-loop conformation, a fragment of Cdks, and the way the ATP binding-site is distorted upon binding of p16(INK4a).