Búsqueda de inhibidores frente a la proteína O-fucosiltransferasa 1 (PoFUT1)

La proteína O-fucosiltransferesa 1 (PoFUT1) es una fucosiltransferasa cuya función consiste en transferir un residuo fucosa a motivos tipo “epidermal growth factor like” (EGF) presentes en la proteína Notch. Esta modificación es esencial en el plegamiento de Notch y en la interacción con sus ligandos Delta y Serrate (Jagged en mamíferos). La vía de señalización de Notch está presente en numerosos procesos biológicos en eucariotas. Estudios recientes han demostrado que Notch aparece sobre-expresada en diversas enfermedades, como por ejemplo en algunos tipos de cáncer. Por tanto, la modificación post-traduccional producida por PoFUT1 es esencial para un correcto comportamiento de la proteína Notch y un correcto funcionamiento de la vía de señalización, lo que convierte a PoFUT1 en una diana terapéutica muy interesante. En nuestro grupo de investigación hemos descrito recientemente la estructura tridimensional de PoFUT1 de la especie Caenorhabditis elegans (CePOFUT1) en complejo con GDP-fucosa y GDP. La elección de esta especie como punto de partida resulta muy acertada, ya que es un enzima con una identidad del 100% con respecto a la humana a nivel de sitio activo, además de poder expresarse en grandes cantidades. En el presente trabajo se proponen cuatro compuestos como posibles inhibidores de la actividad fucosiltransferasa de CePOFUT1. Para ello se ha realizado un cribado de compuestos frente a la proteína utilizando la técnica de fluorescencia térmica. Además, se ha analizado el tipo de unión de los compuestos mediante técnicas de calorimetría, dicroísmo circular y resonancia magnética nuclear y se han obtenido cristales mediante cocristalización de los cuatro compuestos con la proteína. Este trabajo resulta ser novedoso y con gran proyección al ser el punto de partida en la búsqueda de fármacos contra diferentes enfermedades, así como en el estudio del papel de la O-fucosilación en distintas etapas del desarrollo embrionario y en distintas condiciones fisiológicas

Estudio y purificación de los sustratos aceptores de la proteína O-fucosiltransferasa

La O-fucosilación es una modificación post-traduccional que tiene lugar generalmente en un gran número de proteínas de membrana o secretadas. Múltiples estudios han revelado que esta modificación está implicada en la señalización celular y plegamiento de proteínas durante el desarrollo embrionario, así como en el metabolismo del tejido adulto. Entre los diferentes elementos implicados en el proceso de O-fucosilación destacan las glicosiltransferasas (GTs) POFUT1 y POFUT2 las cuales transfieren residuos de fucosa desde GDP-fucosa a sustratos aceptores específicos. Mientras POFUT1 fucosila proteínas conteniendo repeticiones con estructura similar al factor de crecimiento epidermal (EGF). POFUT2 fucosila las repeticiones de tromboespondinas tipo 1 (TSRs). En ambas repeticiones se fucosilan residuos de serina o treonina y están formadas por tres puentes disulfuro. Si bien tanto la estructura cristalina de POFUT2 como la de los dominios TSR ha sido resuelta mediante la difracción por rayos X en múltiples estudios, sigue sin conocerse con precisión el mecanismo de transferencia de cómo la POFUT2 reconoce los TSRs y de por tanto como se fucosilan. En este sentido optimizaremos la expresión y purificación de diferentes dominios TSR para posteriormente caracterizar cualitativa y cuantitativamente la interacción entre estos y la enzima POFUT2. Además, algunos de estos dominios se utilizarán para obtener una estructura cristalina junto con la POFUT2 con el fin de elucidar el mecanismo de reacción de la fucosilación y se cómo esta GT reconoce a sus sustratos aceptores

Structural Insights into the Mechanism of Protein O-Fucosylation

Protein O-fucosylation is an essential post-translational modification, involved in the folding of target proteins and in the role of these target proteins during embryonic development and adult tissue homeostasis, among other things. Two different enzymes are responsible for this modification, Protein O-fucosyltransferase 1 and 2 (POFUT1 and POFUT2, respectively). Both proteins have been characterised biologically and enzymatically but nothing is known at the molecular or structural level. Here we describe the first crystal structure of a catalytically functional POFUT1 in an apo-form and in complex with GDP-fucose and GDP. The enzyme belongs to the GT-B family and is not dependent on manganese for activity. GDP-fucose/GDP is localised in a conserved cavity connected to a large solvent exposed pocket, which we show is the binding site of epidermal growth factor (EGF) repeats in the extracellular domain of the Notch Receptor. Through both mutational and kinetic studies we have identified which residues are involved in binding and catalysis and have determined that the Arg240 residue is a key catalytic residue. We also propose a novel SN1-like catalytic mechanism with formation of an intimate ion pair, in which the glycosidic bond is cleaved before the nucleophilic attack; and theoretical calculations at a DFT (B3LYP/6-31+G(d,p) support this mechanism. Thus, the crystal structure together with our mutagenesis studies explain the molecular mechanism of POFUT1 and provide a new starting point for the design of functional inhibitors to this critical enzyme in the future

Deciphering the Non-Equivalence of Serine and Threonine O-Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody

© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at atomic resolution. For the α-O-GalNAc-Ser derivative, the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also observed in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.Peer Reviewe

Towards a rational design of Saccharomyces cerevisiae Gas2 glycomimetics

Resumen del trabajo presentado a la VII National Conference of the Institute for Biocomputation and Physics of Complex Systems (BIFI), celebrada en Zaragoza (España) del 4 al 6 de febrero de 2015.Glycosyl hydrolases and transglycosylases play a determinating role in fungal cell wall dynamics. Among these enzymes, the transglycosylases family remodeling the β-1,3 glucan have shown to be essential in yeast and in Asp. fumigatus. Therefore, these enzymes are highly interesting as therapeutic targets. To tackle a rationale design of glycomimetics for this family, we chose the Saccharomyces cerevisiae Gas2 (ScGas2) for practical purposes: its structure had been solved and it was very close to the essential Gel4 from Asp. Fumigatus. Even though ScGas2 shows the typical glycosyl hydrolase folding, its activity presents a balance among hydrolysis and transglycosylation depending on the length of the substrate. For example, ScGas2 is inactive against laminaripentaose but it is a pure hydrolase against laminarihexaose and heptaose and becomes a transglycosylase against larger-sized laminarioligosaccharides. In the case of Gel family, laminarinonaose was the minimal tested size to have transglycosylase activity. Here, a combination of docking studies, synthesis of glycomimetics, fluorescence spectroscopy and STD-NMR experiments reveal that a minimum of three glucose units linked via a β-(1,3) linkage and a hydrophobic radical are required to achieve molecular recognition at the binding donor and acceptor sites in ScGas2, respectively. Overall, our data set up the basis for the design of more potent glycomimetics that could be useful to inhibit essential enzymes such as Gel4.Peer reviewe

Synthesis of O- and C-glycosides derived from β-(1,3)-d-glucans

A series of β-(1,3)-d-glucans have been synthesized incorporating structural variations specifically on the reducing end of the oligomers. Both O- and C-glucosides derived from di- and trisaccharides have been obtained in good overall yields and with complete selectivity. Whereas the O-glycosides were obtained via a classical Koenigs-Knorr glycosylation, the corresponding C-glycosides were obtained through allylation of the anomeric carbon and further cross-metathesis reaction. Finally, the compounds were evaluated against two glycosidases and two endo-glucanases and no inhibitory activity was observed. © 2013 Elsevier Ltd. All rights reserved.This study was supported by the Ministerio de Ciencia e Innovación (MICINN) and FEDER Program (Madrid, Spain, projects CTQ2010-19606 and BFU2010-19504), and the Gobierno de Aragón (Zaragoza, Spain. Bioorganic Chemistry Group. E-10). E.M. and J.V.G. thank MEC for FPU and FPI predoctoral grants, respectively.Peer Reviewe

Study of the molecular recognition of novel fungal transglycosylase ligands

Resumen del trabajo presentado a la VII International Conference on Molecular Recognition, celebrada en Zaragoza (España) del 1 al 3 de febrero de 2016.Fungi cell wall remodeling is controlled by the equilibrium between glycoside hydrolases, glycosyltransferases, and transglycosylases. Family 72 glycoside hydrolases (GH72) are ubiquitous in fungal organisms and are known to possess significant transglycosylase activity, producing elongated beta(1-3) glucan chains. Among them are the Gas (in S. cerevisiae), Gel (in A. fumigates) or Phr and Pga (in C. albicans), and all of them with a well conserved catalytic site. The only protein whose structure has been resolved within this family is ScGas2, which will be our model for ligands and inhibitors design. In this communication, they will be presented the design, synthesis and the study of the molecular recognition of novel beta(1-3)glucan-based ligands with ScGas2. To characterize the protein-ligand interactions several techniques were employed, such as saturation transfer difference NMR experiments (STD-NMR), molecular docking, molecular dynamics, non-covalent-interaction calculation (NCI) and X-ray diffraction of protein complexes.Peer reviewe

Long-Term Prognostic Value of Cognitive Impairment on Top of Frailty in Older Adults after Acute Coronary Syndrome

Frailty is a marker of poor prognosis in older adults after acute coronary syndrome. We investigated whether cognitive impairment provides additional prognostic information. The study population consisted of a prospective cohort of 342 older (>65 years) adult survivors after acute coronary syndrome. Frailty (Fried score) and cognitive function (Pfeiffer’s Short Portable Mental Status Questionnaire—SPMSQ) were assessed at discharge. The endpoints were mortality or acute myocardial infarction at 8.7-year median follow-up. Patient distribution according to SPMSQ results was: no cognitive impairment (SPMSQ = 0 errors; n = 248, 73%), mild impairment (SPMSQ = 1–2 errors; n = 52, 15%), and moderate to severe impairment (SPMSQ ≥3 errors; n = 42, 12%). A total of 245 (72%) patients died or had an acute myocardial infarction, and 216 (63%) patients died. After adjustment for clinical data, comorbidities, and Fried score, the SPMSQ added prognostic value for death or myocardial infarction (per number of errors; HR = 1.11, 95%, CI 1.04–1.19, p = 0.002) and death (HR = 1.11, 95% 1.03–1.20, p = 0.007). An SPMSQ with ≥3 errors identified the highest risk subgroup. Geriatric conditions (SPSMQ and Fried score) explained 19% and 43% of the overall chi-square of the models for predicting death or myocardial infarction and death, respectively. Geriatric assessment after acute coronary syndrome should include both frailty and cognitive function. This is particularly important given that cognitive impairment without dementia can be subclinical and thus remain undetected

Inspiratory Muscle Training and Functional Electrical Stimulation for Treatment of Heart Failure With Preserved Ejection Fraction: The TRAINING-HF Trial

SEC 2017: Congreso de las Enfermedades CardiovascularesIntroducción y objetivos No se dispone de tratamientos farmacológicos que demuestren reducir la morbimortalidad asociada en pacientes con insuficiencia cardiaca y función sistólica conservada (IC-FEc). El objetivo del presente estudio fue evaluar si en pacientes con IC-FEc, el entrenamiento de la musculatura inspiratoria (EMI), la electroestimulación muscular funcional (EMF) o la combinación de ambas (EMI + EMF) puede mejorar la capacidad funcional, calidad de vida, parámetros de disfunción diastólica o biomarcadores a las 12 y 24 semanas. Métodos Un total de 61 pacientes estables con IC-FEc (clase funcional de la New York Heart Association II-III) se aleatorizaron (1:1:1:1) a recibir un programa de 12 semanas de EMI, EMF, o EMI + EMF frente a tratamiento médico estándar (control). El objetivo primario fue evaluar el cambio en el consumo máximo de oxígeno. Los objetivos secundarios fueron los cambios en la calidad de vida, parámetros ecocardiográficos y biomarcadores. Se utilizó un modelo lineal mixto para comparar los cambios entre los diferentes grupos. Resultados La edad media fue 74 ± 9 años y la proporción de mujeres fue del 58%. El test de consumo máximo de oxígeno fue de 9,9 ± 2,5ml/min/kg. A las 12 semanas, con respecto al grupo control, el incremento medio de consumo máximo de oxígeno fue de 2,98, 2,93 y 2,47 para EMI, EMF y EMI + EMF, respectivamente (p < 0,001). Este incremento se mantuvo a las 24 semanas (1,95, 2,08 y 1,56, respectivamente; p < 0,001). Resultados similares se observaron en la puntuación del cuestionario de calidad de vida (p < 0,001). Conclusiones En los pacientes con IC-FEc e importante reducción de la capacidad funcional, tanto el EMI como la EMF se asocian con una marcada mejoría de la capacidad funcional y la calidad de vida.Introduction and objectives Despite the prevalence of heart failure with preserved ejection fraction (HFpEF), there is currently no evidence-based effective therapy for this disease. This study sought to evaluate whether inspiratory muscle training (IMT), functional electrical stimulation (FES), or a combination of both (IMT + FES) improves 12- and 24-week exercise capacity as well as left ventricular diastolic function, biomarker profile, and quality of life in HFpEF. Methods A total of 61 stable symptomatic patients (New York Heart Association functional class II-III) with HFpEF were randomized (1:1:1:1) to receive a 12-week program of IMT, FES, or IMT + FES vs usual care. The primary endpoint of the study was to evaluate change in peak exercise oxygen uptake at 12 and 24 weeks. Secondary endpoints were changes in quality of life, echocardiogram parameters, and prognostic biomarkers. We used a mixed-effects model for repeated-measures to compare endpoints changes. Results Mean age and peak exercise oxygen uptake were 74 ± 9 years and 9.9 ± 2.5mL/min/kg, respectively. The proportion of women was 58%. At 12 weeks, the mean increase in peak exercise oxygen uptake (mL/kg/min) compared with usual care was 2.98, 2.93, and 2.47 for IMT, FES, and IMT + FES, respectively (P < .001) and this beneficial effect persisted after 24 weeks (1.95, 2.08, and 1.56; P < .001). Significant increases in quality of life scores were found at 12 weeks (P < .001). No other changes were found. Conclusions In HFpEF patients with low aerobic capacity, IMT and FES were associated with a significant improvement in exercise capacity and quality of life

Clinical Predictors and Prognosis of Myocardial Infarction with Non-Obstructive Coronary Arteries (MINOCA) without ST-Segment Elevation in Older Adults

A non-neglectable percentage of patients with non-ST elevation myocardial infarction (NSTEMI) show non-obstructive coronary arteries (MINOCA). Specific data in older patients are scarce. We aimed to identify the clinical predictors of MINOCA in older patients admitted for NSTEMI and to explore the long-term prognosis of MINOCA. This was a single-center, observational, consecutive cohort study of older (&ge;70 years) patients admitted for NSTEMI between 2010 and 2014 who underwent coronary angiography. Univariate and multivariate Cox regression were performed to analyze the association of variables with MINOCA and all-cause mortality and with major adverse cardiac events (MACE), defined as a combined endpoint of all-cause mortality and nonfatal myocardial infarction and a combined endpoint of cardiovascular mortality, nonfatal myocardial infarction, and unplanned revascularization. The registry included 324 patients (mean age 78.8 &plusmn; 5.4 years), of which 71 (21.9%) were diagnosed with MINOCA. Predictors of MINOCA were female sex, left bundle branch block, pacemaker rhythm, chest pain at rest, peak troponin level, previous MI, Killip &ge;2, and ST segment depression. Regarding prognosis, patients with obstructive coronary arteries (stenosis &ge;50%) and the subgroup of MINOCA patients with plaques &lt;50% had a similar prognosis; while MINOCA patients with angiographically smooth coronary arteries had a reduced risk of MACE. We conclude that the following: (1) in elderly patients admitted for NSTEMI, certain universally available clinical, electrocardiographic, and analytical variables are associated with the diagnosis of MINOCA; (2) elderly patients with MINOCA have a better prognosis than those with obstructive coronary arteries; however, only those with angiographically smooth coronary arteries have a reduced risk of all-cause mortality and MACE