Biología molecular y celular de la alteración de la homeostasis del pirofosfato inorgánico en eucariotas

Texto completo descargado desde TeseoEl PPi es un componente celular que se genera en muchas reacciones anabólicas de biosíntesis de polímeros y metabolitos (Geigenberger, Hajirezaei et al. 1998; Stitt 1998; Rojas-Beltrán, Dubois et al. 1999; Farre, Bachmann et al. 2001; Sonnewald 2001). Su hidrólisis es esencial para que esas reacciones discurran en la dirección correcta y se regenere el Pi necesario para las reacciones de fosforilación. La hidrólisis del PPi es una reacción bioquímica universal catalizada por pirofosfatasas inorgánicas (PPasas, EC 3.6.1.1). Existen algunos estudios que muestran la necesidad de PPasas para el correcto desarrollo del anabolismo celular (Heinonen et al., 2001), así como el efecto negativo de la deficiencia de estas enzimas tanto en procariotas como en eucariotas. Sin embargo, estos estudios no van más allá de postular que la carencia sPPasa genera parada del crecimiento en procariotas (Escherichia coli) (Chen et al., 1990) y es letal en eucariotas (Saccharomyces cerevisiae) (Giaever et al., 2002). En este trabajo de Tesis doctoral, se ha realizado un estudio detallado del mecanismo molecular de la toxicidad del exceso de PPi producido por la deficiencia en la sPPasa en eucariotas, así como sobre el papel fisiológico y bioquímico de dichas enzimas en los diferentes subcompartimentos celulares en los que se localizan. Objetivos: 1. Determinar el mecanismo fisiológico de la toxicidad por exceso de PPi en el modelo eucariota S. cerevisiae. 2. Analizar la relevancia de la localización núcleo-citosólica de la sPPasa Ipp1p en S. cerevisiae. 3. Expresar heterologamente diferentes sPPasas en S. cerevisiae y líneas celulares de mamífero. 4. Reproducir en la levadura el escenario metabólico del PPi citosólico de células vegetales. Conclusiones: 1. La ausencia de la sPPasa Ipp1p en la levaduras S. cerevisiae con metabolismo fermentativo genera muerte celular por autofagia, siendo uno de los mecanismos implicados el desbalance de la razón NAD+/NADH debido a la inhibición de la síntesis de NAD+ por el exceso de PPi. 2. La ausencia de Ipp1p genera parada del ciclo celular en fase S en S. cerevisiae con metabolismo respiratorio. Esta parada es reversible si se retira el exceso de PPi y se reactiva la sPPasa. 3. Ipp1p es una enzima núcleo-citosólica, cuyos niveles tanto de proteína como de actividad catalítica son menores en el compartimento nuclear que en el citosol. Esta diferencia se debe a una estricta regulación de los niveles proteícos de la Ipp1p nuclear, mediante degradación proteasómica. Esta regulación es dependiente de la actividad catalítica de hidrólisis de PPi y no de los niveles de polipéptido en sí. 4. La alta homología tanto en la conformación espacial del sitio activo, como del mecanismo catalítico, permite intercambiar sPPasas de diversas procedencias y diferentes familias en sistemas modelo eucarióticos como levadura o células de mamífero. 5. La compartimentalización nuclear de Ipp1p permite la eficiente complementación en levadura de la deficiencia en V-ATPasa mediante la expresión de una H+-PPasa localizada en la membrana vacuolar, pudiendo ser este un escenario metabólico del PPi similar al que tiene lugar en células vegetales

Intracellular proton pumps as targets in chemotherapy: V-ATPases and cancer

Cancer cells show a metabolic shift that makes them overproduce protons; this has the potential to disturb the cellular acid-base homeostasis. However, these cells show cytoplasmic alkalinisation, increased acid extrusion and endosome-dependent drug resistance. Vacuolar type ATPases (V-ATPases), toghether with other transporters, are responsible to a great extent for these symptoms. These multisubunit proton pumps are involved in the control of cytosolic pH and the generation of proton gradients (positive inside) across endocellular membrane systems like Golgi, endosomes or lysosomes. In addition, in tumours, they have been determined to play an important role in the acidification of the intercellular medium. This importance makes them an attractive target for control of tumour cells. In the present review we portray the major characteristics of this kind of proton pumps, we provide some recent insights on their in vivo regulation, an overview of the consequences that V-ATPase inhibition carries for the tumour cell, such as cell cycle arrest or cell death, and a brief summary of the studies related to cancer made recently with commercially available inhibitors for this kind of proton pump. Some new approaches to affect V-ATPase function are also suggested in the light of recent knowledge on the regulation of this proton pump.Junta de Andalucía PAIDI BIO-261 P07-CVI-3082Ministerio de Ciencia e Innovación BFU2007-61887 BFU2010-1562

Intraorganellar acidification by V-ATPases: a target in cellproliferation and cancer therapy

V-ATPases are multicomponent proton pumps involved in the acidification of singlemembrane intracellular compartments such as endosomes and lysosomes. They couple thehydrolysis of ATP to the translocation of one to two protons across the membrane. Acidification ofthe lumen of single membrane organelles is a necessary factor for the correct traffic of membranesand cargo to and from the different internal compartments of a cell. Also, V-ATPases are involved inregulation of pH at the cytosol and, possibly, extracellular milieu. The inhibition of V-ATPases hasbeen shown to induce apoptosis and cell cycle arrest in tumour cells and, therefore, chemicals thatbehave as inhibitors of this kind of proton pumps have been proposed as putative treatment agentsagainst cancer. The present review will summarize the major types of V-ATPase inhibitors and theirmechanisms of action and put them in relation to the patents registered so far for the treatment of cancer.Junta de Andalucía PAIDI BIO-261 P07-CVI-3082Ministerio de Ciencia e Innovación BMC2007-6188

Nutrición, inflamación y riesgo metabólico en niños y adolescentes europeos

El estudio de los factores de riesgo cardio-metabólicos es importante para intentar prevenir enfermedades futuras. Estos factores de riesgo aparecen cada vez a edades más tempranas como en la adolescencia, o incluso en la infancia, y parecen estar asociados con algunos estilos de vida como la alimentación. Se ha observado que la la inflamación crónica de bajo grado se relaciona con factores de riesgo cardio-metabólicos. Por lo tanto, el estudio del estado inflamatorio en niños y adolescentes es necesario para evaluar esta relación desde sus orígenes y, de esta manera, poder entender sus mecanismos de aparición. Es por ello que el objetivo general de esta Tesis Doctoral es evaluar la relación entre la inflamación, valorada mediante una serie de marcadores inflamatorios, la ingesta y las alteraciones cardio-metabólicas asociadas con la obesidad en niños y adolescentes europeos. Esta memoria se ha realizado por compendio de publicaciones, incluyendo seis artículos.La presente Tesis Doctoral se ha llevado a cabo teniendo en cuenta los resultados de dos grandes estudios europeos: el estudio IDEFICS (Identification and Prevention of Dietary- and Lifestyle- induced Health Effects in Children and Infants) y el estudio HELENA (Healthy Lifestyle in Europe by Nutrition in Adolescence). En el estudio IDEFICS se obtuvo información de más de 16.000 niños, con edades comprendidas entre 2 y 9 años, procedentes de ocho países europeos (Italia, Estonia, Chipre, Bélgica, Suecia, Alemania, Hungría y España). La medida incial se realizó durante el curso 2007-2008. Estos niños fueron re-evaluados dos años después del incio del estudio. Se seleccionaron sujetos de este estudio para valorar la asociación entre la dieta y la proteína C-reactiva de alta sensibilidad (PCR-hs), como marcador de inflamación. En el primer artículo se valoró la asociación entre los ácidos grasos, medidos en sangre total, y la inflamación. Los ácidos grasos son componentes de la dieta que se relacionan con el estado inflamatorio, especialmente en el caso de los ácidos grasos de cadena larga. Se observó que los acidos grasos omega-6 (suma de omega 6 y ácido linoleico) se asociaron con valores bajos de PCR-hs, en chicos, y con valores altos de PCR-hs en chicas (ácido araquidónico, suma de omega 6 altamente insaturados y relación acido araquidónico/linoleico). En el segundo artículo se observó una asociación clara entre la frecuencia de consumo de algunos alimentos y la PCR-hs. Específicamente, la elevada frecuencia de consumo de vegetales se relacionaba inversamente con la inflamación mientras que otros tipos de alimentos, como las bebidas azucaradas o la mayonesa, se relacionaban directamente con la inflamación.En el tercer artículo, tres tipos de patrones dietéticos fueron identificados y mantenidos a lo largo del seguimiento: el patrón ”saludable”, el patrón de “proteína animal y carbohidratos refinados” y el patrón “dulces y alimentos procesados”. En el análisis transversal, realizado al final del seguimiento, se observó que aquellos niños incluidos en el patrón ”dulces y alimentos procesados” mostraban una mayor probabilidad de tener la PCR-hs elevada, en comparación con aquellos asignados a un patrón ”saludable”. De igual manera, se observó que aquellos incluidos en un patrón de “dulces y alimentos procesados” mantenido en el tiempo, es decir, desde la valoración inicial hasta la medida de seguimiento, mostraban mayor probabilidad de tener valores más elevados de la PCR-hs, en comparación con los incluidos en un patrón saludable en las dos valoraciones. En el estudio HELENA, realizado entre 2006 y 2007, se valoraron más de 3.000 adolescentes de 10 ciudades europeas: Atenas, Heraklion, Dortmund, Gante, Lille, Pecs, Roma, Estocolmo, Viena y Zaragoza. Las edades de los adolescentes participantes estaban entre 12,5 y 17,5 años. Con datos de este estudio, se valoraron las asociaciones entre el riesgo cardio-metabólico y la inflamación en la adolescencia. La American Heart Asociation (AHA) ha propuesto un índice de salud cardiovascular ideal (ISCI) que incluye cuatro comportamientos y tres factores saludables. Los criterios relacionados con los comportamientos son: no haber fumado, ser físicamente activo, tener un IMC normal y tener una alimentación saludable, mientras que los factores saludables incluidos son valores normales de: tensión arterial, colesterol total y glucosa. Mediante el uso de este índice se valoró la relación entre la salud cardiovascular y la inflamación, la cual fue medida mediante un índice inflamatorio y, a su vez, mediante los biomarcadores que componían el citado índice individualmente: PCR, el factor C3 y C4 del complemento, leptina y el recuento de glóbulos blancos. En este cuarto artículo, se observó que puntuaciones superiores del índice de salud cardiovascular se relacionaban inversamente con los valores del índice inflamatorio y, además, con algunos de sus componentes individualmente. En el quinto artículo, se observó que la composición corporal juega un papel importante en la relación entre la resistencia a la insulina y la inflamación, medida con varios marcadores inflamatorios. La asociación entre la resistencia a la insulina y el factor C3 del complemento fue especialmente relevante para aquellos adolescentes con mayores niveles de adiposidad. Finalmente, en el último artículo, se valoró la asociación entre la salud metabólica y varios marcadores inflamatorios seleccionados, teniendo en cuenta la presencia o no de sobrepeso/obesidad. La existencia de sobrepeso/obesidad y un estatus metabólico alterado se asocia con marcadores inflamatorios, siendo la PCR, C3 y C4 los marcadores más relacionados con esta condición. C3 y C4 se asociaron consistentemente con la salud cardio-metabólica.En resumen, los resultados de esta Tesis Doctoral confirman la existencia de una asociación entre alimentación y PCR-hs desde la infancia y, a su vez, que los marcadores de riesgo cardio-metabólico están presentes desde la adolescencia y se asocian con distintos marcadores inflamatorios, en esta etapa de la vida. Estos resultados ponen de manifiesto la importancia del desarrollo de estrategias de prevención precoz, teniendo en cuenta la promoción de estilos de vida saludables, para evitar el desarrollo de los factores de riesgo cardio-metabólico y la aparición de un estado inflamatorio crónico de bajo grado, asociado a los mismos.<br /

Conserved gene modules regulate light signals in algae and plants

In the Plant Development Unit (PDU) we aim to discover mechanisms that allowed photosynthetic organisms to reach the level of developmental complexity shown today. Plants are particularly good models as they have been evolving as light autotrophs for millions of years, ever since the first bacteria developed oxygenic photosynthesis and killed 99% of existing species in the process. But light is not only the main source of energy for plants, it is also one of the main regulators of their development, as endo-symbiotic cyanobacteria (chloroplasts) perfected their physiological synchronization with the emerging eukaryotes (1). Another important aspect of plant evolution was the transit to the aerial world and the acquisition of characteristics that allowed them to successfully colonise this new habitat (2). In the PDU we have followed the evolution of the day length response (photoperiod) that coordinates the daily physiological activities of plants and can be also used to regulate seasonal behaviours such as winter recesses or flowering time (3). When gene expression networks from photoperiod experiments from microalgae, bryophytes and higher plants are compared, a common nodular structure is discovered (4). Following these discoveries, we have isolated ancestor algal genes that show the same function as higher plants in the response to photoperiod such as the CONSTANS-DOF module (5). We are currently investigating common regulatory mechanisms in photoperiod sensing such as the effect on the circadian clock, senescence, retrograde signalling (6) and protein stability (7

CONSTANS–FKBP12 interaction contributes to modulation of photoperiodic flowering in Arabidopsis

Flowering time is a key process in plant development. Photoperiodic signals play a crucial role in the floral transition in Arabidopsis thaliana, and the protein CONSTANS (CO) has a central regulatory function that is tightly regulated at the transcriptional and post-translational levels. The stability of CO protein depends on a light-driven proteasome process that optimizes its accumulation in the evening to promote the production of the florigen FLOWERING LOCUS T (FT) and induce seasonal flowering. To further investigate the post-translational regulation of CO protein we have dissected its interactome network employing in vivo and in vitro assays and molecular genetics approaches. The immunophilin FKBP12 has been identified in Arabidopsis as a CO interactor that regulates its accumulation and activity. FKBP12 and CO interact through the CCT domain, affecting the stability and function of CO. fkbp12 insertion mutants show a delay in flowering time, while FKBP12 overexpression accelerates flowering, and these phenotypes can be directly related to a change in accumulation of FT protein. The interaction is conserved between the Chlamydomonas algal orthologs CrCO–CrFKBP12, revealing an ancient regulatory step in photoperiod regulation of plant development.Ministerio de Ciencia BIO2014-52425-P, BIO2017-83629-RJunta de Andalucía P08-AGR-03582, BIO-281European Union GA83831

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Diagnostic assessment of deep learning algorithms for detection of lymph node metastases in women with breast cancer

Importance Application of deep learning algorithms to whole-slide pathology images can potentially improve diagnostic accuracy and efficiency. Objective Assess the performance of automated deep learning algorithms at detecting metastases in hematoxylin and eosin–stained tissue sections of lymph nodes of women with breast cancer and compare it with pathologists’ diagnoses in a diagnostic setting. Design, Setting, and Participants Researcher challenge competition (CAMELYON16) to develop automated solutions for detecting lymph node metastases (November 2015-November 2016). A training data set of whole-slide images from 2 centers in the Netherlands with (n = 110) and without (n = 160) nodal metastases verified by immunohistochemical staining were provided to challenge participants to build algorithms. Algorithm performance was evaluated in an independent test set of 129 whole-slide images (49 with and 80 without metastases). The same test set of corresponding glass slides was also evaluated by a panel of 11 pathologists with time constraint (WTC) from the Netherlands to ascertain likelihood of nodal metastases for each slide in a flexible 2-hour session, simulating routine pathology workflow, and by 1 pathologist without time constraint (WOTC). Exposures Deep learning algorithms submitted as part of a challenge competition or pathologist interpretation. Main Outcomes and Measures The presence of specific metastatic foci and the absence vs presence of lymph node metastasis in a slide or image using receiver operating characteristic curve analysis. The 11 pathologists participating in the simulation exercise rated their diagnostic confidence as definitely normal, probably normal, equivocal, probably tumor, or definitely tumor. Results The area under the receiver operating characteristic curve (AUC) for the algorithms ranged from 0.556 to 0.994. The top-performing algorithm achieved a lesion-level, true-positive fraction comparable with that of the pathologist WOTC (72.4% [95% CI, 64.3%-80.4%]) at a mean of 0.0125 false-positives per normal whole-slide image. For the whole-slide image classification task, the best algorithm (AUC, 0.994 [95% CI, 0.983-0.999]) performed significantly better than the pathologists WTC in a diagnostic simulation (mean AUC, 0.810 [range, 0.738-0.884]; P < .001). The top 5 algorithms had a mean AUC that was comparable with the pathologist interpreting the slides in the absence of time constraints (mean AUC, 0.960 [range, 0.923-0.994] for the top 5 algorithms vs 0.966 [95% CI, 0.927-0.998] for the pathologist WOTC). Conclusions and Relevance In the setting of a challenge competition, some deep learning algorithms achieved better diagnostic performance than a panel of 11 pathologists participating in a simulation exercise designed to mimic routine pathology workflow; algorithm performance was comparable with an expert pathologist interpreting whole-slide images without time constraints. Whether this approach has clinical utility will require evaluation in a clinical setting

8-Dehydrosterols induce membrane traffic and autophagy defects through V-ATPase dysfunction in Saccharomyces cerevisae

8-Dehydrosterols are present in a wide range of biologically relevant situations, from human rare diseases to amine fungicide-treated fungi and crops. However, the molecular bases of their toxicity are still obscure. We show here that 8-dehydrosterols, but not other sterols, affect yeast vacuole acidification through V-ATPases. Moreover, erg2Δ cells display reductions in proton pumping rates consistent with ion-transport uncoupling in vitro. Concomitantly, subunit Vph1p shows conformational changes in the presence of 8-dehydrosterols. Expression of a plant vacuolar H+-pumping pyrophosphatase as an alternative H+-pump relieves Vma−-like phenotypes in erg2Δ-derived mutant cells. As a consequence of these acidification defects, endo- and exo-cytic traffic deficiencies that can be alleviated with a H+-pumping pyrophosphatase are also observed. Despite their effect on membrane traffic, 8-dehydrosterols do not induce endoplasmic reticulum stress or assembly defects on the V-ATPase. Autophagy is a V-ATPase dependent process and erg2Δ mutants accumulate autophagic bodies under nitrogen starvation similar to Vma− mutants. In contrast to classical Atg− mutants, this defect is not accompanied by impairment of traffic through the CVT pathway, processing of Pho8Δ60p, GFP-Atg8p localisation or difficulties to survive under nitrogen starvation conditions, but it is concomitant to reduced vacuolar protease activity. All in all, erg2Δ cells are autophagy mutants albeit some of their phenotypic features differ from classical Atg− defective cells. These results may pave the way to understand the aetiology of sterol-related diseases, the cytotoxic effect of amine fungicides, and may explain the tolerance to these compounds observed in plants.Peer reviewe