Implicación de Caveolina-1 en procesos de mecanotransducción: modulación de la respuesta a cambios en la rigidez de la matriz extracelular a través del control de la actividad

Caveolina-1 (CAV1) es una proteína integral de la membrana plasmática que participa en procesos de endocitosis, modula diferentes vías de señalización celular y participa activamente en la mecanotransducción de señales. CAV1 y caveolas son capaces de responder a diferentes estímulos mecánicos, cómo son el estiramiento, las fuerzas de cizallamiento ejercidas por fluidos sobre la superficie de la célula, o la perdida de adhesión celular. En esta Tesis Doctoral se ha caracterizado la modulación por parte de CAV1 de la respuesta a un estímulo mecánico diferente: la rigidez sustrato extracelular. El regulador transcripcional YAP regula importantes funciones celulares, como proliferación, apoptosis y diferenciación, determinando la homeostasis de tejidos, el control del crecimiento de los órganos y la tumorogénesis. Los estímulos mecánicos controlan la actividad de YAP mediante mecanismos que no están completamente caracterizados todavía. Los resultados presentados en este trabajo indican que CAV1 regula positivamente la actividad de YAP en respuesta a cambios en la rigidez de la matriz extracelular (MEC). Esta regulación de YAP por parte de CAV1 es dependiente de la modulación de la dinámica del citoesqueleto de actina, siendo la actividad de Rho necesaria, pero no suficiente para explicar las diferencias observadas. En el control del eje CAV1-YAP, la fosforilación inhibitoria de YAP tiene un papel determinante, observándose una fosforilación mayor en células que no expresan CAV1, y un rescate de la actividad de YAP al sobreexpresar en estas células un mutante de YAP no fosforilable. Sin embargo, la regulación CAV1-dependiente de YAP no opera a través de quinasas canónicas de la vía Hippo. Estudios de espectrometría de masas para la identificación sistemática de los componentes del interactoma de YAP, y su caracterización funcional posterior, sugieren que la regulación de la actividad de YAP dependiente de CAV1 y actina es ejercida a través de la modulación de la interacción de YAP con la proteína con domino 14-3-3 YWHAH. El silenciamiento de Ywhah rescata el defecto en la actividad de YAP en células sin CAV1. La activación constitutiva de YAP en células sin CAV1 rescata fenotipos asociados a la perdida de Cav1, incluyendo el remodelado de la MEC. El control de YAP mediado por CAV1 se ha validado también en un modelo in vivo de metaplasia acinoductal inducido por pancreatitis. En conjunto, los datos presentados en esta Tesis Doctoral apoyan la existencia y relevancia funcional de un nuevo eje de mecanotransducción a través del control de la actividad de YAP por CAV1 con implicaciones potenciales en procesos fisiológicos y patológicos

Implicación de Caveolina-1 en procesos de mecanotransducción: modulación de la respuesta a cambios en la rigidez de la matriz extracelular a través del control de la actividad de YAP

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 19-11-2018Caveolina-1 (CAV1) es una proteína integral de la membrana plasmática que participa en procesos de endocitosis, modula diferentes vías de señalización celular y participa activamente en la mecanotransducción de señales. CAV1 y caveolas son capaces de responder a diferentes estímulos mecánicos, cómo son el estiramiento, las fuerzas de cizallamiento ejercidas por fluidos sobre la superficie de la célula, o la perdida de adhesión celular. En esta Tesis Doctoral se ha caracterizado la modulación por parte de CAV1 de la respuesta a un estímulo mecánico diferente: la rigidez sustrato extracelular. El regulador transcripcional YAP regula importantes funciones celulares, como proliferación, apoptosis y diferenciación, determinando la homeostasis de tejidos, el control del crecimiento de los órganos y la tumorogénesis. Los estímulos mecánicos controlan la actividad de YAP mediante mecanismos que no están completamente caracterizados todavía. Los resultados presentados en este trabajo indican que CAV1 regula positivamente la actividad de YAP en respuesta a cambios en la rigidez de la matriz extracelular (MEC). Esta regulación de YAP por parte de CAV1 es dependiente de la modulación de la dinámica del citoesqueleto de actina, siendo la actividad de Rho necesaria, pero no suficiente para explicar las diferencias observadas. En el control del eje CAV1-YAP, la fosforilación inhibitoria de YAP tiene un papel determinante, observándose una fosforilación mayor en células que no expresan CAV1, y un rescate de la actividad de YAP al sobreexpresar en estas células un mutante de YAP no fosforilable. Sin embargo, la regulación CAV1-dependiente de YAP no opera a través de quinasas canónicas de la vía Hippo. Estudios de espectrometría de masas para la identificación sistemática de los componentes del interactoma de YAP, y su caracterización funcional posterior, sugieren que la regulación de la actividad de YAP dependiente de CAV1 y actina es ejercida a través de la modulación de la interacción de YAP con la proteína con domino 14-3-3 YWHAH. El silenciamiento de Ywhah rescata el defecto en la actividad de YAP en células sin CAV1. La activación constitutiva de YAP en células sin CAV1 rescata fenotipos asociados a la perdida de Cav1, incluyendo el remodelado de la MEC. El control de YAP mediado por CAV1 se ha validado también en un modelo in vivo de metaplasia acinoductal inducido por pancreatitis. En conjunto, los datos presentados en esta Tesis Doctoral apoyan la existencia y relevancia funcional de un nuevo eje de mecanotransducción a través del control de la actividad de YAP por CAV1 con implicaciones potenciales en procesos fisiológicos y patológico

Improvement of the Antioxidant Properties and Postharvest Life of Three Exotic Andean Fruits by UV-C Treatment

Three Andean fruits naranjilla (Solanum quitoense Lam.), uvilla (Physalis peruviana L.), and mortiño (Vaccinium floribundum Kunth) were subjected to prestorage UV-C treatments (0, 8, or 12.5 kJ m 2) and evaluated weekly to select the most suitable dose for fruit quality maintenance during storage (21 days at 6°C). The highest dose retains quality through lower deterioration index for all three fruits and was selected to further analyze the effects on physicochemical and antioxidant properties during storage. UV- C exposure delayed softening in naranjilla and increased soluble solid content in uvilla. UV-C also improved the maintenance of antioxidant capacity (AC) in mortiño and uvilla. Overall, results indicate that short prestorage UV-C exposure may be an effective nonchemical approach to supplement low temperature storage, maintain quality, and extend the postharvest life of Andean naranjilla, uvilla, and mortiño fruit.Facultad de Ciencias Agrarias y ForestalesCentro de Investigación y Desarrollo en Criotecnología de Alimento

Short UV-C Treatment Prevents Browning and Extends the Shelf-Life of Fresh-Cut Carambola

In this work, we selected a short UV-C treatment for fresh-cut carambola and assessed its efficacy in supplementing the benefits of low temperature storage. UV-C treated (6.0, 10.0, and 12.5 kJm−2) carambola slices showed reduced deterioration compared to control fruit. Treatment with a dose of 12.5 kJm−2 UV-C was more effective in maintaining quality and was selected for subsequent experiments evaluating the combination of UV-C and refrigeration on fruit storability and physical, chemical, and microbiological properties. Short UV-C exposure reduced weight loss and electrolyte leakage. UV-C treated carambola slices presented higher phenolic antioxidants than control after 21 d at 4∘C and showed no alterations in soluble solids or titratable acidity. UV-C exposure also reduced the counts of molds, yeast, and aerobic mesophilic bacteria. UV-C treated fruit showed a fresh-like appearance even after 21 d as opposed to control carambola which presented spoilage and extensive browning symptoms. The reduction of fruit browning in UV-C treated fruit was not due to reduction in phenylalanine-ammonia lyase (PAL) and/or peroxidase (POD), but rather through polyphenol oxidase (PPO) inhibition and improved maintenance of tissue integrity.Facultad de Ciencias Agrarias y Forestales (FCAF

Caveolin-1 is required for TGF-β-induced transactivation of the EGF receptor pathway in hepatocytes through the activation of the metalloprotease TACE/ADAM17

Transforming growth factor-beta (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-β receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-β-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1(-/-) hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-β, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-β-induced survival signals in Cav1(-/-) hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-β was impaired in Cav1(-/-) hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1(-/-) hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1(+/+) cells, which was not the case in Cav1(-/-) cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-β treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-β-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-β pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-β in liver cancer cells.This work was supported by grants from: (1) the Ministry of Economy and Competitiveness (MINECO), Spain (BFU2012-35538 and ISCIII-RTICC: RD12-0036-0029 to IF; SAF2013-43713 to PM-S; BFU2012-33932 to GE; SAF2011-25047 and CSD2009-00016 to MAdP); (2) AGAUR-Generalitat de Catalunya (2009SGR-312 to IF); and (3) People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. PITN-GA-2012-316549 (IT LIVER) to IF JM-C and RM-V were recipients of pre-doctoral fellowships from the FPU program (Ministry of Education, Culture and Sport, Spain) and the FPI program (associated to SAF201125047, MINECO, Spain), respectively. We acknowledge the review and suggestions of Dr. Christoph Meyer (University of Heidelberg, Mannheim, Germany).S

Caveolin-1 is required for TGF-β-induced transactivation of the EGF receptor pathway in hepatocytes through the activation of the metalloprotease TACE/ADAM17

Transforming growth factor-beta (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-β receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-β-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1−/− hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-β, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-β-induced survival signals in Cav1−/− hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-β was impaired in Cav1−/− hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1−/− hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1+/+ cells, which was not the case in Cav1−/− cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-β treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-β-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-β pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-β in liver cancer cells

Analysis of Killer Immunoglobulin-Like Receptor Genes in Colorectal Cancer

Natural killer cells (NK cells) play a major role in the immune response to cancer. An important element of NK target recognition is the binding of human leucocyte antigen (HLA) class I molecules by killer immunoglobulin-like receptors (KIRs). Colorectal carcinoma (CRC) is one of the most common types of inflammation-based cancer. The purpose of the present study was to investigate the presence of KIR genes and HLA class I and II alleles in 1074 CRC patients and 1272 controls. We imputed data from single-nucleotide polymorphism (SNP) Illumina OncoArray to identify associations at HLA (HLA-A, B, C, DPB1, DQA1, DQB1, and DRB1) and KIRs (HIBAG and KIR*IMP, respectively). For association analysis, we used PLINK (v1.9), the PyHLA software, and R version 3.4.0. Only three SNP markers showed suggestive associations (p < 10(-3); rs16896742, rs28367832, and rs9277952). The frequency of KIR2DS3 was significantly increased in the CRC patients compared to healthy controls (p < 0.005). Our results suggest that the implication of NK cells in CRC may not act through allele combinations in KIR and HLA genes. Much larger studies in ethnically homogeneous populations are needed to rule out the possible role of allelic combinations in KIR and HLA genes in CRC risk

Unraveling the role of the secretor antigen in human rotavirus attachment to histo-blood group antigens

Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P[8] genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P[8] VP4 spike protein to its cellular receptor, the secretory H type-1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc; H1), and to its precursor lacto-N-biose (Gal-β1,3-GlcNAc; LNB) have been determined. The resolution of P[8] VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P[8] VP8* protein through a binding pocket shared with other members of the P[II] genogroup (i.e.: P[4], P[6] and P[19]). Our results show that the L-fucose moiety from H1 only displays indirect contacts with P[8] VP8*. However, the induced conformational changes in the LNB moiety increase the ligand affinity by two-fold, as measured by surface plasmon resonance (SPR), providing a molecular explanation for the different susceptibility to rotavirus infection between secretor and non-secretor individuals. The unexpected interaction of P[8] VP8* with LNB, a building block of type-1 human milk oligosaccharides, resulted in inhibition of rotavirus infection, highlighting the role and possible application of this disaccharide as an antiviral. While key amino acids in the H1/LNB binding pocket were highly conserved in members of the P[II] genogroup, differences were found in ligand affinities among distinct P[8] genetic lineages. The variation in affinities were explained by subtle structural differences induced by amino acid changes in the vicinity of the binding pocket, providing a fine-tuning mechanism for glycan binding in P[8] rotavirus

Improvement of the Antioxidant Properties and Postharvest Life of Three Exotic Andean Fruits by UV-C Treatment

Three Andean fruits naranjilla (Solanum quitoense Lam.), uvilla (Physalis peruviana L.), and mortiño (Vaccinium floribundum Kunth) were subjected to prestorage UV-C treatments (0, 8, or 12.5 kJ m 2) and evaluated weekly to select the most suitable dose for fruit quality maintenance during storage (21 days at 6°C). The highest dose retains quality through lower deterioration index for all three fruits and was selected to further analyze the effects on physicochemical and antioxidant properties during storage. UV- C exposure delayed softening in naranjilla and increased soluble solid content in uvilla. UV-C also improved the maintenance of antioxidant capacity (AC) in mortiño and uvilla. Overall, results indicate that short prestorage UV-C exposure may be an effective nonchemical approach to supplement low temperature storage, maintain quality, and extend the postharvest life of Andean naranjilla, uvilla, and mortiño fruit.Facultad de Ciencias Agrarias y ForestalesCentro de Investigación y Desarrollo en Criotecnología de Alimento