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Los ribosomas son las estructuras subcelulares encargadas de llevar a cabo la síntesis proteica. La biogénesis ribosómica es un proceso muy complejo que comienza con la transcripción del ARN ribosómico por la ARN polimerasa I en el nucléolo. Este tránscrito inicial sufre un largo procesamiento que implica su plegamiento y asociación con las distintas proteínas ribosómicas. Además, el proceso de biogénesis requiere de la participación de un grupo heterogéneo de proteínas denominadas factores de ensamblaje ribosómicos. Se trata de proteínas que ejercen un papel esencial en el proceso de biogénesis ribosómica pero no forman parte del ribosoma maduro. Muchas de estas proteínas establecen interacciones binarias que son esenciales para su correcto funcionamiento. Las células cancerosas deben mantener una elevada tasa de biogénesis ribosómica a fin de proliferar con mayor rapidez que las células sanas. Por esta razón la biogénesis ribosómica constituye una importante diana terapéutica para el desarrollo de fármacos antitumorales. Ya han sido desarrollados fármacos basados en la inhibición específica de la ARN polimerasa I. Sin embargo, el uso de estos fármacos presenta inconvenientes tales como el desarrollo de resistencias o su carácter genotóxico. Una estrategia novedosa en este campo es el uso de péptidos de interferencia dirigidos contra interaccione específicas entre factores de ensamblaje. El grupo dirigido por el Dr. Bravo ha desarrollado un conjunto de péptidos capaces de bloquear la interacción entre los factores de ensamblaje Erb1 y Ytm1. Estos péptidos se derivan de la secuencia de Erb1 en su zona de interacción con Ytm1. En este trabajo hemos ampliado estos resultados preliminares mediante experimentos in vitro empleando células tumorales humanas. Hemos empleado formas equivalentes de los péptidos de interés derivadas de la secuencia de Bop1, la proteína homóloga de Erb1 en humanos. Estos péptidos actúan in vitro provocando citotoxicidad y apoptosis en células tumorales humanas. Estos efectos son independientes a la expresión de p53. Se sugiere así que los péptidos desarrollados podrían ser utilizados en el tratamiento de tumores en los que p53 se encuentre mutado, lo cual es un fenómeno frecuente en oncología. Los péptidos desarrollados pueden ser combinados in vitro con oxaliplatino, un fármaco quimioterapéutico. Esta estrategia permite disminuir la concentración de oxaliplatino necesaria para alcanzar un efecto citotóxico sobre las células tumorales, lo cual contribuiría a disminuir los efectos secundarios asociados al uso de oxaliplatino en la clínica. Además, estos péptidos podrían resultar eficaces en contextos de quimiorresistencia. Por último, en este trabajo hemos evaluado otros factores de ensamblaje como posibles dianas para el desarrollo de péptidos de interferencia. En conjunto, nuestros resultados muestran el potencial de la biogénesis ribosómica como diana terapéutica para el desarrollo de nuevos tratamientos antitumorales. Ribosomes are the subcellular structures responsible for carrying out protein synthesis. Ribosomal biogenesis is a very complex process that begins with the transcription of ribosomal RNA by RNA polymerase I in the nucleolus. This initial transcript undergoes complex post-transcriptional processing involving its folding and association with the various ribosomal proteins. In addition, the process of ribosome biogenesis requires the participation of a heterogeneous group of proteins called ribosomal assembly factors. These are proteins that play an essential role in the promotion of ribosomal biogenesis but are not part of the mature ribosome. Many of these proteins establish binary interactions that are essential for their proper function. Cancer cells must maintain a high rate of ribosomal biogenesis in order to divide faster than healthy cells. For this reason, ribosomal biogenesis is an important therapeutic target for the development of anti-cancer drugs. Drugs based on specific inhibition of RNA polymerase I have already been developed. However, the use of these drugs has drawbacks, such as the development of resistance or their genotoxic nature. A novel strategy in this field is the use of interference peptides directed against specific interactions between assembly factors. The group led by Dr. Bravo has recently developed a set of peptides able to block the interaction between the assembly factors Erb1 and Ytm1. These peptides are derived from the sequence of Erb1 in its interaction area with Ytm1. In this work, we have extended these preliminary results by performing in vitro experiments using human tumor cells. We have used equivalent forms of the peptides of interest derived from the sequence of Bop1, the homologue protein of Erb in Homo sapiens. These peptides produce in vitro cytotoxicity and apoptosis in human tumor cells. These effects are independent of p53 expression. It is thus suggested that the developed peptides could be used in the treatment of tumors in which p53 is mutated, which is a frequent phenomenon in oncology. The developed peptides can be combined in vitro with oxaliplatin, a chemotherapeutic drug. This strategy allows for a lower concentration of oxaliplatin needed to achieve a cytotoxic effect on tumor cells, which would help reduce the side effects associated with the use of this drug in the clinic. Furthermore, these peptides could be effective in the context of chemoresistance. Finally, in this work, we have evaluated other assembly factors as potential targets for the development of interfering peptides. Taken together, our results show the potential of ribosomal biogenesis as a therapeutic target for the development of new anti-tumor therapies.

We have previously shown the delivery of phosphatase of regenerating liver-1 (PRL-1) to the immunological synapse (IS) and proposed a regulatory role of the catalytic activity of PRLs (PRL-1, PRL-2 and PRL-3) in antigen-induced IL-2 production. Nonetheless, the expression in T cells and delivery to the IS of the highly homologous PRL-3, as well as the role of the catalytic activity of PRLs in antigen-induced early signaling, has not been investigated. Here, the expression of PRL-3 protein was detected in primary CD4 T cells and in the CD4 T cell line Jurkat (JK), in which an overexpressed GFP-PRL-3 fluorescent fusion protein trafficked through the endosomal recycling compartment and co-localized with PLCγ1 signaling sites at the IS. Pharmacological inhibition was used to compare the role of the catalytic activity of PRLs in antigen-induced early signaling and late IL-2 production. Although the phosphatase activity of PRLs was not critical for early signaling triggered by antigen, it seemed to regulate signaling dynamics and was necessary for proper IL-2 production. We propose that enzymatic activity of PRLs has a higher significance for cytokine production than for early signaling at the IS. However, further research will be necessary to deeply understand the regulatory role of PRLs during lymphocyte activation and effector function.

Trabajo presentado en la ALTANT Conference: Innate Host Defence and Infections | From basic science to applications, celebrada en Utrecht (Países Bajos), del 18 al 20 de mayo de 2016, Pulmonary surfactant is a complex network that covers the alveolar epithelium, which is composed of 90% lipids and 10% proteins (SP-A, SP-B, SP-C, and SP-D). The two principal surfactant components involved in innate immunity are SP-A and SP-D. The role of surfactant membranes with or without the hydrophobic proteins SP-B and SP-C during infection has been relatively unexplored. Non-typable Haemophilus influenzae (NTHi) is the respiratory pathogen most frequently isolated during chronic obstructive pulmonary disease (COPD) exacerbations. It causes chronic infections likely due to its capacity to invade and survive within alveolar epithelial cells. In this study we evaluate whether surfactant membranes with or without SP-B and SP-C: i) protect pneumocytes against invasion of NTHi (clinical strains obtained from patients with COPD and otitis media); and ii) limit NTHi infection in vivo. We found that surfactant multilamellar vesicles with or without SP-B and SP-C (MLVs, >1microm) prevented adhesion of NTHi to pneumocytes. Furthermore, endocytosis of surfactant large unilamellar vesicles (LUVs, 100 nm) by pneumocytes inhibited invasion of NTHi clinical strains but not bacterial adhesion to the pneumocyte surface. Confocal microscopy confirmed that endocytosis of surfactant LUVs reduced the percentage of fluorescent bacteria that co-localize with late endosomes. Bacterial internalization inhibition correlates with the inhibitory effect of intracellular surfactant LUVs on Akt phosphorylation induced by NTHi, which is essential for the NTHi invasion process. An antibody that blocks the scavenger receptor class B type 1 inhibited NTHiinduced Akt phosphorylation much like surfactant LUVs. Finally, intratracheal administration of lung surfactant membranes containing SP-B and SP-C significantly reduced bacterial burden after 24h of NTHi infection in mice lungs. These results suggest that surfactant lipid vesicles play an important role in controlling respiratory infections by NTHi.