Rv2577 of mycobacterium tuberculosis Is a virulence factor with dual phosphatase and phosphodiesterase functions

Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide affecting mainly developing countries. Mtb can persist and survive inside infected cells through modulation of host antibacterial attack, i.e., by avoiding the maturation of phagosome containing mycobacteria to more acidic endosomal compartment. In addition, bacterial phosphatases play a central role in the interplay between host cells and Mtb. In this study, we characterized the Rv2577 of Mtb as a potential alkaline phosphatase/phosphodiesterase enzyme. By an in vitro kinetic assay, we demonstrated that purified Rv2577 expressed in Mycobacterium smegmatis displays both enzyme activities, as evidenced by using the artificial substrates p-NPP and bis-(p-NPP). In addition, a three-dimensional model of Rv2577 allowed us to define the catalytic amino acid residues of the active site, which were confirmed by site-directed mutagenesis and enzyme activity analysis, being characteristic of a member of the metallophosphatase superfamily. Finally, a mutation introduced in Rv2577 reduced the replication of Mtb in mouse organs and impaired the arrest of phagosomes containing mycobacteria in early endosomes; which indicates Rv2577 plays a role in Mtb virulence

Rv2577 of Mycobacterium tuberculosis is a virulence factor with dual phosphatase and phosphodiesterase functions

Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide affecting mainly developing countries. Mtb can persist and survive inside infected cells through modulation of host antibacterial attack, i.e., by avoiding the maturation of phagosome containing mycobacteria to more acidic endosomal compartment. In addition, bacterial phosphatases play a central role in the interplay between host cells and Mtb. In this study, we characterized the Rv2577 of Mtb as a potential alkaline phosphatase/phosphodiesterase enzyme. By an in vitro kinetic assay, we demonstrated that purified Rv2577 expressed in Mycobacterium smegmatis displays both enzyme activities, as evidenced by using the artificial substrates p-NPP and bis-(p-NPP). In addition, a three-dimensional model of Rv2577 allowed us to define the catalytic amino acid residues of the active site, which were confirmed by site-directed mutagenesis and enzyme activity analysis, being characteristic of a member of the metallophosphatase superfamily. Finally, a mutation introduced in Rv2577 reduced the replication of Mtb in mouse organs and impaired the arrest of phagosomes containing mycobacteria in early endosomes; which indicates Rv2577 plays a role in Mtb virulence.Instituto de BiotecnologíaFil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marrero Diaz de Villegas, Rubén. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vazquez, Cristina Lourdes. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Yaneff, Agustín. Universidad de Buenos Aires. Instituto de Investigaciones Farmacológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Elizabeth Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gutierrez, Maximiliano Gabriel. The Francis Crick Institute, Host-Pathogen Interactions in Tuberculosis Laboratory; Reino UnidoFil: Durán, Rosario. Institut Pasteur de Montevideo; Uruguay. Instituto de Investigaciones Biológicas Clemente Estable; UruguayFil: Villarino, Andrea. Universidad de la República (UdelaR). Facultad de Ciencias. Sección Bioquímica; UruguayFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de investigaciones Científicas y Tecnológicas; Argentin

Multidrug transporter MRP4/ABCC4 as a key determinant of pancreatic cancer aggressiveness

Recent findings show that MRP4 is critical for pancreatic ductal adenocarcinoma (PDAC) cell proliferation. Nevertheless, the significance of MRP4 protein levels and function in PDAC progression is still unclear. The aim of this study was to determine the role of MRP4 in PDAC tumor aggressiveness. Bioinformatic studies revealed that PDAC samples show higher MRP4 transcript levels compared to normal adjacent pancreatic tissue and circulating tumor cells express higher levels of MRP4 than primary tumors. Also, high levels of MRP4 are typical of high-grade PDAC cell lines and associate with an epithelial-mesenchymal phenotype. Moreover, PDAC patients with high levels of MRP4 depict dysregulation of pathways associated with migration, chemotaxis and cell adhesion. Silencing MRP4 in PANC1 cells reduced tumorigenicity and tumor growth and impaired cell migration. Transcriptomic analysis revealed that MRP4 silencing alters PANC1 gene expression, mainly dysregulating pathways related to cell-to-cell interactions and focal adhesion. Contrarily, MRP4 overexpression significantly increased BxPC-3 growth rate, produced a switch in the expression of EMT markers, and enhanced experimental metastatic incidence. Altogether, our results indicate that MRP4 is associated with a more aggressive phenotype in PDAC, boosting pancreatic tumorigenesis and metastatic capacity, which could finally determine a fast tumor progression in PDAC patients.Fil: Sahores, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Carozzo, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: May, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Gomez, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Di Siervi, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: de Sousa Serro, Maximiliano Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Yaneff, Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Rodriguez Gonzalez, Angela Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Abba, Martín Carlos. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Shayo, Carina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Davio, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentin

Cyclic AMP efflux through MRP4 regulates actin dynamics signalling pathway and sperm motility in bovines

Previously we demonstrated that multidrug resistance-associated protein 4 transporter (MRP4) mediates cAMP efflux in bovine spermatozoa and that extracellular cAMP (ecAMP) triggers events associated to capacitation. Here, we deepen the study of the role of MRP4 in bovine sperm function by using MK571, an MRP4 inhibitor. The incubation of spermatozoa with MK571 during 45 min inhibited capacitation-associated events. MRP4 was localized in post-acrosomal region and mid-piece at 15 min capacitation, while at 45 min it was mainly located in the acrosome. After 15 min, MK571 decreased total sperm motility (TM), progressive motility (PM) and several kinematic parameters. The addition of ecAMP rescued MK571 effect and ecAMP alone increased the percentage of motile sperm and kinematics parameters. Since actin cytoskeleton plays essential roles in the regulation of sperm motility, we investigated if MRP4 activity might affect actin polymerization. After 15 min capacitation, an increase in F-actin was observed, which was inhibited by MK571. This effect was reverted by the addition of ecAMP. Furthermore, ecAMP alone increased F-actin levels while no F-actin was detected with ecAMP in the presence of PKA inhibitors. Our results support the importance of cAMP efflux through MRP4 in sperm capacitation and suggest its involvement in the regulation of actin polymerization and motility.Fil: Chiarante, Nicolás Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Alonso, Carlos A. I.. McGill University; CanadáFil: Plaza, Jessica. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Lottero Leconte, Raquel María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Arroyo Salvo, Camila Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Yaneff, Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Osycka Salut, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Davio, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Miragaya, Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Perez Martinez, Silvina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Multidrug transporter MRP4/ABCC4 as a key determinant of pancreatic cancer aggressiveness

Recent fndings show that MRP4 is critical for pancreatic ductal adenocarcinoma (PDAC) cell proliferation. Nevertheless, the signifcance of MRP4 protein levels and function in PDAC progression is still unclear. The aim of this study was to determine the role of MRP4 in PDAC tumor aggressiveness. Bioinformatic studies revealed that PDAC samples show higher MRP4 transcript levels compared to normal adjacent pancreatic tissue and circulating tumor cells express higher levels of MRP4 than primary tumors. Also, high levels of MRP4 are typical of high-grade PDAC cell lines and associate with an epithelial-mesenchymal phenotype. Moreover, PDAC patients with high levels of MRP4 depict dysregulation of pathways associated with migration, chemotaxis and cell adhesion. Silencing MRP4 in PANC1 cells reduced tumorigenicity and tumor growth and impaired cell migration. Transcriptomic analysis revealed that MRP4 silencing alters PANC1 gene expression, mainly dysregulating pathways related to cell-to-cell interactions and focal adhesion. Contrarily, MRP4 overexpression signifcantly increased BxPC-3 growth rate, produced a switch in the expression of EMT markers, and enhanced experimental metastatic incidence. Altogether, our results indicate that MRP4 is associated with a more aggressive phenotype in PDAC, boosting pancreatic tumorigenesis and metastatic capacity, which could fnally determine a fast tumor progression in PDAC patients.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada