RepA-WH1 prionoid: Clues from bacteria on factors governing phase transitions in amyloidogenesis

10 p.-1 fig.In bacterial plasmids, Rep proteins initiate DNA replication by undergoing a structural transformation coupled to dimer dissociation. Amyloidogenesis of the ‘winged-helix’ N-terminal domain of RepA (WH1) is triggered in vitro upon binding to plasmid-specific DNA sequences, and occurs at the bacterial nucleoid in vivo. Amyloid fibers are made of distorted RepA-WH1 monomers that assemble as single or double intertwined tubular protofilaments. RepA-WH1 causes in E. coli an amyloid proteinopathy, which is transmissible from mother to daughter cells, but not infectious, and enables conformational imprinting in vitro and in vivo; i.e. RepA-WH1 is a ‘prionoid’. Microfluidics allow the assessment of the intracellular dynamics of RepA-WH1: bacterial lineages maintain two types (strains-like) of RepA-WH1 amyloids, either multiple compact cytotoxic particles or a single aggregate with the appearance of a fluidized hydrogel that it is mildly detrimental to growth. The Hsp70 chaperone DnaK governs the phase transition between both types of RepA-WH1 aggregates in vivo, thus modulating the vertical propagation of the prionoid. Engineering chimeras between the Sup35p/[PSI*] prion and RepA-WH1 generates [REP-PSI*], a synthetic prion exhibiting strong and weak phenotypic variants in yeast. These recent findings on a synthetic, self-contained bacterial prionoid illuminate central issues of protein amyloidogenesis.Research on RepA-WH1 amyloids at CIBCSIC is currently financed by Spanish MINECO grants BIO2012-30852 and CSD2009-00088.Peer reviewe

Interrelaciones pedagógicas para el desarrollo y fortalecimiento de competencias básicas

El proyecto de investigación-acción “Formar en competencias: un desafío pedagógico” (Jornadas Profesorado de Enfermería. San Sebastián, Diciembre 2007) es el punto de partida para evidenciar el nivel de competencias básicas (Literacia) con las que accede el alumnado a la titulación Enfermera. Durante 2005-2007 (primera fase) se aplicó una prueba a alumnos de la UPV. La percepción de las docentes de esta titulación, respecto a la falta de comprensión del alumnado ante la información, quedó patente ya que el 72% de los alumnos estaban por debajo del nivel mínimo deseado. En 2007-2008 (segunda fase) se aplica la misma prueba de Literacia a estudiantes de primero. Los resultados indican un aumento del porcentaje de alumnos que están por debajo de ese nivel. Con la colaboración de profesoras de otras Escuelas de Enfermería, percibida la dificultad en sus alumnos para manejar la información; aplicamos la prueba y constatamos un déficit similar en distintos lugares de nuestra geografía. Esto nos compromete a intervenir; lo hacemos en la UPV con un grupo experimental para comprobar cómo a través de una Acción Formativa de Literacia mejoran sus competencias básicas. Por ello, ofrecemos intervenciones pedagógico-formativas análogas (gestión información, comunicación, razonamiento lógico, trabajo en equipo, actitud proactiva…) para mejorar el nivel de competencias básicas de otros estudiantes. Si un sistema educativo quiere garantizar la calidad “deberá dotar a las personas de un conjunto de competencias básicas que aumenten su empleabilidad y su aptitud para participar como ciudadano en la vida pública y en la sociedad del conocimiento”.SIN FINANCIACIÓNNo data 200

Intercellular transmission of a synthetic bacterial cytotoxic prion-like protein in mammalian cells

18 p.-6 fig.RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein,horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1.Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepAWH1(WT)] and assayed its response to exposure to in vitro-assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria,protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways.IMPORTANCE. Proteotoxic amyloid seeds can be transmitted between mammalian cells,arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Escherichia coli. Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol,following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.This work was financed with grants from the Spanish State Research Agency (AEI) (BIO2012-30852 and BIO2015-68730-R) to R.G. A.R.-G. was a recipient of an AEI shortterm fellowship (EEBB-I-17-12294) to work at the laboratory of I.M.V.Peer reviewe

RepA-WH1: A fully bacterial model of amyloid disease

Trabajo presentado en el 3rd Iberian Congress on Prions, celebrado en Zaragoza (España) del 02 al 03 de diciembre de 2014.Peer reviewe

Mad Bacteria: Deconstructing neurodegenerative disease in E. coli with a synthetic prionoid

Trabajo presentado en EMBO Symposium: New approaches and concepts in Microbiology, celebrado en Heidelberg (Alemania) del 11 al 14 de octubre de 2015

Synthetic bacterial and cellular models of amyloid disease

Trabajo presentado al XXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia (España) del 07 al 10 de septiembre de 2015.Peer reviewe