Metacercarial proteins interacting with WD40-repeat protein of Clonorchis sinensis

The WD40-repeat proteins serve as a platform coordinating partner proteins and are involved in a range of regulatory cellular functions. A WD40-repeat protein (CsWD1) of Clonorchis sinensis previously cloned is expressed stage-specifically in the tegumental syncytium of C. sinensis metacercariae. In the present study, interacting proteins with the CsWD1 protein was purified by immunoprecipitation and 2 dimension gel electrophoresis from the C. sinensis metacercaria soluble extract, and tryptic peptides were analyzed by LC/ESI-MS. Putative partner proteins were annotated to be actin-2, glyceraldehyde-3-phosphate dehydrogenase, and hypothetical and unmanned proteins. The CsWD1 protein was predicted to contain 3 conserved actin-interacting residues on its functional surface. With these results, the CsWD1 protein is suggested to be an actin-interacting protein of C. sinensis.

Molecular and biochemical characterization of a novel actin bundling protein in Acanthamoeba

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 micrometer of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.

Proteínas de unión a actina en Plasmodium falciparum

La invasión del Plasmodium falciparaum, el agente causante de la malaria en humanos, implica un movimiento de penetración del parásito a los eritrocitos. Con el objetivo de determinar proteínas de unión a actina, se utilizaron dos estrategias diferentes: la primera consistió en preparar columnas de afinidad con actina polimerizada (actina F), para cromatografiar extractos de parásito marcados radioactivamente con L-[Superíndice 35S] metionina, y seleccionar proteínas de unión a actina que posteriormente, fueron identificadas por medio de anticuerpos específicos. En la segunda se utilizó la Reacción en Cadena de la Polimerasa (PCR) y oligonucleótidos generados, diseñados con base en secuencias consenso de miosinas, para buscar un fragmento de este gen en el ADN genómico (ADNg) del parásito y posteriormente clonarlo y secuenciarlo. Los resultados obtenidos sugieren la existencia de un motor molecular basado en actina-miosina, en la invasión del parásito a los eritrocitos. Esta afirmación esta sustentanda en primer lugar por el hallazgo de un gen de miosina en P. falciparum, que por homología de su secuencia parcial se clasifica dentro de la clase I de las miosinas no convencionales, caracterizadas por su capacidad de generar movimientos sobre filamentos de actina en diferentes células. Adicionalmente por medio de Western Blot y anticuerpos específicos, anti alfa actina, tripomiosina, espectrina, miosina y actina se determinó la exitencia de estas proteínas en el parásito