RESUMEN
Poly-alanine (Ala) tract expansions in transcription factors have been shown to be associated with human birth defects such as malformations of the brain, the digits, and other structures. Expansions of a poly-Ala tract from 15 to 22 (+7)-29 (+14) Ala in Hoxd13, for example, result in the limb malformation synpolydactyly in humans and in mice [synpolydactyly homolog (spdh)]. Here, we show that an increase of the Ala repeat above a certain length (22 Ala) is associated with a shift in the localization of Hoxd13 from nuclear to cytoplasmic, where it forms large amorphous aggregates. We observed similar aggregates for expansion mutations in SOX3, RUNX2 and HOXA13, pointing to a common mechanism. Cytoplasmic aggregation of mutant Hoxd13 protein is influenced by the length of the repeat, the level of expression and the efficacy of degradation by the proteasome. Heat shock proteins Hsp70 and Hsp40 co-localize with the aggregates and activation of the chaperone system by geldanamycin leads to a reduction of aggregate formation. Furthermore, recombinant mutant Hoxd13 protein forms aggregates in vitro demonstrating spontaneous misfolding of the protein. We analyzed the mouse mutant spdh, which harbors a +7 Ala expansion in Hoxd13 similar to the human synpolydactyly mutations, as an in vivo model and were able to show a reduction of mutant Hoxd13 and, in contrast to wt Hoxd13, a primarily cytoplasmic localization of the protein. Our results provide evidence that poly-Ala repeat expansions in transcription factors result in misfolding, degradation and cytoplasmic aggregation of the mutant proteins.
Asunto(s)
Expansión de las Repeticiones de ADN , Proteínas de Homeodominio/genética , Péptidos/genética , Polidactilia/genética , Factores de Transcripción/genética , Animales , Células COS , Núcleo Celular/química , Chlorocebus aethiops , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Citoplasma/química , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/análisis , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/análisis , Proteínas de Choque Térmico/metabolismo , Proteínas del Grupo de Alta Movilidad/análisis , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Ratones Mutantes , Mutación/genética , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Polidactilia/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Secuencias Repetitivas de Aminoácido/genética , Factores de Transcripción SOXB1 , Factores de Transcripción/análisis , Factores de Transcripción/metabolismoRESUMEN
Robinow syndrome (RS) is a human dwarfism syndrome characterized by mesomelic limb shortening, vertebral and craniofacial malformations and small external genitals. We have analyzed Ror2(-/-) mice as a model for the developmental pathology of RS. Our results demonstrate that vertebral malformations in Ror2(-/-) mice are due to reductions in the presomitic mesoderm and defects in somitogenesis. Mesomelic limb shortening in Ror2(-/-) mice is a consequence of perturbed chondrocyte differentiation. Moreover, we show that the craniofacial phenotype is caused by a midline outgrowth defect. Ror2 expression in the genital tubercle and its reduced size in Ror2(-/-) mice makes it likely that Ror2 is involved in genital development. In conclusion, our findings suggest that Ror2 is essential at multiple sites during development. The Ror2(-/-) mouse provides a suitable model that may help to explain many of the underlying developmental malformations in individuals with Robinow syndrome.
