RESUMO
Lafora disease is the most severe teenage-onset progressive epilepsy, a unique form of glycogenosis with perikaryal accumulation of an abnormal form of glycogen, and a neurodegenerative disorder exhibiting an unusual generalized organellar disintegration. The disease is caused by mutations of the EPM2A gene, which encodes two isoforms of the laforin protein tyrosine phosphatase, having alternate carboxyl termini, one localized in the cytoplasm (endoplasmic reticulum) and the other in the nucleus. To date, all documented disease mutations, including the knockout mouse model deletion, have been in the segment of the protein common to both isoforms. It is therefore not known whether dysfunction of the cytoplasmic, nuclear, or both isoforms leads to the disease. In the present work, we identify six novel mutations, one of which, c.950insT (Q319fs), is the first mutation specific to the cytoplasmic laforin isoform, implicating this isoform in disease pathogenesis. To confirm this mutation's deleterious effect on laforin, we studied the resultant protein's subcellular localization and function and show a drastic reduction in its phosphatase activity, despite maintenance of its location at the endoplasmic reticulum.
Assuntos
Citoplasma/química , Doença de Lafora/genética , Proteínas Tirosina Fosfatases/deficiência , Proteínas Tirosina Fosfatases/genética , Adulto , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Células COS , Linhagem Celular , Chlorocebus aethiops , Citoplasma/genética , Fosfatases de Especificidade Dupla , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação/genética , Mutação de Sentido Incorreto/genética , Linhagem , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Fosfatases não ReceptorasRESUMO
We have identified an interacting partner protein (encoded by the human EPM2AIP1 gene (approved symbol)) for laforin, the product of the EPM2A gene, which is mutated in an autosomal recessive form of adolescent progressive myoclonus epilepsy. The EPM2AIP1 gene was identified in a screen for laforin-interacting proteins with a human brain cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by coimmunoprecipitation of in vivo-transfected protein and by using EPM2A deletion constructs. Subcellular colocalization of laforin and EPM2AIP1 protein was also demonstrated. The human EPM2AIP1 gene, corresponding to the KIAA0766 cDNA clone in the databases, was characterized and shown, like EPM2A, to be ubiquitously expressed. The gene, which comprises one large exon 1824 nucleotides in length and has alternative 3' untranslated regions, maps to human chromosome 3p22.1. The function is currently not known and extensive analyses do not reveal any homology to other proteins or any obvious structural motifs. Because genetic heterogeneity in Lafora disease has been described, mutational analysis of the EPM2AIP1 gene was performed on non-EPM2A patients, but no mutations were found. The identification of this first binding partner for laforin promises to be an important step toward unraveling the underlying pathogenesis of this severest form of teenage-onset epilepsy.
