RESUMO
Using a phage-displayed peptide library, we have identified the epitope recognized by a new panel of five monoclonal antibodies (mAbs) raised against full-length recombinant human lamin A. The mAbs were found to recognize both lamin A and C by Western blotting and immunolocalization at the nuclear rim. A nine-amino acid consensus sequence PLLTYRFPP in the common immunoglobulin-like (Ig-like) domain of lamin A/C contains the binding site for all five mAbs. Three-dimensional structure of the Ig-like domain of lamin A/C shows this sequence is a complete beta-strand. This sequence includes arginine-482 (R482) which is mutated in most cases of Dunnigan-type familial partial lipodystrophy (FPLD). R482 may be part of an interaction site on the surface of lamin A/C for lamin-binding proteins associated with lipodystrophy.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos , Lamina Tipo A/genética , Lamina Tipo A/imunologia , Lipodistrofia/genética , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Células Cultivadas , Mapeamento de Epitopos , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Lamina Tipo A/metabolismo , Lipodistrofia/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Emerin is a nuclear membrane protein that interacts with lamin A/C at the nuclear envelope. Mutations in either emerin or lamin A/C cause Emery-Dreifuss muscular dystrophy (EDMD). The functions of emerin are poorly understood, but EDMD affects mainly skeletal and cardiac muscle. We used a high-stringency yeast two-hybrid method to screen a human heart cDNA library, with full-length emerin as bait. Four out of five candidate interactors identified were nuclear proteins: lamin A, splicing factor YT521-B, proteasome subunit PA28 gamma and transcription factor vav-1. Specific binding between emerin and the functional C-terminal domain of YT521-B was confirmed by pull-down assays and biomolecular interaction analysis (BIAcore). Inhibition by emerin of YT521-B-dependent splice site selection in vivo suggests that the interaction is physiologically significant. A 'bipartite' binding site for YT521-B in emerin was identified using alanine substitution or disease-associated mutations in emerin. The transcription factor GCL (germ cell-less) has previously been shown to bind to the same site. The results are consistent with an emerging view that lamins and lamina-associated proteins, like emerin, have a regulatory role, as well as a structural role in the nucleus. YT521-B joins a growing list of candidates for a role in a gene expression model of the pathogenesis of EDMD.