RESUMEN
Human Ankrd2 transcript encodes a 37-kDa protein that is similar to mouse Ankrd2 recently shown to be involved in hypertrophy of skeletal muscle. These novel ankyrin-rich proteins are related to C-193/CARP/MARP, a cardiac protein involved in the control of cardiac hypertrophy. A human genomic region of 14,300 bp was sequenced revealing a gene organization similar to mouse Ankrd2 with nine exons, four of which encode ankyrin repeats. The intracellular localization of Ankrd2 was unknown since no protein studies had been reported. In this paper we studied the intracellular localization of the protein and its expression on differentiation using polyclonal and monoclonal antibodies produced to human Ankrd2. In adult skeletal muscle Ankrd2 is found in slow fibers; however, not all of the slow fibers express Ankrd2 at the same level. This is particularly evident in dystrophic muscles, where the expression of Ankrd2 in slow fibers seems to be severely reduced.
Asunto(s)
Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Regiones Promotoras Genéticas , Animales , Repetición de Anquirina , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Peso Molecular , Proteínas Musculares/análisis , Músculo Esquelético/citología , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Proteínas Nucleares , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Represoras , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , TATA Box , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
We report the identification and characterization of a novel 32-kDa protein expressed in skeletal muscle and located in the Z-disc of the sarcomere. We found that this protein binds to three other Z-disc proteins; therefore, we have named it FATZ, gamma-filamin/ABP-L, alpha-actinin and telethonin binding protein of the Z-disc. From yeast two-hybrid experiments we are able to show that the SR3-SR4 domains of alpha-actinin 2 are required to bind the COOH-terminal region of the FATZ as does gamma-filamin/ABP-L. Furthermore, by using a glutathione S-transferase overlay assay we find that FATZ also binds telethonin. The level of FATZ protein in muscle cells increases during differentiation, being clearly detectable before the onset of myosin. Although FATZ has no known interaction domains, it would appear to be involved in a complex network of interactions with other Z-band components. On the basis of the information known about its binding partners, we could envisage a central role for FATZ in the myofibrillogenesis. After screening our muscle expressed sequence tag data base and the public expressed sequence tag data bases, we were able to assemble two other muscle transcripts that show a high level of identity with FATZ in two different domains. Therefore, FATZ may be the first member of a small family of novel muscle proteins.
Asunto(s)
Actinina/metabolismo , Proteínas Portadoras/análisis , Proteínas Contráctiles/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/análisis , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Diferenciación Celular , Células Cultivadas , Clonación Molecular , Conectina , Filaminas , Humanos , Ratones , Datos de Secuencia MolecularRESUMEN
We have cloned and sequenced a cDNA from a human adult skeletal muscle cDNA library, encoding for a novel isoform of alpha-tubulin (tuba8) that is preferentially expressed in heart, skeletal muscle, and testis. A genomic DNA sequence from the chromosomal region 22q11 allowed us to determine the complete structure of the TUBA8 gene that mirrors the canonical exon/intron organization of the vertebrate alpha-tubulin genes. We also cloned and sequenced the cDNA of its murine homologue (MMU-TUBA8). The latter encodes for a protein that differs from its human counterpart in only three amino acids, revealing an extreme rate of conservation that is even extended to both the 3' and 5' UTRs of the mRNAs. Sequence comparison of these novel isoforms with other known alpha tubulins shows that tuba8 is the most divergent member of the mammalian alpha-tubulin family. The sequence peculiarity of the human and murine tuba8 strongly suggests that they might have functional significance and, according to the multi-tubulin hypothesis, that they might play specific functional roles in the cell cytoskeleton.
Asunto(s)
Cromosomas Humanos Par 22 , Músculo Esquelético/metabolismo , Tubulina (Proteína)/genética , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Humanos , Mamíferos , Ratones , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Tubulina (Proteína)/químicaRESUMEN
PDZ motifs are modular protein-protein interaction domains, consisting of 80-120 amino acid residues, whose function appears to be the direction of intracellular proteins to multiprotein complexes. In skeletal muscle, there are a few known PDZ-domain proteins, which include neuronal nitric oxide synthase and syntrophin, both of which are components of the dystrophin complex, and actinin-associated LIM protein, which binds to the spectrin-like repeats of alpha-actinin-2. Here, we report the identification and characterization of a new skeletal muscle protein containing a PDZ domain that binds to the COOH-terminal region of alpha-actinin-2. This novel 31-kD protein is specifically expressed in heart and skeletal muscle. Using antibodies produced to a fragment of the protein, we can show its location in the sarcomere at the level of the Z-band by immunoelectron microscopy. At least two proteins, 32 kD and 78 kD, can be detected by Western blot analysis of both heart and skeletal muscle, suggesting the existence of alternative forms of the protein. In fact, several forms were found that appear to be the result of alternative splicing. The transcript coding for this Z-band alternatively spliced PDZ motif (ZASP) protein maps on chromosome 10q22.3-10q23.2, near the locus for infantile-onset spinocerebellar ataxia.