Dystrophin muscle enhancer 1 is implicated in the activation of non-muscle isoforms in the skeletal muscle of patients with X-linked dilated cardiomyopathy.

X-linked dilated cardiomyopathy (XLDC) is a dystrophinopathy characterized by severe cardiomyopathy with no skeletal muscle involvement. Several XLDC patients have been described with mutations that abolish dystrophin muscle (M) isoform expression. The absence of skeletal muscle degeneration normally associated with loss of dystrophin function was shown to be due to increased expression of brain (B) and cerebellar Purkinje (CP) isoforms of the gene exclusively in the skeletal muscle of these patients. This suggested that the B and CP promoters have an inherent capacity to function in skeletal muscle or that they are up-regulated by a skeletal muscle-specific enhancer unaffected by the mutations in these patients. In this work we have analyzed the deletion breakpoints of two XLDC patients with deletions removing the M promoter and exon 1, but not affecting the B and CP promoters. Despite the presence of several muscle-specific regulatory motifs, the B and CP promoters were found to be essentially inactive in muscle cell lines and primary cultures. As dystrophin muscle enhancer 1 (DME1), the only known muscle-specific enhancer within the dystrophin gene, is preserved in these patients, we tested its ability to up-regulate the B and CP promoters in muscle cells. B and CP promoter activity was significantly increased in the presence of DME1, and more importantly, activation was observed exclusively in cells presenting a skeletal muscle phenotype. These results point to a role for DME1 in the induction of B and CP isoform expression in the skeletal muscle of XLDC patients defective for M isoform expression.

The b and delta subunits of the Escherichia coli ATP synthase interact via residues in their C-terminal regions.

An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms of b24-156, in which one or four residues from the C terminus were removed, competed poorly for F1 binding, suggesting that these residues play an important role in b-F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b24-156 resulted in a disruption of its association with the purified delta subunit of the enzyme. To determine whether these residues interact directly with delta, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and delta were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b24-156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue delta subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and delta subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0.