Coiled-coil interactions modulate multimerization, mitochondrial binding and kinase activity of myotonic dystrophy protein kinase splice isoforms

The myotonic dystrophy protein kinase polypeptide repertoire in mice and humans consists of six different splice isoforms that vary in the nature of their C-terminal tails and in the presence or absence of an internal Val-Ser-Gly-Gly-Gly motif. Here, we demonstrate that myotonic dystrophy protein kinase isoforms exist in high-molecular-weight complexes controlled by homo- and heteromultimerization. This multimerization is mediated by coiled-coil interactions in the tail-proximal domain and occurs independently of alternatively spliced protein segments or myotonic dystrophy protein kinase activity. Complex formation was impaired in myotonic dystrophy protein kinase mutants in which three leucines at positions a and d in the coiled-coil heptad repeats were mutated to glycines. These coiled-coil mutants were still capable of autophosphorylation and transphosphorylation of peptides, but the rates of their kinase activities were significantly lowered. Moreover, phosphorylation of the natural myotonic dystrophy protein kinase substrate, myosin phosphatase targeting subunit, was preserved, even though binding of the myotonic dystrophy protein kinase to the myosin phosphatase targeting subunit was strongly reduced. Furthermore, the association of myotonic dystrophy protein kinase isoform C to the mitochondrial outer membrane was weakened when the coiled-coil interaction was perturbed. Our findings indicate that the coiled-coil domain modulates myotonic dystrophy protein kinase multimerization, substrate binding, kinase activity and subcellular localization characteristics.     

Delineation of CTG repeats and clinical features in a Taiwanese myotonic dystrophy family

Myotonic dystrophy (DM) is an inherited, autosomal dominant muscular disease which is primarily caused by a CTG trinucleotide expansion mutation on chromosome 19q13.3. The size of this trinucleotide repeat is related both to the age of onset and to the severity of the clinical manifestation. This disease is very rare in Taiwan, and clinical and genetic study on DM has not yet been documented in this area. Here, we present both clinical features and degrees of CTG expansion for a Taiwanese DM family. All of the DM patients examined in this family showed obvious clinical manifestations by age 30, which included facial and limb muscle weakness with atrophy, myotonia, and ptosis. In addition, individual DM members also exhibited variable phenotypes, which may reflect the complexity of the pathogenic mechanism. Because the collection of blood specimens was considered to be an invasive procedure, a genetic study on this DM family was performed using buccal cells. Our results confirmed that four members showing classic symptoms of DM had CTG repeat expansion in the DMI locus, and that one member with ptosis and minor muscle weakness in the right foot was a normal homozygote for CTG repeat. These data demonstrate that buccal cells can provide clear and reliable results, and thus, are suitable for a family study of DM.     

Myotonic dystrophy protein kinase (DMPK) and its role in the pathogenesis of myotonic dystrophy 1

Myotonic dystrophy 1 (DM1) is an autosomal, dominant inherited, neuromuscular disorder. The DM1 mutation consists in the expansion of an unstable CTG-repeat in the 3'-untranslated region of a gene encoding DMPK (myotonic dystrophy protein kinase). Clinical expression of DM1 is variable, presenting a progressive muscular dystrophy that affects distal muscles more than proximal and is associated with the inability to relax muscles appropriately (myotonia), cataracts, cardiac arrhythmia, testicular atrophy and insulin resistance. DMPK is a Ser/Thr protein kinase homologous to the p21-activated kinases MRCK and ROCK/rho-kinase/ROK. The most abundant isoform of DMPK is an 80 kDa protein mainly expressed in smooth, skeletal and cardiac muscles. Decreased DMPK protein levels may contribute to the pathology of DM1, as revealed by gene target studies. Here we review current understanding of the structural, functional and pathophysiological characteristics of DMPK.     

Mutagenic stress modulates the dynamics of CTG repeat instability associated with myotonic dystrophy type 1

The molecular basis of the myotonic dystrophy type 1 is the expansion of a CTG repeat at the DMPK locus. The expanded disease-associated repeats are unstable in both somatic and germ lines, with a high tendency towards expansion. The rate of expansion is directly related to the size of the pathogenic allele, increasing the size heterogeneity with age. It has also been suggested that additional factors, including as yet unidentified environmental factors, might affect the instability of the expanded CTG repeats to account for the observed CTG size dynamics over time. To investigate the effect of environmental factors in the CTG repeat instability, three lymphoblastoid cell lines were established from two myotonic dystrophy patients and one healthy individual, and parallel cultures were concurrently expanded in the presence or absence of the mutagenic chemical mitomycin C for a total of 12 population doublings. The new alleles arising along the passages were analysed by radioactive small pool PCR and sequencing gels. An expansion bias of the stepwise mutation was observed in a (CTG)₁₂₄ allele of a cell line harbouring two modal alleles of 28 and 124 CTG repeats. Interestingly, this expansion bias was clearly enhanced in the presence of mitomycin C. The effect of mitomycin C was also evident in the normal size alleles in two cell lines with alleles of 13/13 and 12/69 repeats, where treated cultures showed new longer alleles. In conclusion, our results indicate that mitomycin C modulates the dynamics of myotonic dystrophy-associated CTG repeats in LBCLs, enhancing the expansion bias of long-pathogenic repeats and promoting the expansion of normal length repeats.     

Altered tracheal smooth muscle activities in an animal model of human myotonic dystrophy.

Although the 20,25 Diazacholesterol-treated rat exhibits several pathological changes involving skeletal muscle and other tissues resembling those seen in human myotonic dystrophy patients, this animal has not hitherto been examined as a model for studying smooth muscle involvement in this human disease. Rats were therefore treated chronically with 20, 25-D and comparisons made with control animals of dose-response curves of tracheal strip preparations to carbamylcholine, before and after partial irreversible blockade of cholinergic receptors by Dibenamine. Tracheas from myotonic animals exhibited diminished contractility and an increased ED₅₀. The dissociation constant for the carbamylcholine-cholinergic receptor interaction was decreased and the percentage receptor occupancy for a given contractile response was increased. These results indicate that tracheal smooth muscle function is altered after 20,25-D administration and suggest that such animals may constitute a model for studying smooth muscle function in human myotonic dystrophy.     

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.     

Haploinsuffciency for Znf9 in Znf9+/- mice is associated with multiorgan abnormalities resembling myotonic dystrophy

Myotonic dystrophy type 2 is caused by a (CCTG)/(CCUG)n repeat expansion in the first intron of the ZNF9 gene. The pathomechanism for the myotonic dystrophies is not well understood and the role of ZNF9 in myotonic dystrophy type 2 pathogenesis has not been fully clarified. We characterized Znf9+/- mice, in which the expression of Znf9 was significantly decreased, and found that their phenotype reflects many of the features of myotonic dystrophy, including muscle histological morphology, and myotonic discharges and heart conduction abnormalities, shown by electromyography and electrocardiogram analysis, respectively. Znf9 is normally highly expressed in heart and skeletal muscle, where skeletal muscle chloride channel 1 (Clc1) plays an important role. Clc1 expression was dramatically decreased in Znf9+/- mice. Znf9 transgenic mice raised Znf9 and Clc1 expression and rescued the myotonic dystrophy phenotype in Znf9+/- mice. Our results suggest that the Znf9 haploinsufficiency contributes to the myotonic dystrophy phenotype in Znf9+/- mice.     

Intracellular protein modification associated with altered T cell functions in autoimmunity

Posttranslational protein modifications influence a number of immunologic responses ranging from intracellular signaling to protein processing and presentation. One such modification, termed isoaspartyl (isoAsp), is the spontaneous nonenzymatic modification of aspartic acid residues occurring at physiologic pH and temperature. In this study, we have examined the intracellular levels of isoAsp residues in self-proteins from MRL(+/+), MRL/lpr, and NZB/W F(1) mouse strains compared with nonautoimmune B10.BR mice. In contrast to control B10.BR or NZB/W mice, the isoAsp content in MRL autoimmune mice increased and accumulated with age in erythrocytes, brain, kidney, and T lymphocytes. Moreover, T cells that hyperproliferate to antigenic stimulation in MRL mice also have elevated intracellular isoAsp protein content. Protein l-isoaspartate O-methyltransferase activity, a repair enzyme for isoAsp residues in vivo, remains stable with age in all strains of mice. These studies demonstrate a role for the accumulation of intracellular isoAsp proteins associated with T cell proliferative defects of MRL autoimmune mice.     

Multiple defects in translation associated with altered ribosomal protein L4

The ribosomal proteins L4 and L22 form part of the peptide exit tunnel in the large ribosomal subunit. In Escherichia coli, alterations in either of these proteins can confer resistance to the macrolide antibiotic, erythromycin. The structures of the 30S as well as the 50S subunits from each antibiotic resistant mutant differ from wild type in distinct ways and L4 mutant ribosomes have decreased peptide bond-forming activity. Our analyses of the decoding properties of both mutants show that ribosomes carrying the altered L4 protein support increased levels of frameshifting, missense decoding and readthrough of stop codons during the elongation phase of protein synthesis and stimulate utilization of non-AUG codons and mutant initiator tRNAs at initiation. L4 mutant ribosomes are also altered in their interactions with a range of 30S-targeted antibiotics. In contrast, the L22 mutant is relatively unaffected in both decoding activities and antibiotic interactions. These results suggest that mutations in the large subunit protein L4 not only alter the structure of the 50S subunit, but upon subunit association, also affect the structure and function of the 30S subunit.     

Structure and dynamics of the DNA hairpins formed by tandemly repeated CTG triplets associated with myotonic dystrophy.

Anomalous expansion of the DNA triplet (CTG)n causes myotonic dystrophy. Structural studies have been carried out on (CTG)n repeats in an attempt to better understand the molecular mechanism of repeat expansion. NMR and gel electrophoretic studies demonstrate the presence of hairpin structures for (CTG)5 and (CTG)6 in solution. The monomeric hairpin structure remains invariant over a wide range of salt concentrations (10-200 mM NaCl), DNA concentrations (micromolar to millimolar in DNA strand) and pH (6.0-7.5). The (CTG)n hairpin contains three bases in the loop when n is odd and four bases when n is even. For both odd and even n the stacking and pairing in the stem remain the same, i.e, two hydrogen bond T.T pairs stack with the neighboring G.C pairs. All the nucleotides in (CTG)5 and (CTG)6 adopt C2'-endo, anti conformations. Full-relaxation matrix analysis has been performed to derive the NOE distance constraints from NOESY experiments at seven different mixing times (25, 50, 75, 100, 125, 200 and 500 ms). NOESY-derived distance constraints were subsequently used in restrained molecular dynamics simulations to obtain a family of structures consistent with the NMR data. The theoretical order parameters are computed for H5-H6(cytosines) and H2'-H2" dipolar correlations for both (CTG)5 and (CTG)6 by employing the Lipari-Szabo formalism. Experimental data show that the cytosine in the loop of the (CTG)5 hairpin is slightly more flexible than those in the stem. The cytosine in the loop of the (CTG)6 hairpin is extremely flexible, implying that the dynamics of the four base loop is intrinsically different from that of the three base loop.