Abstract
Mutations in the C terminus of titin, situated at the M-band of the striated muscle sarcomere, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy (LGMD) type 2J. Mutations in the protease calpain 3 (CAPN3), in turn, lead to LGMD2A, and secondary CAPN3 deficiency in LGMD2J suggests that the pathomechanisms of the diseases are linked. Yeast two-hybrid screens carried out to elucidate the molecular pathways of TMD/LGMD2J and LGMD2A resulted in the identification of myospryn (CMYA5, cardiomyopathy-associated 5) as a binding partner for both M-band titin and CAPN3. Additional yeast two-hybrid and coimmunoprecipitation studies confirmed both interactions. The interaction of myospryn and M-band titin was supported by localization of endogenous and transfected myospryn at the M-band level. Coexpression studies showed that myospryn is a proteolytic substrate for CAPN3 and suggested that myospryn may protect CAPN3 from autolysis. Myospryn is a muscle-specific protein of the tripartite motif superfamily, reported to function in vesicular trafficking and protein kinase A signaling and implicated in the pathogenesis of Duchenne muscular dystrophy. The novel interactions indicate a role for myospryn in the sarcomeric M-band and may be relevant for the molecular pathomechanisms of TMD/LGMD2J and LGMD2A.
Highlights
Mutations in the extreme C terminus of titin, situated in the periphery of the M-band, underlie two muscle diseases
When present on one allele, mutations lead to tibial muscular dystrophy (TMD,2 MIM #600334), a late-adult-onset distal myopathy typically restricted to the anterior muscles of the lower leg [6, 7]
The downstream mechanisms leading to muscular dystrophy remain unknown, but in the absence of major ultrastructural defect in the sarcomere [6, 11], the pathogenesis is likely to depend on altered regulatory functions of M-band titin
Summary
Yeast Two-hybrid Constructs—The titin bait constructs pGBKT7-M10 WT and FINmaj were produced by cloning the corresponding cDNA sequences to the pGBKT7 vector of the Matchmaker 3 system (Clontech). To generate the bait construct for the CAPN3 interaction screen, the cDNA sequence encoding Thr417– Ser643 of human CAPN3 isoform a (Fig. 1B) was cloned into the pB27 plasmid as a LexA C-terminal fusion [28]. Cultured neonatal rat cardiomyocytes, fixed onto culture dishes, were permeabilized with 0.2% Triton X-100/PBS for 5 min, washed with PBS, and stained with antibodies diluted in 1% BSA/Gold buffer (20 mM Tris-HCl, pH 7.5, 155 mM NaCl, 2 mM EGTA, 2 mM MgCl2). The sections were blocked with Duolink blocking reagent, incubated with appropriate primary antibodies (Des122 alone or together with T51 or T41) for 1–2 h room temperature, probed with anti-rabbit plus and anti-mouse minus PLA probes (at 1:10) for 1 h at 37 °C, and stained with the Duolink fluorescent detection kit 563. Protein Modeling—A structural model of the C-terminal FN3 domain of myospryn was generated by homology modeling at the Swiss-Model workspace [38], using the Protein Data Bank structure 2dmk as template
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