Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein-C slow.
ABSTRACT: Myosin binding protein C (MyBP-C) is expressed in striated muscles, where it plays key roles in the modulation of actomyosin cross-bridges. Slow MyBP-C (sMyBP-C) consists of multiple variants sharing common domains but also containing unique segments within the NH2 and COOH termini. Two missense mutations in the NH2 terminus (W236R) and COOH terminus (Y856H) of sMyBP-C have been causally linked to the development of distal arthrogryposis-1 (DA-1), a severe skeletal muscle disorder. Using a combination of in vitro binding and motility assays, we show that the COOH terminus mediates binding of sMyBP-C to thick filaments, while the NH2 terminus modulates the formation of actomyosin cross-bridges in a variant-specific manner. Consistent with this, a recombinant NH2-terminal peptide that excludes residues 34-59 reduces the sliding velocity of actin filaments past myosin heads from 9.0 ± 1.3 to 5.7 ± 1.0 ?m/s at 0.1 ?M, while a recombinant peptide that excludes residues 21-59 fails to do so. Notably, the actomyosin regulatory properties of sMyBP-C are completely abolished by the presence of the DA-1 mutations. In summary, our studies are the first to show that the NH2 and COOH termini of sMyBP-C have distinct functions, which are regulated by differential splicing, and are compromized by the presence of missense point mutations linked to muscle disease.
Project description:Myosin Binding Protein-C slow (sMyBP-C) comprises a complex family of proteins expressed in slow and fast type skeletal muscles. Similar to its fast and cardiac counterparts, sMyBP-C functions to modulate the formation of actomyosin cross-bridges, and to organize and stabilize sarcomeric A- and M-bands. The slow form of MyBP-C was originally classified as a single protein, however several variants encoded by the single MYBPC1 gene have been recently identified. Alternative splicing of the 5' and 3' ends of the MYBPC1 transcript has led to the differential expression of small unique segments interspersed between common domains. In addition, the NH2-terminus of sMyBP-C undergoes complex phosphorylation. Thus, alternative splicing and phosphorylation appear to regulate the functional activities of sMyBP-C. sMyBP-C proteins are not restricted to slow twitch muscles, but they are abundantly expressed in fast twitch muscles, too. Using bioinformatic tools, we herein perform a systematic comparison of the known human and mouse sMyBP-C variants. In addition, using single fiber westerns and antibodies to a common region of all known sMyBP-C variants, we present a detailed and comprehensive characterization of the expression profile of sMyBP-C proteins in the slow twitch soleus and the fast twitch flexor digitorum brevis (FDB) mouse muscles. Our studies demonstrate for the first time that distinct sMyBP-C variants are co-expressed in the same fiber, and that their expression profile differs among fibers. Given the differential expression of sMyBP-C variants in single fibers, it becomes apparent that each variant or combination thereof may play unique roles in the regulation of actomyosin cross-bridges formation and the stabilization of thick filaments.
Project description:A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats. Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly. We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle. The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C. alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides. NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein. The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat. It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs. The C-terminal sequence of MyBP-C is present in a 14-kD peptide which contains only the last C2 repeat. We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments. Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity. The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin. We have expressed and purified the 14-kD peptide in Escherichia coli. The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM). These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C. The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components. A comparison of the highly conserved Ig C2 domains present at the COOH-terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.
Project description:Myosin binding protein-C slow (sMyBP-C) comprises a family of accessory proteins in skeletal muscles that bind both myosin and actin filaments. Herein, we examined the role of sMyBP-C in adult skeletal muscles using in vivo gene transfer and clustered regularly interspaced short palindromic repeats technology to knock down all known sMyBP-C variants. Our findings, confirmed in two different skeletal muscles, demonstrated efficient knockdown (KD) of sMyBP-C (>70%) resulting in notably decreased levels of thick, but not thin, filament proteins ranging from ?50% for slow and fast myosin to ?20% for myomesin. Consistent with this, A bands were selectively distorted, and sarcomere length was significantly reduced. Contrary to earlier in vitro studies showing that addition of recombinant sMyBP-C slows down the formation of actomyosin crossbridges, our work demonstrates that KD of sMyBP-C in intact myofibers results in decreased contraction and relaxation kinetics under no-load conditions. Similarly, KD muscles develop markedly reduced twitch and tetanic force and contraction velocity. Taken together, our results show that sMyBP-C is essential for the regular organization and maintenance of myosin filaments into A bands and that its structural role precedes its ability to regulate actomyosin crossbridges.-Geist, J., Ward, C. W., Kontrogianni-Konstantopoulos, A. Structure before function: myosin binding protein-C slow is a structural protein with regulatory properties.
Project description:Muscle contraction relies on interaction between myosin-based thick filaments and actin-based thin filaments. Myosin binding protein C (MyBP-C) is a key regulator of actomyosin interactions. Recent studies established that the N'-terminal domains (NTDs) of MyBP-C can either activate or inhibit thin filaments, but the mechanism of their collective action is poorly understood. Cardiac MyBP-C (cMyBP-C) harbors an extra NTD, which is absent in skeletal isoforms of MyBP-C, and its role in regulation of cardiac contraction is unknown. Here we show that the first two domains of human cMyPB-C (i.e., C0 and C1) cooperate to activate the thin filament. We demonstrate that C1 interacts with tropomyosin via a positively charged loop and that this interaction, stabilized by the C0 domain, is required for thin filament activation by cMyBP-C. Our data reveal a mechanism by which cMyBP-C can modulate cardiac contraction and demonstrate a function of the C0 domain.
Project description:OBJECTIVE:To define a distinct, dominantly inherited, mild skeletal myopathy associated with prominent and consistent tremor in two unrelated, three-generation families. METHODS:Clinical evaluations as well as exome and panel sequencing analyses were performed in affected and nonaffected members of two families to identify genetic variants segregating with the phenotype. Histological assessment of a muscle biopsy specimen was performed in 1 patient, and quantitative tremor analysis was carried out in 2 patients. Molecular modeling studies and biochemical assays were performed for both mutations. RESULTS:Two novel missense mutations in MYBPC1 (p.E248K in family 1 and p.Y247H in family 2) were identified and shown to segregate perfectly with the myopathy/tremor phenotype in the respective families. MYBPC1 encodes slow myosin binding protein-C (sMyBP-C), a modular sarcomeric protein playing structural and regulatory roles through its dynamic interaction with actin and myosin filaments. The Y247H and E248K mutations are located in the NH2 -terminal M-motif of sMyBP-C. Both mutations result in markedly increased binding of the NH2 terminus to myosin, possibly interfering with normal cross-bridge cycling as the first muscle-based step in tremor genesis. The clinical tremor features observed in all mutation carriers, together with the tremor physiology studies performed in family 2, suggest amplification by an additional central loop modulating the clinical tremor phenomenology. INTERPRETATION:Here, we link two novel missense mutations in MYBPC1 with a dominant, mild skeletal myopathy invariably associated with a distinctive tremor. The molecular, genetic, and clinical studies are consistent with a unique sarcomeric origin of the tremor, which we classify as "myogenic tremor." ANN NEUROL 2019.
Project description:Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein, localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle type-specific manner. Are the expression, localization, and Ca<sup>2+</sup>-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague-Dawley rats? By mass spectrometry, 4 MyBP-C isoforms (1 fast-type MyBP-C and 3 N-terminally spliced slow-type MyBP-C) were expressed in EDL, but only the 3 slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that, only in the C-zone, MyBP-C Ca<sup>2+</sup> sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-C's N terminus as N-terminal proteolysis attenuated MyBP-C's functional capacities. To determine each MyBP-C isoform's contribution to thin filament Ca<sup>2+</sup> sensitization and slowing in the C-zone, we used a combination of in vitro motility assays using expressed recombinant N-terminal fragments and in silico mechanistic modeling. Our results suggest that each skeletal MyBP-C isoform's N terminus is functionally distinct and has modulatory capacities that depend on the muscle type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.
Project description:Myosin binding protein C (MyBP-C) consists of a family of thick filament associated proteins. Three isoforms of MyBP-C exist in striated muscles: cardiac, slow skeletal, and fast skeletal. To date, most studies have focused on the cardiac form, due to its direct involvement in the development of hypertrophic cardiomyopathy. Here we focus on the slow skeletal form, discuss past and current literature, and present evidence to support that: (i) MyBP-C slow comprises a subfamily of four proteins, resulting from complex alternative shuffling of the single MyBP-C slow gene, (ii) the four MyBP-C slow isoforms are expressed in variable amounts in different skeletal muscles, (iii) at least one MyBP-C slow isoform is preferentially found at the periphery of M-bands and (iv) the MyBP-C slow subfamily may play important roles in the assembly and stabilization of sarcomeric M- and A-bands and regulate the contractile properties of the actomyosin filaments.
Project description:The role and utility of intrinsically disordered regions (IDRs) is reviewed for two groups of sarcomeric proteins, such as members of tropomodulin/leiomodin (Tmod/Lmod) protein homology group and myosin binding protein C (MyBP-C). These two types of sarcomeric proteins represent very different but strongly interdependent functions, being responsible for maintaining structure and operation of the muscle sarcomere. The role of IDRs in the formation of complexes between thin filaments and Tmods/Lmods is discussed within the framework of current understanding of the thin filament length regulation. For MyBP-C, the function of IDRs is discussed in the context of MYBP-C-dependent sarcomere contraction and actomyosin activation.
Project description:Obscurin is a multidomain protein composed of adhesion and signaling domains that plays key roles in the organization of contractile and membrane structures in striated muscles. Overexpression of the second immunoglobulin domain of obscurin (Ig2) in developing myotubes inhibits the assembly of A- and M-bands, but not Z-disks or I-bands. This effect is mediated by the direct interaction of the Ig2 domain of obscurin with a novel isoform of myosin binding protein-C slow (MyBP-C slow), corresponding to variant-1. Variant-1 contains all the structural motifs present in the known forms of MyBP-C slow, but it has a unique COOH terminus. Quantitative reverse transcription-polymerase chain reaction indicated that MyBP-C slow variant-1 is expressed in skeletal muscles both during development and at maturity. Immunolabeling of skeletal myofibers with antibodies to the unique COOH terminus of variant-1 demonstrated that, unlike other forms of MyBP-C slow that reside in the C-zones of A-bands, variant-1 preferentially concentrates around M-bands, where it codistributes with obscurin. Overexpression of the Ig2 domain of obscurin or reduction of expression of obscurin inhibited the integration of variant-1 into forming M-bands in skeletal myotubes. Collectively, our experiments identify a new ligand of obscurin at the M-band, MyBP-C slow variant-1 and suggest that their interaction contributes to the assembly of M- and A-bands.
Project description:The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region.