Identification of the A-band localization domain of myosin binding proteins C and H (MyBP-C, MyBP-H) in skeletal muscle

Although major constituents of the thick filaments of vertebrate striated muscles, the myosin binding proteins (MyBP-C and MyBP-H) are still of uncertain function. Distributed in the cross-bridge bearing zone of the A-bands of myofibrils, in a series of transverse 43 nm stripes, the proteins are constructed of a tandem series of small globular domains, each composed of approximately 90–100 amino acids, which have sequence similarities to either the C2-set of immunoglobulins (IgC2) and the fibronectin type III (FnIII) motifs. MyBP-C is composed of ten globular domains (approximately 130 kDa) whereas MyBP-H is smaller (approximately 58 kDa) and consists of a unique N-terminal segment followed by four globular domains, the order of which is identical to that of MyBP-C (FnIII-IgC2-FnIII-IgC2). To improve our understanding of this protein family we have characterized the domains in each of these two proteins which are required for targeting the proteins to their native site(s) in the sarcomere during myogenesis. Cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding mutant constructs of the MyBPs bearing an N-terminal myc epitope, and their localization to the A-band examined by immunofluorescence microscopy. Based on the clarity and intensity of the myc A-band signals we concluded that constructs encoding the four C-terminal motifs of MyBP-C and MyBP-H (approximately 360 amino acids) were all that was necessary to efficiently localize each of these peptides to the A-band. Truncation mutants lacking one of these 4 domains were less efficiently targeted to the C-zone of the sarcomere. Deletion of the last C-terminal motif of MyBP-H, its myosin binding domain, abolished all localization to the A-band. A chimeric construct, HU-3C10, in which the C-terminal motif of MyBP-H was replaced by the myosin binding domain of MyBP-C, efficiently localized to the A-band. Taken together, these observations indicate that MyBP-C and MyBP-H are localized to the A-band by the same C-terminal domain, composed of two IgC2 and two FnIII motifs. A model has been proposed for the interaction and positioning of the MyBPs in the thick filament through a ternary complex of the four C-terminal motifs with the myosin rods and titin.

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The carboxyl terminus of myosin binding protein C (MyBP-C, C-protein) specifies incorporation into the A-band of striated muscle

Journal of Cell Science ◽

10.1242/jcs.109.1.101 ◽

1996 ◽

Vol 109 (1) ◽

pp. 101-111 ◽

Cited By ~ 3

Author(s):

R. Gilbert ◽

M.G. Kelly ◽

T. Mikawa ◽

D.A. Fischman

Keyword(s):

Amino Acids ◽

Protein C ◽

Binding Protein ◽

Thick Filament ◽

Binding Domain ◽

C Protein ◽

Amino Terminal ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

Myosin binding protein-C (MyBP-C), also known as C-protein, is a major constituent of the thick filaments of vertebrate striated muscles. The protein, approximately 130 kDa, consists of a series of 10 globular motifs (numbered I to X) each of approximately 90–100 amino acids, bearing resemblance to the C2-set of immunoglobins (Ig C2) and to the fibronectin type III (FnIII) motifs. Using pure preparations of myosin and MyBP-C, it has been demonstrated that the major myosin binding domain of MyBP-C resides within the C-terminal Ig C2 motif (motif X). However, in the context of the in vivo thick filament, it is uncertain if the latter domain is sufficient to target MyBP-C correctly to the A-band or if other regions of the molecule are required for this process. To answer this question, cultures of skeletal muscle myoblasts were transfected with expression plasmids encoding seven truncation mutants of MyBP-C, and their targeting to the A-band investigated by immunofluorescence microscopy. To distinguish the recombinant proteins from endogenous MyBP-C, a myc epitope was inserted at each amino terminus. Recombinant MyBP-C exhibited an identical distribution in the sarcomere to that of native MyBP-C; i.e. it was found exclusively in the C-zone of the A-band. A mutant encoding the C-terminal 372 amino acids, but lacking motifs I-VI (termed delta 1–6), also targeted correctly to the A-band. This fragment, which is composed of two Ig C2 and two FnIII motifs, was the minimal protein fragment required for correct A-band incorporation. Larger amino-terminal deletions or deletion of motif X, the myosin binding domain, abolished all localization to the A-band. One construct (delta 10) lacking only motif X strongly inhibited myofibril assembly. We conclude that the myosin binding domain of MyBP-C, although essential, is not sufficient for correct incorporation into the A-band and that motifs VII to IX are required for this process. The data suggest a topological model in which MyBP-C is associated with the thick filament through its C terminus.

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The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif.

The Journal of Cell Biology ◽

10.1083/jcb.123.3.619 ◽

1993 ◽

Vol 123 (3) ◽

pp. 619-626 ◽

Cited By ~ 143

Author(s):

T Okagaki ◽

F E Weber ◽

D A Fischman ◽

K T Vaughan ◽

T Mikawa ◽

...

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Variable Number ◽

Terminal Sequence ◽

C Protein ◽

C2 Domains ◽

Type Iii ◽

Myosin Filaments ◽

Fibronectin Type Iii ◽

Myosin Binding

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.

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Characterization of two Arabidopsis thaliana myb-like proteins showing affinity to telomeric DNA sequence

Genome ◽

10.1139/g03-136 ◽

2004 ◽

Vol 47 (2) ◽

pp. 316-324 ◽

Cited By ~ 44

Author(s):

Petra Schrumpfová ◽

Milan Kuchař ◽

Gabriela Miková ◽

Lenka Skříšovská ◽

Tatiana Kubičárová ◽

...

Keyword(s):

Amino Acids ◽

Arabidopsis Thaliana ◽

Dna Binding ◽

Dna Sequence ◽

Binding Proteins ◽

Binding Domain ◽

Dna Binding Domain ◽

Telomeric Dna ◽

Telomeric Repeats ◽

Two Hybrid

Telomere-binding proteins participate in forming a functional nucleoprotein structure at chromosome ends. Using a genomic approach, two Arabidopsis thaliana genes coding for candidate Myb-like telomere binding proteins were cloned and expressed in E. coli. Both proteins, termed AtTBP2 (accession Nos. T46051 (protein database) and GI:638639 (nucleotide database); 295 amino acids, 32 kDa, pI 9.53) and AtTBP3 (BAB08466, GI:9757879; 299 amino acids, 33 kDa, pI 9.88), contain a single Myb-like DNA-binding domain at the N-terminus, and a histone H1/H5-like DNA-binding domain in the middle of the protein sequence. Both proteins are expressed in various A. thaliana tissues. Using the two-hybrid system interaction between the proteins AtTBP2 and AtTBP3 and self interactions of each of the proteins were detected. Gel-retardation assays revealed that each of the two proteins is able to bind the G-rich strand and double-stranded DNA of plant telomeric sequence with an affinity proportional to a number of telomeric repeats. Substrates bearing a non-telomeric DNA sequence positioned between two telomeric repeats were bound with an efficiency depending on the length of interrupting sequence. The ability to bind variant telomere sequences decreased with sequence divergence from the A. thaliana telomeric DNA. None of the proteins alone or their mixture affects telo merase activity in vitro. Correspondingly, no interaction was observed between any of two proteins and the Arabidopsis telo merase reverse transcriptase catalytic subunit TERT (accession No. AF172097) using two-hybrid assay.Key words: plant telomere-binding protein, two-hybrid assay, protein expression, telomerase.

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Myosin Binding Protein-C Slow: An Intricate Subfamily of Proteins

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/652065 ◽

2010 ◽

Vol 2010 ◽

pp. 1-10 ◽

Cited By ~ 16

Author(s):

Maegen A. Ackermann ◽

Aikaterini Kontrogianni-Konstantopoulos

Keyword(s):

Skeletal Muscles ◽

Protein C ◽

Binding Protein ◽

Thick Filament ◽

Striated Muscles ◽

Present Evidence ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Associated Proteins ◽

Myosin Binding

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 ofM-bands and (iv) the MyBP-C slow subfamily may play important roles in the assembly and stabilization of sarcomericM- andA-bands and regulate the contractile properties of the actomyosin filaments.

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Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

The Journal of Cell Biology ◽

10.1083/jcb.132.1.211 ◽

1996 ◽

Vol 132 (1) ◽

pp. 211-226 ◽

Cited By ~ 146

Author(s):

A M Belkin ◽

N I Zhidkova ◽

F Balzac ◽

F Altruda ◽

D Tomatis ◽

...

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Map Kinases ◽

Neuromuscular Junctions ◽

Critical Role ◽

Functional Characterization ◽

Focal Adhesions ◽

Integrin Subunit ◽

Striated Muscles ◽

Adult Skeletal Muscle

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

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SKELETAL MyBP-C ISOFORMS TUNE THE MOLECULAR CONTRACTILITY OF DIVERGENT SKELETAL MUSCLE SYSTEMS

10.1101/676601 ◽

2019 ◽

Author(s):

Amy Li ◽

Shane Nelson ◽

Sheema Rahmanseresht ◽

Filip Braet ◽

Anabelle S. Cornachione ◽

...

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Thick Filament ◽

Muscle Contractility ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

N Terminus ◽

Slow Type ◽

Myosin Binding ◽

Slow Twitch

ABSTRACTSkeletal 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 Ca2+-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, four MyBP-C isoforms (one fast-type MyBP-C and three N-terminally spliced slow-type MyBP-C) were expressed in EDL but only the three 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 Ca2+-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 Ca2+-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.SIGNIFICANCEMyosin-binding protein C (MyBP-C) is a critical component of the skeletal muscle sarcomere, muscle’s smallest contractile unit. MyBP-C’s importance is evident by genetic mutations leading to human myopathies, such as distal arthrogryposis (i.e. club foot). However, the molecular basis of MyBP-C’s functional impact on skeletal muscle contractility is far from certain. Complicating matters further is the expression of fast- and slow-type MyBP-C isoforms that depend on whether the muscle is fast- or slow-twitch. Using multi-scale proteomic, biophysical and mathematical modeling approaches, we define the expression, localization, and modulatory capacities of these distinct skeletal MyBP-C isoforms in rat skeletal muscles. Each MyBP-C isoform appears to modulate muscle contractility differentially; providing the capacity to fine-tune muscle’s mechanical performance as physiological demands arise.

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Multiple regions of junctin drive interaction with calsequestrin-1 and localization at triads in skeletal muscle

Journal of Cell Science ◽

10.1242/jcs.259185 ◽

2021 ◽

Author(s):

Daniela Rossi ◽

Stefania Lorenzini ◽

Enrico Pierantozzi ◽

Filip Van Petegem ◽

David Osamwonuyi Amadsun ◽

...

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Ryanodine Receptor ◽

Transmembrane Protein ◽

Sr Proteins ◽

Receptor Interaction ◽

Striated Muscles ◽

Charged Amino Acids ◽

Multiple Regions ◽

Junctional Sarcoplasmic Reticulum

Junctin is a transmembrane protein of striated muscles, localized at the junctional sarcoplasmic reticulum (j-SR). It is characterized by a luminal C-terminal tail, through which it functionally interacts with calsequestrin and the ryanodine receptor. Interaction with calsequestrin was ascribed to the presence of stretches of charged amino acids. However, the regions able to bind calsequestrin have not been defined in detail. We report here that, in non-muscle cells, junctin and calsequestrin assemble in long linear regions within the endoplasmic reticulum, mirroring the formation of calsequestrin polymers. In differentiating myotubes, the two proteins co-localize at triads, where they assemble with other j-SR proteins. By performing GST pull-down assays with distinct regions of the junctin tail, we identified two KEKE motifs able to bind calsequestrin. In addition, stretches of charged amino acids downstream these motifs were found to be also able to bind calsequestrin and the ryanodine receptor. Deletion of even one of these regions impaired the ability of junctin to localize at the j-SR, suggesting that interaction with other proteins at this site represents a key element in junctin targeting.

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Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.5.c1085 ◽

1993 ◽

Vol 264 (5) ◽

pp. C1085-C1095 ◽

Cited By ~ 441

Author(s):

H. L. Sweeney ◽

B. F. Bowman ◽

J. T. Stull

Keyword(s):

Skeletal Muscle ◽

Light Chain ◽

Myosin Light Chain ◽

Striated Muscle ◽

Low Frequency ◽

Force Production ◽

Thick Filament ◽

Striated Muscles ◽

Wide Range ◽

Cross Bridges

The regulatory light chain of myosin (RLC) is phosphorylated in striated muscles by Ca2+/calmodulin-dependent myosin light chain kinase. Unique biochemical and cellular properties of this phosphorylation system in fast-twitch skeletal muscle maintain RLC in the phosphorylated form for a prolonged period after a brief tetanus or during low-frequency repetitive stimulation. This phosphorylation correlates with potentiation of the rate of development and maximal extent of isometric twitch tension. In skinned fibers, RLC phosphorylation increases force production at low levels of Ca2+ activation, via a leftward shift of the force-pCa relationship, and increases the rate of force development over a wide range of activation levels. In heart and slow-twitch skeletal muscle, the functional consequences of RLC phosphorylation are probably similar, and the primary physiological determinants are phosphorylation and dephosphorylation properties unique to each muscle. The mechanism for these physiological responses probably involves movement of the phosphorylated myosin cross bridges away from the thick-filament backbone. The movement of cross bridges may also contribute to the regulation of myosin interactions with actin in vertebrate smooth and invertebrate striated muscles.

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Distribution of c-protein isoforms in sarcomeres of developing chick skeletal muscle

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010010319x ◽

1988 ◽

Vol 46 ◽

pp. 226-227

Author(s):

D. A. Fischman ◽

J. E. Dennis ◽

T. Obinata ◽

H. Takano-Ohmuro

Keyword(s):

Skeletal Muscles ◽

Thick Filament ◽

Protein Isoforms ◽

Actin Binding ◽

Striated Muscles ◽

C Protein ◽

Sarcomeric Protein ◽

Thick Filaments ◽

Cross Bridges ◽

Bridge Bearing

C-protein is a 150 kDa protein found within the A bands of all vertebrate cross-striated muscles. By immunoelectron microscopy, it has been demonstrated that C-protein is distributed along a series of 7-9 transverse stripes in the medial, cross-bridge bearing zone of each A band. This zone is now termed the C-zone of the sarcomere. Interest in this protein has been sparked by its striking distribution in the sarcomere: the transverse repeat between C-protein stripes is 43 nm, almost exactly 3 times the 14.3 nm axial repeat of myosin cross-bridges along the thick filaments. The precise packing of C-protein in the thick filament is still unknown. It is the only sarcomeric protein which binds to both myosin and actin, and the actin-binding is Ca-sensitive. In cardiac and slow, but not fast, skeletal muscles C-protein is phosphorylated. Amino acid composition suggests a protein of little or no αhelical content. Variant forms (isoforms) of C-protein have been identified in cardiac, slow and embryonic muscles.

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