Maegen A. Ackermann

Obscurin Interacts with a Novel Isoform of MyBP-C Slow at the Periphery of the Sarcomeric M-Band and Regulates Thick Filament Assembly

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.

Myosin Binding Protein-C Slow is a Novel Substrate for Protein Kinase A (PKA) and C (PKC) in Skeletal Muscle

Myosin Binding Protein-C slow (MyBP-C slow), a family of thick filament-associated proteins, consists of four alternatively spliced forms, namely variants 1-4. Variants 1-4 share common structures and sequences; however, they differ in three regions: variants 1 and 2 contain a novel 25-residue long insertion at the extreme NH(2)-terminus, variant 3 carries an 18-amino acid long segment within immunoglobulin (Ig) domain C7, and variant 1 contains a unique COOH-terminus consisting of 26-amino acids, while variant 4 does not possess any of these insertions. Variants 1-4 are expressed in variable amounts among skeletal muscles, exhibiting different topographies and potentially distinct functions. To date, the regulatory mechanisms that modulate the activities of MyBP-C slow are unknown. Using an array of proteomic approaches, we show that MyBP-C slow comprises a family of phosphoproteins. Ser-59 and Ser-62 are substrates for PKA, while Ser-83 and Thr-84 are substrates for PKC. Moreover, Ser-204 is a substrate for both PKA and PKC. Importantly, the levels of phosphorylated skeletal MyBP-C proteins (i.e., slow and fast) are notably increased in mouse dystrophic muscles, even though their overall amounts are significantly decreased. In brief, our studies are the first to show that the MyBP-C slow subfamily undergoes phosphorylation, which may regulate its activities in normalcy and disease.

Obscurins: Goliaths and Davids take over non-muscle tissues.

Obscurins comprise a family of proteins originally identified in striated muscles, where they play essential roles in myofibrillogenesis, cytoskeletal organization, and Ca2+ homeostasis. They are encoded by the single OBSCN gene, and are composed of tandem adhesion domains and signaling motifs. To date, two giant obscurin isoforms have been described in detail that differ only at the extreme COOH-terminus; while obscurin-A (∼720 kDa) contains a non-modular COOH-terminus that harbors binding sites for the adaptor proteins ankyrins, obscurin-B (∼870 kDa) contains two COOH-terminal serine-threonine kinase domains preceded by adhesion motifs. Besides the two known giant obscurins, a thorough search of transcript databases suggests that complex alternative splicing of the obscurin transcript results in the generation of additional giant as well as small isoforms with molecular masses ranging between ∼50–970 kDa. These novel isoforms share common domains with the characterized isoforms, but also contain unique regions. Using a panel of highly specific antibodies directed against epitopes spanning the entire length of giant obscurins, we employed western blotting and immunohistochemistry to perform a systematic and comprehensive characterization of the expression profile of obscurins in muscle and non-muscle tissues. Our studies demonstrate for the first time that obscurins are not restricted to striated muscles, but are abundantly expressed in several tissues and organs including brain, skin, kidney, liver, spleen, and lung. While some obscurin isoforms are ubiquitously expressed, others are preferentially present in specific tissues and organs. Moreover, obscurins are present in select structures and cell types where they assume nuclear, cytosolic, and membrane distributions. Given the ubiquitous expression of some obscurins, along with the preferential expression of others, it becomes apparent that obscurins may play common and unique roles, respectively, in the regulation and maintenance of cell homeostasis in various tissues and organs throughout the body.

Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein-C slow

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 μm/s at 0.1 μM, 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.—Ackermann, M. A., Patel, P. D., Valenti, J., Takagi, Y., Homsher, E., Sellers, J. R., Kontrogiannni‐Konstantopoulos, A., Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein‐C slow. FASEB J. 27, 3217–3228 (2013). www.fasebj.org

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

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.

Integrity of the network sarcoplasmic reticulum in skeletal muscle requires small ankyrin 1.

Small ankyrin 1 (sAnk1; Ank1.5) is a ~20 kDa protein of striated muscle that concentrates in the network compartment of the sarcoplasmic reticulum (nSR). We used siRNA targeted to sAnk1 to assess its role in organizing the sarcoplasmic reticulum (SR) of skeletal myofibers in vitro. siRNA reduced sAnk1 mRNA and protein levels and disrupted the organization of the remaining sAnk1. Sarcomeric proteins were unchanged, but two other proteins of the nSR, SERCA and sarcolipin, decreased significantly in amount and segregated into distinct structures containing sarcolipin and sAnk1, and SERCA, respectively. Exogenous sAnk1 restored SERCA to its normal distribution. Ryanodine receptors and calsequestrin in the junctional SR, and L-type Ca2+ channels in the transverse tubules were not reduced, although their striated organization was mildly altered. Consistent with the loss of SERCA, uptake and release of Ca2+ were significantly inhibited. Our results show that sAnk1 stabilizes the nSR and that its absence causes the nSR to fragment into distinct membrane compartments.

Obscurins: Unassuming giants enter the spotlight

Discovered about a decade ago, obscurin (∼720 kDa) is a member of a family of giant proteins expressed in striated muscle that are essential for normal muscle function. Much of what we understand about obscurin stems from its functions in cardiac and skeletal muscle. However, recent evidence has indicated that variants of obscurin (“obscurins”) are expressed in diverse cell types, where they contribute to distinct cellular processes. Dysfunction or abrogation of obscurins has also been implicated in the development of several pathological conditions, including cardiac hypertrophy and cancer. Herein, we present an overview of obscurins with an emphasis on novel findings that demonstrate their heretofore‐unsuspected importance in cell signaling and disease progression.

Electrostatic interactions mediate binding of obscurin to small ankyrin 1: biochemical and molecular modeling studies.

Small ankyrin 1 (sAnk1; also known as Ank1.5) is an integral protein of the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle cells, where it is thought to bind to the C-terminal region of obscurin, a large modular protein that surrounds the contractile apparatus. Using fusion proteins in vitro, in combination with site-directed mutagenesis and surface plasmon resonance measurements, we previously showed that the binding site on sAnk1 for obscurin consists, in part, of six lysine and arginine residues. Here we show that four charged residues in the high-affinity binding site on obscurin for sAnk1 (between residues 6316 and 6345), consisting of three glutamates and a lysine, are necessary, but not sufficient, for this site on obscurin to bind to sAnk1 with high affinity. We also identify specific complementary mutations in sAnk1 that can partially or completely compensate for the changes in binding caused by charge-switching mutations in obscurin. We used molecular modeling to develop structural models of residues 6322-6339 of obscurin bound to sAnk1. The models, based on a combination of Brownian and molecular dynamics simulations, predict that the binding site on sAnk1 for obscurin is organized as two ankyrin-like repeats, with the last α-helical segment oriented at an angle to nearby helices, allowing lysine 6338 of obscurin to form an ionic interaction with aspartate 111 of sAnk1. This prediction was validated by double-mutant cycle experiments. Our results are consistent with a model in which electrostatic interactions between specific pairs of side chains on obscurin and sAnk1 promote binding and complex formation.

Characterization and Comparison of Two Binding Sites on Obscurin for Small Ankyrin 1

Obscurin A, an ∼720 kDa modular protein of striated muscles, binds to small ankyrin 1 (sAnk1, Ank 1.5), an integral protein of the sarcoplasmic reticulum, through two distinct carboxy-terminal sequences, Obsc(6316-6436) and Obsc(6236-6260). We hypothesized that these sequences differ in affinity but that they compete for the same binding site on sAnk1. We show that the sequence within Obsc(6316-6436) that binds to sAnk1 is limited to residues 6316-6345. Comparison of Obsc(6231-6260) to Obsc(6316-6345) reveals that Obsc(6316-6345) binds sAnk1 with an affinity (133 ± 43 nM) comparable to that of the Obsc(6316-6436) fusion protein, whereas Obsc(6231-6260) binds with lower affinity (384 ± 53 nM). Oligopeptides of each sequence compete for binding with both sites at half-maximal inhibitory concentrations consistent with the affinities measured directly. Five of six site-directed mutants of sAnk1 showed similar reductions in binding to each binding site on obscurin, suggesting that they dock to many of the same residues of sAnk1. Circular dichroism (CD) analysis of the synthetic oligopeptides revealed a 2-fold greater α-helical content in Obsc(6316-6346), ∼35%, than Obsc(6231-6260,) ∼17%. Using these data, structural prediction algorithms, and homology modeling, we predict that Obsc(6316-6345) contains a bent α-helix of 12 amino acids, flanked by short disordered regions, and that Obsc(6231-6260) has a short, N-terminal α-helix of 4-5 residues followed by a long disordered region. Our results are consistent with a model in which both sequences of obscurin differ significantly in structure but bind to the ankyrin-like repeat motifs of sAnk1 in a similar though not identical manner.

Myosin binding protein-C slow: a multifaceted family of proteins with a complex expression profile in fast and slow twitch skeletal muscles

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.