"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan muscles: evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud'ko, G.G. Matusovskaya, T.V. Permyakova, O.S. Matusovsky, Arch. Biochem. Biophys. 432 (2004) 269-277]. In this paper, we proposed a scheme for a possible catch mechanism involving twitchin links and regulated thin filaments. The experimental evidence in support of the scheme is provided. It was found that twitchin can interact not only with mussel myosin and rabbit F-actin but also with the paramyosin core of thick filaments, myorod, mussel thin filaments, "natural" F-actin from mussel, and skeletal myosin from rabbit. No difference was revealed in binding of twitchin with mussel and rabbit myosin. The capability of twitchin to interact with all thick filament proteins suggests that putative twitchin links can be attached to any site of thick filaments. Addition of twitchin to a mixture of actin and paramyosin filaments, or to a mixture of Ca(2+)-regulated actin and myosin filaments under relaxing conditions caused in both cases similar changes in the optical properties of suspensions, indicating an interaction and aggregation of the filaments. The interaction of actin and myosin filaments in the presence of twitchin under relaxing conditions was not accompanied by an appreciable increase in the MgATPase activity. We suggest that in both cases aggregation of filaments was caused by formation of twitchin links between the filaments. We also demonstrate that native thin filaments from the catch muscle of the mussel Crenomytilus grayanus are Ca(2+)-regulated. Twitchin inhibits the ability of thin filaments to activate myosin MgATPase in the presence of Ca(2+). We suggest that twitchin inhibition of the actin-myosin interaction is due to twitchin-induced switching of the thin filaments to the inactive state.     

Phosphorylation of myorod (catchin) by kinases tightly associated to molluscan and vertebrate smooth muscle myosins

Myorod, also known as catchin, a newly discovered component of molluscan smooth muscle thick filaments, is an alternative product of the myosin heavy chain gene. It contains a C-terminal rod part that is identical to that part of myosin and a unique N-terminal domain that is very small relative to the myosin head domain. The role of myorod in contraction or relaxation of this muscle type is unknown. In the present study we demonstrated that myorod was phosphorylated not only by a kinase endogenous to molluscan myosin and twitchin but also to vertebrate smooth muscle myosin light chain kinase (MLCK). The rates and maximal levels of phosphorylation were up to threefold higher than those observed by protein kinase A with clear optima at the physiological salt concentrations. Using a mild digestion with chymotrypsin we isolated an 11 kDa phosphopeptide and showed that the phosphorylation site was located at the N-terminal domain of myorod at Thr 141 position. The sequence around this site exhibited a high degree of similarity to that expected for the substrate recognition site of MLCK. The phosphorylation rates strongly depended on the ionic conditions indicating that this site could be readily sterically blocked during myorod polymerization. Another component of the thick filaments involved in regulation of the catch state, twitchin, was phosphorylated by MLCK and exhibited endogenous myorod kinase and MLCK activities. A possible role of these phosphorylation reactions in the regulation of molluscan smooth muscles is discussed.     

Twitchin, a thick-filament protein from molluscan catch muscle, interacts with F-actin in a phosphorylation-dependent way

Twitchin belongs to the titin-like giant proteins family, it is co-localized with thick filaments in molluscan catch muscles and regulates the catch state depending on its level of phosphorylation. The mechanism by which twitchin controls the catch state remains to be established. We report for the first time the ability of twitchin to interact with F-actin. The interaction is observed at low and physiological ionic strengths, irrespective of the presence or absence of Ca(2+). It was demonstrated by viscosity and turbidity measurements, low- and high-speed co-sedimentation, and with the light-scattering particle size analysis revealing the specific twitchin-actin particles. The twitchin-actin interaction is regulated by twitchin phosphorylation: in vitro phosphorylated twitchin does not interact with F-actin. We speculate that the catch muscle twitchin might provide a mechanical link between thin and thick filaments, which contributes to catch force maintenance.     

Xin-repeats and nebulin-like repeats bind to F-actin in a similar manner

Xin and nebulette are striated muscle-specific actin-binding proteins that both contain multiple actin-binding repeats. The nature of these repeats is different: nebulette has nebulin-like repeats, while Xin contains its own unique repeats. However, the suggestion was made from biochemical data that the Xin-repeats may bind to multiple sites on the actin molecule as was found for nebulin. We have used electron microscopy and the iterative helical real space reconstruction to visualize complexes of F-actin with Xin fragments containing either three or six Xin-repeats, and with the CN5-nebulette fragment, containing five nebulin-like repeats. Our results indicate that Xin and nebulette fragments bind to F-actin in a similar manner and in two distinct modes: in one mode actin subdomain 1 is bound, while in the second mode the binding bridges between a different site on actin subdomains 1/2 of one protomer and subdomains 3/4 of an adjacent actin protomer. Taken together with published data about nebulin, tropomyosin and ADF/cofilin, our results suggest that the ability to bind in multiple modes to the actin protomer is a general property of many actin-binding proteins.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate     

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with ¹⁵N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.     

Adaptation of mouse skeletal muscle to a novel functional overload test: changes in myosin heavy chains and SERCA and physiological consequences

We have used a new approach to study the effects of overload on skeletal muscle phenotype in mice. The method used avoids any traumatising contact with muscles and the inflammatory reaction that this may provoke. Blocks of lead embedded in silicone were inserted under the skin of the lower part of the back. After 1 month, a 17% hypertrophy was found to have occurred in the tonic soleus muscle, but no change was observed in the fast-twitch extensor digitorum longus (EDL) muscle. The main effects on the contractile properties of the soleus muscle were a decrease in the tetanic relaxation rate and a reduction in the maximal velocity of shortening. Immunohistological analysis of the soleus muscles revealed an increase in the proportion of fibres that express myosin heavy chain (MHC) 1, from 54.2% to 73.9%, with a reduction in the proportion of MHC2a-positive fibres, from 45.8% to 30.2%. These changes were accompanied by an increase in the proportion of fibres that express the slow type of sarcoplasmic reticulum calcium pump (SERCA2a), from 61.8% to 84.7%. In EDL muscles, overload induced only minor changes. Thus, this method of overload affected the soleus muscle in particular. The observed changes in the control of muscle contraction were significantly larger than the changes in typical myofibrillar properties that were observed. These results indicate that there is a temporal dissociation between the relative expression of MHCs and SERCAs.     

Centipede (Scolopendra subspinipes mutilans) venom toxin peptide exhibits cytotoxic and cell growth effects in a concentration-dependent manner

Centipede venom contains a variety of proteins, peptides, and enzymes. However, the biological actions of toxin peptides in centipede venom remain largely unknown. In this study, we identified a centipede (Scolopendra subspinipes mutilans) venom toxin peptide (SsmTP) that was shown to act as a cell growth factor at low concentrations-in vitro. SsmTP was found to consist of 66 amino acids that display seven cysteine residues, which exhibited high similarity to the predicted neurotoxin. SsmTP was expressed in the venom gland of S. s. mutilans. A recombinant SsmTP peptide of approximately 5.2 kDa was produced in baculovirus-infected insect cells. Interestingly, SsmTP exhibited cytotoxicity in murine cells in a concentration-dependent manner but displayed a cell growth effect at low concentrations. SsmTP at a low concentration also protected murine cells against oxidative damage through the inhibition of caspase-1 and apoptosis. Our data indicate that SsmTP acts as a cell growth factor and a toxin peptide in a concentration-dependent manner. Consequently, our results provide evidence that the identification of SsmTP, a centipede toxin peptide, may not only elucidate the biological action of toxin peptides but also offer additional insight into the pharmacological applications of centipede venoms.     

Support for a trimeric collar of myosin binding protein C in cardiac and fast skeletal muscle, but not in slow skeletal muscle

Myosin-binding protein C (MyBPC) is proposed to take on a trimeric collar arrangement around the thick filament backbone in cardiac muscle, based on interactions between cardiac MyBPC domains C5 and C8. We have now determined, using yeast two-hybrid and in vitro binding assays, that the C5:C8 interaction is not dependent on the 28-residue cardiac-specific insert in C5. Furthermore, an interaction of similar affinity occurs between domains C5 and C8 of fast skeletal muscle MyBPC, but not between these domains of the slow skeletal muscle protein. These data have implications for the role and quaternary structure of MyBPC in skeletal muscle.     

Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle

Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction-associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinase, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin-linked contractile machinery in smooth muscle.     

Trim32 is a ubiquitin ligase mutated in limb girdle muscular dystrophy type 2H that binds to skeletal muscle myosin and ubiquitinates actin

Trim32 belongs to the tripartite motif (TRIM) protein family, which is characterized by a common domain structure composed of a RING-finger, a B-box, and a coiled-coil motif. In addition to these motifs, Trim32 possesses six C-terminal NHL-domains. A point mutation in one NHL domain (D487N) has been linked to two forms of muscular dystrophy called limb girdle muscular dystrophy type 2H and sarcotubular myopathy. In the present study we demonstrate that Trim32 is an E3 ubiquitin ligase that acts in conjunction with ubiquitin-conjugating enzymes UbcH5a, UbcH5c, and UbcH6. Western blot analysis showed that Trim32 is expressed primarily in skeletal muscle, and revealed its differential expression from one muscle to another. The level of Trim32 expression was elevated significantly in muscle undergoing remodeling due to changes in weight bearing. Furthermore, expression of Trim32 was induced in myogenic differentiation. Thus, variability in Trim32 expression in different skeletal muscles could be due to induction of Trim32 expression upon changes in physiological conditions. We show that Trim32 associates with skeletal muscle thick filaments, interacting directly with the head and neck region of myosin. Our data indicate that myosin is not a substrate of Trim32; however, Trim32 was found to ubiquitinate actin in vitro and to cause a decrease in the level of endogenous actin when transfected into HEK293 cells. In conclusion, our results demonstrate that Trim32 is a ubiquitin ligase that is expressed in skeletal muscle, can be induced upon muscle unloading and reloading, associates with myofibrils and is able to ubiquitinate actin, suggesting its likely participation in myofibrillar protein turnover, especially during muscle adaptation.     

Ibuprofen loading and release in amphiphilic peptide FA32 and its derivatives: a coarse-grained molecular dynamics simulation study

A molecular dynamics simulation study is reported to investigate the loading and release of ibuprofen (IBU) in amphiphilic peptide (AF)₆H₅K₁₅ (FA32) and its derivatives (F₁₂H₅K₁₅ and F₁₆H₅K₁₅). The peptides are represented by the MARTINI coarse-grained model, and a similar model is developed here for IBU. Upon the loading of IBU in FA32, quasi-spherical core/shell structured micelles are formed. IBU is predominantly located in the hydrophobic core and covered by Phe and Ala residues, while Lys is in the hydrophilic shell. With increasing concentration of IBU, the micelles become larger due to increased hydrophobic interactions. In FA32 derivatives, the loading of IBU leads to different morphologies; particularly, a well-structured nanofibre is formed in F₁₆H₅K₁₅. Upon pH change, the release of IBU from FA32 micelles is found to be slower than from F₁₆H₅K₁₅ nanofibre, suggesting the former is better in controlled release. The simulation study reveals that IBU-loaded morphology can be altered by changing the type of peptide and has a significant effect on IBU release profile. This bottom-up insight might be useful in the rational design of carriers for efficient drug loading and release.     

Binding of HIV-1 TAR mRNA to a peptide nucleic acid oligomer and its conjugates with metal-ion-binding multidentate ligands

A peptide nucleic acid (PNA) oligomer and a series of PNA conjugates featuring covalently attached pendant 1,4,7,10-tetraazacyclododecane (cyclen) or bis((pyridin-2-yl)methyl)amine (DPA) moieties have been synthesized that are complementary to regions of the HIV-1 TAR messenger RNA stem-loop. Thermal denaturation studies, in conjunction win with native gel shift assays, suggest that the PNAs “invade” TAR to produce a mixture of two 1:1 PNA–TAR adducts, tentatively assigned as an “open-duplex” structure, in which the TAR stem-loop dissociates and the PNA hybridizes with its RNA complement via Watson–Crick base-pairing, and a triplex-type structure, in which the initially displaced RNA segment is bound to the PNA:RNA duplex through Hoogsteen base-pairing. Thermal denaturation experiments with the TAR sequence and single-stranded RNA and DNA oligonucleotides, both in the presence and in the absence of Zn²⁺ ions, show that the introduction of cyclen or DPA ligand arms into the PNA oligomer leads to a small but reproducible increase in the T _m values. This is attributed to hydrogen-bonding and/or electrostatic interactions between protonated forms of cyclen/DPA and the cognate RNA or DNA oligonucleotide targets. Contrary to expectations, the addition of Zn²⁺ ions did not further enhance duplex formation through binding of Zn(II)–cyclen or Zn(II)–DPA moieties to the complementary RNA or DNA. Native gel shift assays further confirmed the stability increase of the metal-free cyclen- and DPA-modified PNA hybrids as compared with a control PNA sequence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of atrial natriuretic peptide receptor-A antagonizes the mitogen-activated protein kinases (Erk2 and P38MAPK) in cultured human vascular smooth muscle cells

To understand the signaling mechanisms of atrial natriuretic peptide (ANP) receptor-A (NPRA), we studied the effect of the ANP/NPRA system on mitogen-activated protein kinases (MAPKs), with particular emphasis on the extracellular-regulated kinase (Erk2) and stress-activated protein kinase (p38^MAPK) in cultured human vascular smooth muscle cells (HVSMC). Angiotensin II (ANG II) and platelet-derived growth factor (PDGF) stimulated the immunoreactive Erk2 and p38^MAPK activities and their protein levels by 2–4 fold. The pretreatment of cells with ANP significantly inhibited the agonist-stimulated Erk2 and p38^MAPK activities and protein expression by 65–75% in HVSMC transiently transfected with NPRA, as compared with only 18–22% inhibition in vector-transfected cells. The pretreatment of cells with KT5823, an inhibitor of cGMP-dependent protein kinase (PKG), reversed the inhibitory effects of ANP on MAPK activities and protein expression by 90–95%. PD98059, which inhibits Erk2 by directly inhibiting the MAPK-kinase (MEK), and SB202192, a selective antagonist of p38^MAPK, blocked the Erk2 and p38^MAPK activities, respectively. Interestingly, ANP stimulated the MAPK-phosphatase-3 (MKP-3) protein levels by more than 3-fold in HVSMC over-expressing NPRA, suggesting that ANP-dependent inhibition of MAPKs may also proceed by stimulating the phosphatase cascade. These present findings provide the evidence that ANP exerts inhibitory effects on agonist-stimulated MAPKs (Erk2 and p38^MAPK) activities and protein levels in a 2-fold manner: by antagonizing the upstream signaling pathways and by activation of MKP-3 to counter-regulate MAPKs in a cGMP and PKG-dependent manner. Our results identify a signal transduction pathway in HVSMC that could contribute to vascular remodeling and structural changes in human hypertension.     

(-)-[3H]Desmethoxyverapamil, a novel Ca2+ channel probe. Binding characteristics and target size analysis of its receptor in skeletal muscle

(-)-[3H]Desmethoxyverapamil (2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan- 7-aza-9-(3-methoxyphenyl)-nonanhydrochloride) was used to label putative Ca2+ channels in guinea pig skeletal muscle. The binding sites for (-)-[3H]desmethoxyverapamil co-purified with t-tubule membrane markers in an established subcellular fractionation procedure. (-)-[3H]Desmethoxyverapamil bound to partially purified t-tubule membranes with a KD of 2.2 +/- 0.1 nM and a Bmax of 18 +/- 4 pmol/mg membrane protein at 25 degrees C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non-competitive fashion by the benzothiazepine Ca2+ antagonist d-cis-diltiazem and the 1,4-dihydropyridine Ca2+ antagonist (+)-PN 200-110. Target size analysis of the (-)-[3H]desmethoxyverapamil drug receptor site revealed a molecular mass of 107 +/- 2 kDa. In contrast, the target size of the allosterically coupled benzothiazepine drug receptor site, labelled by d-cis-[3H]diltiazem, was 130.5 +/- 4 kDa (p less than 0.01) and of the 1,4-dihydropyridine binding site 179 kDa, when labelled with [3H]nimodipine. It is concluded that (-)-[3H]desmethoxyverapamil is an extremely useful radioligand for the phenylalkylamine-selective receptor site of the t-tubule localized Ca2+ channel which is allosterically linked to two other distinct drug receptor sites.     

Design, synthesis, and immunochemical characterization of a chimeric glycopeptide corresponding to the Shigella flexneri Y O-polysaccharide and its peptide mimic MDWNMHAA

Two glycopeptide chimeras corresponding to the Shigella flexneri Y O-polysaccharide and its peptide mimic were designed in an attempt to improve the binding affinity by increasing the entropy of binding relative to the original octapeptide mimic of the O-polysaccharide. The design was based on the X-ray crystal structures of a monoclonal antibody SYA/J6 in complex with its cognate ligands, a pentasaccharide corresponding to the S. flexneri Y O-polysaccharide and the octapeptide mimic, MDWNMHAA. Both chimeric molecules consist of a rhamnose trisaccharide linked through an α- or β-thioglycosidic linkage to a MDW moiety in which the W unit has been modified. We predicted that omission of the NMHAA moiety would obviate the bound water molecules that provided complementarity with the antibody-combining site, and the conformational restriction resulting from imposition of an α-turn at the C-terminus of the peptide. The glycopeptides were then docked into the active site of SYA/J6 using the program autodock 3.0, and the structures were optimized. The best models obtained in each case showed that the chimeric molecules, with either an α- or β-thioglycosidic linkage, might be reasonable surrogate ligands for the antibody. We report here the synthesis of the α-glycopeptide employing solution and solid-phase strategies. Immunochemical characterization indicated that the α-glycopeptide unfortunately did not inhibit binding of SYA/J6 to the S. flexneri Y lipopolysaccharide.     

New multiple labelling method for improved satellite cell identification in human muscle: application to a cohort of power-lifters and sedentary men

Presently applied methods to identify and quantify human satellite cells (SCs) give discrepant results. We introduce a new immunofluorescence method that simultaneously monitors two SC markers (NCAM and Pax7), the basal lamina and nuclei. Biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects were re-examined. Significantly different results from those with staining for NCAM and nuclei were observed. There were three subtypes of SCs; NCAM⁺/Pax7⁺ (94%), NCAM⁺/Pax7⁻ (4%) and NCAM⁻/Pax7⁺ (1%) but large individual variability existed. The proportion of SCs per nuclei within the basal lamina of myofibres (SC/N) was similar for all groups reflecting a balance between the number of SCs and myonuclei to maintain homeostasis. We emphasise that it is important to quantify both SC/N and the number of SCs per fibre. Our multiple marker method is more reliable for SC identification and quantification and can be used to evaluate other markers of muscle progenitor cells.