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In the original version of this article, the name of one of the authors is not correct. The correct name should be W. A. Linke, which is shown correctly in the authorgroup section above.
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The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5-10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180 °C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.
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OBJECTIVE: Mutations in TPM3, encoding Tpm3.12, cause a clinically and histopathologically diverse group of myopathies characterized by muscle weakness. We report two patients with novel de novo Tpm3.12 single glutamic acid deletions at positions ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with congenital muscle stiffness, rather than weakness, and respiratory failure in one patient. METHODS: The effect of the Tpm3.12 deletions on the contractile properties in dissected patient myofibers was measured. We used quantitative in vitro motility assay to measure Ca(2+) sensitivity of thin filaments reconstituted with recombinant Tpm3.12 ΔE218 and ΔE224. RESULTS: Contractility studies on permeabilized myofibers demonstrated reduced maximal active tension from both patients with increased Ca(2+) sensitivity and altered cross-bridge cycling kinetics in ΔE224 fibers. In vitro motility studies showed a two-fold increase in Ca(2+) sensitivity of the fraction of filaments motile and the filament sliding velocity concentrations for both mutations. INTERPRETATION: These data indicate that Tpm3.12 deletions ΔE218 and ΔE224 result in increased Ca(2+) sensitivity of the troponin-tropomyosin complex, resulting in abnormally active interaction of the actin and myosin complex. Both mutations are located in the charged motifs of the actin-binding residues of tropomyosin 3, thus disrupting the electrostatic interactions that facilitate accurate tropomyosin binding with actin necessary to prevent the on-state. The mutations destabilize the off-state and result in excessively sensitized excitation-contraction coupling of the contractile apparatus. This work expands the phenotypic spectrum of TPM3-related disease and provides insights into the pathophysiological mechanisms of the actin-tropomyosin complex.
Sujet(s)
Contraction musculaire , Fibres musculaires squelettiques/anatomopathologie , Maladies musculaires/génétique , Tropomyosine/génétique , Enfant d'âge préscolaire , Exome , Femelle , Humains , Mâle , Maladies musculaires/anatomopathologie , Maladies musculaires/physiopathologie , Mutation , Phénotype , Insuffisance respiratoire , Délétion de séquenceSujet(s)
Arthrite infectieuse/diagnostic , Arthroplastie prothétique , Infections dues aux prothèses/diagnostic , Algorithmes , Arthrite infectieuse/sang , Protéine C-réactive/métabolisme , Maladie chronique , Tests hématologiques , Humains , Infections dues aux prothèses/sang , Manipulation d'échantillons , Synovie/cytologieSujet(s)
Actines/génétique , Hypertonie musculaire/génétique , Mutation/génétique , Myopathies némaline/diagnostic , Myopathies némaline/génétique , Issue fatale , Humains , Nourrisson , Mâle , Hypertonie musculaire/complications , Hypertonie musculaire/diagnostic , Myopathies némaline/complicationsRÉSUMÉ
This review is devoted to critical analysis of data concerning the structure and functions of small heat shock proteins with apparent molecular mass 20 kD (Hsp20). We describe the structure of Hsp20, its phosphorylation by different protein kinases, interaction of Hsp20 with other small heat shock proteins, and chaperone activity of Hsp20. The distribution of Hsp20 in different animal tissues and the factors affecting expression of Hsp20 are also described. Data on the possible involvement of Hsp20 in regulation of platelet aggregation and glucose transport are presented and analyzed. Special attention is paid to literature data describing probable regulatory effect of Hsp20 on contraction of smooth muscle. Two hypotheses postulating direct effect of Hsp20 on actomyosin interaction or its effect on cytoskeleton are compared and analyzed. The most recent data on the effect of Hsp20 on apoptosis and contractile activity of cardiomyocytes are also presented.
Sujet(s)
Protéines du choc thermique/physiologie , Contraction musculaire/physiologie , Phosphoprotéines/physiologie , Animaux , Apoptose/physiologie , Protéines du choc thermique HSP20 , Protéines du choc thermique/composition chimique , Protéines du choc thermique/métabolisme , Humains , Masse moléculaire , Muscles lisses/physiologie , Myocytes cardiaques/physiologie , Phosphoprotéines/composition chimique , Phosphoprotéines/métabolisme , Phosphorylation , Agrégation plaquettaire/physiologie , Distribution tissulaireRÉSUMÉ
The effect of applying an external load to actin filaments moving in the in vitro motility assay is studied. Bead-tailed actin filaments were made by polymerising actin onto 2.8 microm diameter Dynabeads conjugated with gelsolin-G actin. These were introduced into a motility cell coated with 100 microg/ml rabbit fast skeletal myosin in the presence of ATP and 0.5% methylcellulose. The motility cell was inserted between the pole-pieces of an electromagnet and the fluorescent beads and filaments were observed. The force-current relationship of the electromagnet was determined from the velocity of free beads in viscous solution and Stokes' equation. The magnet produced up to 6 pN force on the Dynabeads at 1 A. Many bead-tailed actin filaments stuck to the surface, but the beads that did move moved at the same speed as unloaded f-actin in the same cell. Bead-tailed filaments slowed down under an increasing magnetic load, eventually stalled and then slid backward under increasing load before detaching from the surface. Single-filament force-velocity curves were constructed and a stalling force of about 0.6 pN/mm of actin filament estimated.
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Magnétisme , Micromanipulation/méthodes , Moteurs moléculaires/composition chimique , Myosines/composition chimique , Nanotechnologie/méthodes , Cytosquelette d'actine/ultrastructure , Animaux , Élasticité , Champs électromagnétiques , Micromanipulation/instrumentation , Moteurs moléculaires/ultrastructure , Myosines/ultrastructure , Nanotechnologie/instrumentation , Liaison aux protéines , Lapins , Contrainte mécaniqueRÉSUMÉ
The modern classification of small heat shock proteins (sHsp) is presented and peculiarities of their primary structure and the mechanism of formation of oligomeric complexes are described. Data on phosphorylation of sHsp by different protein kinases are presented and the effect of phosphorylation on oligomeric state and chaperone activity of sHsp is discussed. Intracellular location of sHsp under normal and stress conditions is described and it is emphasized that under certain condition sHsp interact with different elements of cytoskeleton. The literature concerning the effect of sHsp on polymerization of actin in vitro is analyzed. An attempt is made to compare effects of sHsp on polymerization of actin in vitro with the results obtained on living cells under normal conditions and after heat shock or hormone action. The literature concerning possible effects of sHsp on cell motility is also analyzed.
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Protéines du cytosquelette/métabolisme , Protéines du choc thermique/composition chimique , Protéines du choc thermique/métabolisme , Séquence d'acides aminés , Animaux , Mouvement cellulaire , Humains , Muscles/physiologie , Phosphorylation , Structure quaternaire des protéinesRÉSUMÉ
Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments' interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506-793) and CaDH2 (residues 683-767) on the structure of actin-tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin-tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle 'ghost' fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)-phalloidin or fluorescein-5'-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Phi(A), Phi(E), Theta(1/2) and Nu were calculated. Phi(A) and Phi(E) are angles between the fiber axis and the absorption and emission dipoles, respectively; Theta(1/2) is the angle between the F-actin filament axis and the fiber axis; Nu is the relative number of randomly oriented fluorophores. Actin-tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the 'on' conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca(2+)) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca(2+)) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between 'off' (caldesmon and CaDH1) and 'on' (S-1 and CaDH2) states in a manner analogous to troponin.
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Actines/composition chimique , Actines/métabolisme , Protéines de liaison à la calmoduline/composition chimique , Muscles lisses/métabolisme , Adenosine triphosphatases/métabolisme , Animaux , Sites de fixation , Calcium/métabolisme , Protéines de liaison à la calmoduline/métabolisme , Relation dose-effet des médicaments , Activation enzymatique , Muscles squelettiques/métabolisme , Muscles lisses/cytologie , Sous-fragments de myosine/métabolisme , Peptides/composition chimique , Conformation des protéines , Structure tertiaire des protéines , Lapins , Spectrométrie de fluorescence , Facteurs temps , Tropomyosine/composition chimique , Tropomyosine/métabolisme , Troponine/métabolismeRÉSUMÉ
The inherited muscle diseases, skeletal muscle nemaline myopathy and cardiac muscle hypertrophic myopathy (HCM) have been recognised for decades. Recently it has become apparent that mutations in almost any protein component of the sarcomere could cause myopathy. Thus changes in many sarcomeric protein genes can produce a common phenotype. Several recent publications indicate the opposite property: mutations in one sarcomeric protein can produce different muscle disease phenotypes. The most dramatic example of this property is actin, mutations in which are associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, nemaline myopathy and actin myopathy.
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Actines/génétique , Cardiomyopathie dilatée/génétique , Cardiomyopathie hypertrophique familiale/génétique , Protéines du muscle/génétique , Myopathies némaline/génétique , Cytosquelette d'actine/ultrastructure , Actines/composition chimique , Actines/physiologie , Substitution d'acide aminé , Cardiomyopathie dilatée/métabolisme , Cardiomyopathie dilatée/anatomopathologie , Cardiomyopathie hypertrophique familiale/anatomopathologie , Prédisposition génétique à une maladie , Génotype , Humains , Modèles moléculaires , Protéines du muscle/composition chimique , Myopathies némaline/anatomopathologie , Phénotype , Mutation ponctuelle , Conformation des protéines , Sarcomères/ultrastructure , Relation structure-activité , Tropomyosine/composition chimique , Tropomyosine/génétique , Tropomyosine/physiologie , Troponine I/composition chimique , Troponine I/génétique , Troponine I/physiologie , Troponine T/composition chimique , Troponine T/génétique , Troponine T/physiologieRÉSUMÉ
The interaction of smooth muscle caldesmon with synthetic calmodulin (SynCam) and its five mutants with replacement of Lys-75 was analyzed by means of intrinsic Trp fluorescence, zero-length crosslinking and by caldesmon-induced inhibition of actomyosin ATPase activity. SynCam and its double mutant with replacement K75P and simultaneous insertion of KGK between residues 80 and 81 have a comparably low affinity to caldesmon and the probability of crosslinking of this mutant to caldesmon was the lowest among all mutants analyzed. SynCam and its double mutant (K75P+KGK) induced nearly complete reversion of caldesmon inhibition of actomyosin ATPase activity with half-maximal reversion achieved at about 1 microM. Two mutants, K75A and K75V, with partially stabilized less positive central domain have higher affinity to caldesmon. These mutants induce 80-85% reversion of caldesmon inhibition of actomyosin ATPase and the half-maximal reversion was achieved at about 0.3-0.4 microM. Two last mutants, K75P and K75E, with distorted central domain have high affinity to caldesmon and the probability of crosslinking of K75P to caldesmon was the highest among calmodulin mutants tested. These mutants induced complete reversion of caldesmon inhibition with half-maximal effect observed at 0.3-0.4 microM. We suggest that the length, flexibility and charge of the central domain affect binding of calmodulin mutants and their ability to reverse caldesmon-induced inhibition of actomyosin ATPase activity.
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Protéines de liaison à la calmoduline/métabolisme , Calmoduline/métabolisme , Lysine/métabolisme , Muscles lisses/métabolisme , Substitution d'acide aminé , Animaux , Calmoduline/composition chimique , Bovins , Canards , Myosines/antagonistes et inhibiteurs , Lapins , Spectrométrie de fluorescenceRÉSUMÉ
The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight caldesmon (l-caldesmon) was studied. The N-terminal myosin-binding domain of caldesmon N152 colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-caldesmon, suggesting it may represent a regulatory mechanism for actin-caldesmon interaction in vivo.
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Actomyosine/métabolisme , Protéines de liaison à la calmoduline/métabolisme , Fibroblastes/métabolisme , Actines/métabolisme , Animaux , Fixation compétitive/effets des médicaments et des substances chimiques , Fixation compétitive/physiologie , Protéines de liaison à la calmoduline/génétique , Protéines de liaison à la calmoduline/pharmacologie , Cellules cultivées , Poulets , Réactifs réticulants/pharmacologie , Cytosquelette/métabolisme , Fibroblastes/cytologie , Cellules HeLa/cytologie , Cellules HeLa/métabolisme , Humains , Mitogen-Activated Protein Kinases/métabolisme , Mitogen-Activated Protein Kinases/pharmacologie , Mutagenèse dirigée , Myosines/métabolisme , Fragments peptidiques/génétique , Fragments peptidiques/pharmacologie , Phosphorylation/effets des médicaments et des substances chimiques , Structure tertiaire des protéines/physiologie , TransfectionRÉSUMÉ
PURPOSE: To document the outcomes of arthroscopic stabilization of Snyder type II SLAP (superior labrum, anterior and posterior) lesions, using a bioabsorbable tack. TYPE OF STUDY: A case series. METHODS: Twenty-five SLAP lesions were repaired arthroscopically using a bioabsorbable tack. There were 22 recreational, 2 high school, and 1 professional athlete in this group. Shoulder function was surveyed at a mean follow-up of 35 months (range, 24 to 51 months) using the UCLA and ASES shoulder scoring algorithms. RESULTS: Shoulder function improved in 24 of the 25 cases. Follow-up UCLA scores averaged 32 points with 9 patients scoring as excellent, 13 good, 2 fair, and 1 poor, for an overall success rate of 88%. ASES shoulder scores similarly improved from a preoperative average of 45 points to a postoperative average of 92. All but 2 of the athletes had returned to their preinjury level of sports participation. CONCLUSIONS: Detachment of the superior labrum from the glenoid is recognized as a problematic injury in throwing athletes and others who engage in repetitive overhead activities. We conclude from our experience that using an absorbable tack to repair type II SLAP lesions is an effective treatment, even in athletes with high demands and expectations for shoulder function.
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Implant résorbable , Arthroscopie , Traumatismes sportifs/chirurgie , Lésions de l'épaule , Articulation glénohumérale/chirurgie , Plaies non pénétrantes/chirurgie , Adolescent , Adulte , Algorithmes , Vis orthopédiques , Femelle , Études de suivi , Football américain/traumatismes , Humains , Mâle , Adulte d'âge moyen , Résultat thérapeutique , Plaies non pénétrantes/diagnosticRÉSUMÉ
We have studied the effect of an internal load on the movement of actin filaments over a bed of heavy meromyosin (HMM) in the in vitro motility assay. Immobilized alpha-actinin can bind to actin filaments reversibly and ultimately stop the filaments from moving. Above a critical concentration of alpha-actinin, thin filament velocity rapidly diminished to zero. The fraction of thin motile filaments decreased linearly to zero with increasing alpha-actinin concentration. The concentration of alpha-actinin needed to stop all filaments from moving (0.8 microg/ml with actin) was very consistent both within and between experiments. In the present study we have defined the 'index of retardation' as the concentration of alpha-actinin needed to stop all filament movement, and we propose that this index is a measure of the isometric force exerted by HMM on actin filaments. When we measured the effect of immobilized alpha-actinin on motility in the presence of 10 mM P(i) we found that the index of retardation was 0.62+/-0.07 (n=3) times that in the absence of P(i). This observation is in agreement with the reduction of isometric tension in chemically-skinned muscle due to P(i). In a series of comparative experiments we observed that tropomyosin and troponin increase the index of retardation and that the degree of increase depends upon the tropomyosin isoform studied. The index of retardation of actin is increased 1.8-fold by skeletal-muscle tropomyosin, and 3-fold by both cardiac-muscle and smooth-muscle tropomyosin. In the presence of troponin the index of retardation is 2.9-3.4-fold greater than that of actin with all tropomyosin isoforms.
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Actines/physiologie , Myosines/physiologie , Tropomyosine/physiologie , Troponine/physiologie , Animaux , Contraction musculaire , Muscles squelettiques/physiologie , LapinsRÉSUMÉ
Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca2+-binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca2+-regulated at 25 degrees C/50 mM KCl. We isolated the Ca2+-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25 degrees C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca2+]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.
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Cytosquelette d'actine/métabolisme , Calcium/métabolisme , Protéines de liaison à la calmoduline/métabolisme , Calmoduline/composition chimique , Calmoduline/isolement et purification , Contraction musculaire/physiologie , Muscles lisses/métabolisme , Cytosquelette d'actine/ultrastructure , Actines/métabolisme , Animaux , Données de séquences moléculaires , Muscles lisses/ultrastructure , Isoformes de protéines/composition chimique , Isoformes de protéines/isolement et purification , Structure tertiaire des protéines/physiologie , Similitude de séquences d'acides aminés , Tropomyosine/métabolismeRÉSUMÉ
TRITC-phalloidin or FITC-labeled F-actin of ghost muscle fibers was bound to tropomyosin and C-terminal recombinant fragments of caldesmon CaDH1 (residues 506-793) or CaDH2 (residues 683-767). After that the fibers were decorated with myosin subfragment 1. In the absence of caldesmon fragments, subfragment 1 interaction with F-actin caused changes in parameters of polarized fluorescence, that were typical of "strong" binding of myosin heads to F-actin and of the "switched on" conformational state of actin. CaDH1 inhibited, whereas CaDH2 activated the effect of subfragment 1. It is suggested that C-terminal part of caldesmon may modulate the transition of F-actin subunits from the "switched on" to the "switched off" state.
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Actines/métabolisme , Protéines de liaison à la calmoduline/métabolisme , Myosines/métabolisme , Animaux , Sites de fixation , Protéines de liaison à la calmoduline/composition chimique , Polarisation de fluorescence , Colorants fluorescents , Liaison aux protéines , Conformation des protéines , LapinsRÉSUMÉ
The ability of caldesmon to inhibit actomyosin ATPase activity involves the interaction of three nonsequential segments of caldesmon domain 4 (amino acids 600-756) with actin. Two of these contacts are located in the C-terminal half of this region of caldesmon which has been designated domain 4b (658-756). To investigate the spatial relationship between the two sites and to determine whether their corresponding contacts on actin are sequentially distinct, we have used NMR spectroscopy to compare the actin binding properties of the minimal inhibitory peptide LW30 comprising residues 693-722 with those of the recombinant domain 4b constructs 658C (658-756) and Cg1 (a mutant of 658C in which the sequence (691)Glu-Trp-Leu-Thr-Lys-Thr(696) is changed to Pro-Gly-His-Tyr-Asn-Asn). Cg1 retains dual-sited actin attachment but displays lowered actin affinity. In the presence of tropomyosin, domain 4b-actin contacts were stronger but not qualitatively different, indicating that tropomyosin affected the conformational equilibrium of caldesmon binding. Simultaneous dual-sited attachment of domain 4b to actin is enabled by the conformational properties of the site-spanning sequence common to 658C, Cg1, and LW30 as reflected in the corresponding NOE and other NMR spectral parameters. A backbone turn region ((713)Gly-Asp-Val-Ser(716)) preceded by an extended segment (Ser(702)-Pro-Ala-Pro-Lys-Pro) acts to constrain the relative disposition of the flanking actin contact sites of domain 4b. In tests with a library of actin peptides, only the C-terminus, 350-375, bound to 658C and LW30. The use of Cu(2+) as a paramagnetic spectral probe bound to the unique His-371 provided evidence of a well-defined geometry for the complex between LW30 and actin residues 350-375 with the N-terminal, site B of domain 4b close to the C-terminal residues of actin. The data are discussed in the context of the potentiation of inhibitory activity by tropomyosin.
Sujet(s)
Actines/composition chimique , Protéines de liaison à la calmoduline/composition chimique , Fragments peptidiques/composition chimique , Actines/métabolisme , Séquence d'acides aminés , Animaux , Sites de fixation , Protéines de liaison à la calmoduline/génétique , Protéines de liaison à la calmoduline/métabolisme , Poulets , Modèles moléculaires , Données de séquences moléculaires , Mutagenèse dirigée , Résonance magnétique nucléaire biomoléculaire/méthodes , Fragments peptidiques/génétique , Fragments peptidiques/métabolisme , Conformation des protéines , Structure tertiaire des protéines/génétique , Lapins , Tropomyosine/composition chimique , Tropomyosine/métabolismeRÉSUMÉ
The effect of direct phosphorylation by recombinant p44erk1 mitogen-activated protein kinase on the inhibitory activity of caldesmon and its C-terminal fragment H1 was studied in vitro. Neither inhibition of actin-tropomyosin activated ATPase of heavy meromyosin by caldesmon or H1, nor inhibition of the actin-tropomyosin motility over heavy meromyosin by H1 was significantly affected by the phosphorylation while only a moderate effect on the actin-activated component of heavy meromyosin ATPase inhibition was observed. Phosphopeptide mapping of caldesmon immunoprecipitated from [32P]PO4-labelled intact gizzard strips revealed that it is predominantly phosphorylated at mitogen-activated protein kinase sites in unstimulated tissue and that it is stimulated for 1 h with phorbol 12,13-dibutyrate. We find that phorbol 12,13-dibutyrate also induces a transitory phosphorylation of caldesmon peaking at 15 min after addition and this phosphorylation is not attributed to mitogen-activated protein kinase, protein kinase C, Ca2+/calmodulin-dependent kinase II or casein kinase II. We suggest that a yet unidentified kinase, rather than mitogen-activated protein kinase, may be involved in regulation of the caldesmon function in vivo.
Sujet(s)
Calcium-Calmodulin-Dependent Protein Kinases/métabolisme , Protéines de liaison à la calmoduline/métabolisme , Protéines de liaison à la calmoduline/pharmacologie , Mitogen-Activated Protein Kinase 1/métabolisme , Mitogen-Activated Protein Kinases , Muscles lisses/enzymologie , Protein kinases/métabolisme , Animaux , Protéines de liaison à la calmoduline/composition chimique , Poulets , Gésier/enzymologie , Cinétique , Mitogen-Activated Protein Kinase 3 , Cartographie peptidique , 12,13-Dibutyrate de phorbol/pharmacologie , Phosphopeptides/composition chimique , Phosphopeptides/isolement et purification , Phosphorylation , Protéine kinase C/métabolismeRÉSUMÉ
We studied the effect of caldesmon peptides on the regulation of shortening of single guinea-pig taenia coli cells permeabilised with saponin. When contraction was initiated by Ca2+ and MgATP shortening rate at pCa 4.5 was 0.17+/-0.04 cell lengths s-1 and half-maximal rate was at pCa 5.6. Following thiophosphorylation with 1 mM adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) at pCa 4.5 for 10 min, on addition of ATP these cells contracted at of 0.25+/-0.04 cell lengths s-1 independently of pCa. If thiophosphorylated cells were preincubated with H1 (domains 3 and 4 of caldesmon), shortening speed was reduced (ID50=2 microM). Shortening speed was also reduced by 658C (domain 4b) at higher concentrations (ID50=400 microM). H13 (domain 4a), which does not block weak binding but inhibits actin-tropomyosin, inhibited cell shortening (ID50=6 microM). H2, which blocks weak binding but does not inhibit actin-tropomyosin, did not inhibit shortening. Western blots of the cells showed that the peptides were tightly bound within the cell but the native caldesmon was not displaced. These results indicate that exogenous caldesmon peptides added to smooth muscle cells may be incorporated into the thin filaments and produce effects on shortening, as expected if it were involved in tropomyosin-dependent inhibition of the actin filament in the cell.