RESUMEN
Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine-binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by â¼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor V/efectos de los fármacos , Factor Xa/efectos de los fármacos , Músculo Esquelético/química , Miocardio/química , Miosinas/farmacología , Fosfolípidos/farmacología , Animales , Antígenos de Superficie/farmacología , Miosinas Cardíacas/aislamiento & purificación , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/farmacología , Bovinos , Contaminación de Medicamentos , Factor VIIa/metabolismo , Factor Xa/metabolismo , Humanos , Lipoproteínas/farmacología , Proteínas de la Leche/farmacología , Miosinas/aislamiento & purificación , Miosinas/metabolismo , Fosfolipasas A2/farmacología , Conejos , Tromboplastina/farmacologíaRESUMEN
The myosin family of motor proteins is an attractive target of therapeutic small-molecule protein inhibitors and modulators. Milligrams of protein quantities are required to conduct proper biophysical and biochemical studies to understand myosin functions. Myosin protein expression and purification represent a critical starting point towards this goal. Established utilization of Dictyostelium discoideum, Drosophila melanogaster, insect and mouse cells for myosin expression and purification is limited, cost, labor and time inefficient particularly for (full-length) human myosins. Here we are presenting detailed protocols for production of several difficult-to-purify recombinant human myosins in efficient quantities up to 1â¯mg of protein per liter of cell culture. This is the first time that myosins have been purified in large scales from suspension adapted transiently and stably expressing human cells. The method is also useful for expressing other human proteins in quantities sufficient to perform extensive biochemical and biophysical characterization.
Asunto(s)
Miosinas/aislamiento & purificación , Miosinas/metabolismo , Animales , Técnicas de Cultivo de Célula , Dictyostelium/metabolismo , Células HEK293 , Humanos , Ratones , Miosinas/genética , Regiones Promotoras Genéticas , TransfecciónRESUMEN
Myosins are actin-based molecular motors which are enzymatically adapted for their cellular functions such as transportation and membrane tethering. Human Myo19 affects mitochondrial motility, and promotes their localization to stress-induced filopodia. Therefore, studying Myo19 enzymology is essential to understand how this motor may facilitate mitochondrial motility. Towards this goal, we have purified Myo19 motor domain (Myo19-3IQ) from a human-cell expression system and utilized transient kinetics to study the Myo19-3IQ ATPase cycle. We found that Myo19-3IQ exhibits noticeable conformational changes (isomerization steps) preceding both ATP and ADP binding, which may contribute to nucleotide binding regulation. Notably, the ADP isomerization step and subsequent ADP release contribute significantly to the rate-limiting step of the Myo19-3IQ ATPase cycle. Both the slow ADP isomerization and ADP release prolong the time Myo19-3IQ spend in the strong actin binding state and hence contribute to its relatively high duty ratio. However, the predicted duty ratio is lower than required to support motility as a monomer. Therefore, it may be that several Myo19 motors are required to propel mitochondria movement on actin filaments efficiently. Finally, we provide a model explaining how Myo19 translocation may be regulated by the local ATP/ADP ratio, coupled to the mitochondria presence in the filopodia.
Asunto(s)
Mitocondrias/metabolismo , Miosinas/metabolismo , Seudópodos/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico , Biomarcadores , Activación Enzimática , Expresión Génica , Humanos , Hidrólisis , Cinética , Mitocondrias/genética , Mutación , Miosinas/genética , Miosinas/aislamiento & purificación , Unión Proteica , Seudópodos/genéticaRESUMEN
Myosin is an enzyme that utilizes ATP to produce a conformational change generating a force. The kinetics of the myosin reverse recovery stroke depends on the metal cation complexed with ATP. The reverse recovery stroke is slow for MgATP and fast for MnATP. The metal ion coordinates the γ phosphate of ATP in the myosin active site. It is accepted that the reverse recovery stroke is correlated with the phosphate release; therefore, magnesium "holds" phosphate tighter than manganese. Magnesium and manganese are similar ions in terms of their chemical properties and the shell complexation; hence, we propose to use these ions to study the mechanism of the phosphate release. Analysis of octahedral complexes of magnesium and manganese show that the partial charge of magnesium is higher than that of manganese and the slightly larger size of manganese ion makes its ionic potential smaller. We hypothesize that electrostatics play a role in keeping and releasing the abstracted γ phosphate in the active site, and the stronger electric charge of magnesium ion holds γ phosphate tighter. We used stable myosin-nucleotide analog complex and Raman spectroscopy to examine the effect of the metal cation on the relative position of γ phosphate analog in the active site. We found that in the manganese complex, the γ phosphate analog is 0.01 nm further away from ADP than in the magnesium complex. We conclude that the ionic potential of the metal cation plays a role in the retention of the abstracted phosphate.
Asunto(s)
Adenosina Difosfato/química , Adenosina Trifosfato/química , Magnesio/química , Manganeso/química , Miosinas/química , Fosfatos/química , Animales , Dominio Catalítico , Cationes Bivalentes , Músculo Esquelético/química , Miosinas/aislamiento & purificación , Conformación Proteica , Conejos , Espectrometría Raman , Electricidad EstáticaRESUMEN
Porcine myocardium is regarded as a byproduct in slaughterhouses and is rarely used as a food source due to its unsuitability for processing and consumption. In this study, we sought to develop an efficient ultrasonication method to extract protein from porcine myocardium. Comparisons of protein yield using various ultrasonication conditions with porcine myocardium revealed that treatment with 0.2 M NaCl, with pH 8.0, at an extraction temperature of less than 40 °C and an amplitude of 60% to 80% was optimal, yielding an extraction rate of 90%. In addition, SDS-PAGE analysis showed that increasing the time interval for ultrasonication increased the presence of myosin heavy chain and actin protein content. Functional analysis of the physiological properties of the isolated proteins using an ATPase assay showed that Ca and Mg ATPase activity was virtually undetectable in the early stages of ultrasonic treatment and that the proteins denatured rapidly. An analysis of protein digestion also showed that the digestive capacity of proteins treated by ultrasonication methods was greater. These results demonstrate that the ultrasonication method is effective for high-yield protein extraction from cardiac myofibrils of porcine myocardium with low salt concentrations, low Ca and Mg ATPase activities, and high digestive capacities.
Asunto(s)
Actinas/aislamiento & purificación , Proteínas en la Dieta/análisis , Manipulación de Alimentos/métodos , Miocardio/química , Miofibrillas/química , Miosinas/aislamiento & purificación , Ondas Ultrasónicas , Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Calcio/metabolismo , Humanos , Magnesio/metabolismo , Miocardio/citología , Miosinas/metabolismo , PorcinosRESUMEN
We studied the modulating role of cardiac myosin-binding protein C (cMyBP-C) in tropomyosin regulation of the actin-myosin interaction. The effect of cMyBP-C on the velocity of actin-tropomyosin filament sliding over cardiac and slow skeletal myosins was evaluated using in vitro motility assay. The effect of cMyBP-C on the actin-tropomyosin filaments sliding depended on the type of myosin. The regulatory effect of cMyBP-C differs for cardiac and slow skeletal myosin because of the presence of specific essential light chain (LC1sa) in slow skeletal myosin isoform.
Asunto(s)
Actinas/química , Proteínas Portadoras/farmacología , Miosinas/química , Tropomiosina/química , Actinas/aislamiento & purificación , Actinas/metabolismo , Animales , Bioensayo , Tampones (Química) , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Bovinos , Pollos , Expresión Génica , Humanos , Movimiento (Física) , Músculo Esquelético/química , Músculo Esquelético/fisiología , Miocardio/química , Miocardio/metabolismo , Miosinas/aislamiento & purificación , Miosinas/metabolismo , Especificidad de Órganos , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Conejos , Soluciones , Tropomiosina/aislamiento & purificación , Tropomiosina/metabolismoRESUMEN
Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.
Asunto(s)
Dineínas/aislamiento & purificación , Cinesinas/aislamiento & purificación , Microscopía Fluorescente/métodos , Miosinas/aislamiento & purificación , Dineínas/química , Humanos , Cinesinas/química , Microscopía de Interferencia/métodos , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Miosinas/químicaRESUMEN
While more than 70 genes have been linked to deafness, most of which are expressed in mechanosensory hair cells of the inner ear, a challenge has been to link these genes into molecular pathways. One example is Myo7a (myosin VIIA), in which deafness mutations affect the development and function of the mechanically sensitive stereocilia of hair cells. We describe here a procedure for the isolation of low-abundance protein complexes from stereocilia membrane fractions. Using this procedure, combined with identification and quantitation of proteins with mass spectrometry, we demonstrate that MYO7A forms a complex with PDZD7, a paralog of USH1C and DFNB31. MYO7A and PDZD7 interact in tissue-culture cells, and co-localize to the ankle-link region of stereocilia in wild-type but not Myo7a mutant mice. Our data thus describe a new paradigm for the interrogation of low-abundance protein complexes in hair cell stereocilia and establish an unanticipated link between MYO7A and PDZD7.
Asunto(s)
Proteínas Portadoras/análisis , Membranas/química , Miosinas/análisis , Estereocilios/química , Animales , Proteínas Portadoras/aislamiento & purificación , Espectrometría de Masas , Ratones , Miosina VIIa , Miosinas/aislamiento & purificación , Unión ProteicaRESUMEN
Numerous female reproductive abnormalities are consequences of disorders in uterus smooth muscle (myometrium) contractile function. In this work, we described activators of ATPase, which could be used for development of effective treatments for correcting this dysfunction. Myosin ATPase localized in the catalytic domain of myosin subfragment-1 transforms a chemical energy deposited in macroergic bonds of ATP into mechanical movement. It was shown that Ñalix[4]arene C-90 and its structural analogs functionalized at the upper rim of macrocycle with four or at least two N-phenylsulfonÑltrifluoroacetamidine groups, are able to activate ATP hydrolysis catalyzed by myometrium myosin subfragment-1. It was shown with the method of computer modeling that N-phenylsulfonÑltrifluoroacetamidine groups of calix[4]arene C-90 interact with responsible for binding, coordination and the hydrolysis of ATP amino acid residues of myosin subfragment-1. The results can be used for further research aimed at using calix[4]arene C-90 and its analogs as pharmacological compounds that can effectively normalize myometrium contractile hypofunction.
Asunto(s)
Adenosina Trifosfato/química , Calixarenos/química , Miometrio/química , Subfragmentos de Miosina/química , Miosinas/química , Fenoles/química , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Calixarenos/síntesis química , Dominio Catalítico , Activación Enzimática , Femenino , Hidrólisis , Cinética , Simulación del Acoplamiento Molecular , Miometrio/enzimología , Subfragmentos de Miosina/agonistas , Subfragmentos de Miosina/aislamiento & purificación , Subfragmentos de Miosina/metabolismo , Miosinas/aislamiento & purificación , Miosinas/metabolismo , Fenoles/síntesis química , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Sulfonas/química , PorcinosRESUMEN
Structures similar to blood vessels in location, morphology, flexibility, and transparency have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty osteonal network after degradation of original organic components. Here, we test the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence. Two lines of evidence support this hypothesis. First, peptide sequencing of Brachylophosaurus canadensis blood vessel extracts is consistent with peptides comprising extant archosaurian blood vessels and is not consistent with a bacterial, cellular slime mold, or fungal origin. Second, proteins identified by mass spectrometry can be localized to the tissues using antibodies specific to these proteins, validating their identity. Data are available via ProteomeXchange with identifier PXD001738.
Asunto(s)
Vasos Sanguíneos/anatomía & histología , Vasos Sanguíneos/metabolismo , Dinosaurios/anatomía & histología , Dinosaurios/metabolismo , Fósiles/anatomía & histología , Actinas/genética , Actinas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Vasos Sanguíneos/microbiología , Huesos/irrigación sanguínea , Pollos , Dinosaurios/genética , Técnica del Anticuerpo Fluorescente/métodos , Espectrometría de Masas , Modelos Biológicos , Datos de Secuencia Molecular , Miosinas/genética , Miosinas/aislamiento & purificación , Filogenia , Proteómica/métodos , Alineación de Secuencia , Especificidad de la Especie , Struthioniformes , Tropomiosina/genética , Tropomiosina/aislamiento & purificación , Tubulina (Proteína)/genética , Tubulina (Proteína)/aislamiento & purificaciónRESUMEN
Myosin is a mechano-enzyme that hydrolyzes ATP in order to move unidirectionally along actin filaments. Here we show by single molecule imaging that myosin V motion can be activated by local heat. We constructed a dark-field microscopy that included optical tweezers to monitor 80 nm gold nanoparticles (GNP) bound to single myosin V molecules with nanometer and submillisecond accuracy. We observed 34 nm processive steps along actin filaments like those seen when using 200 nm polystyrene beads (PB) but dwell times (ATPase activity) that were 4.5 times faster. Further, by using DNA nanotechnology (DNA origami) and myosin V as a nanometric thermometer, the temperature gradient surrounding optically trapped GNP could be estimated with nanometer accuracy. We propose our single molecule measurement system should advance quantitative analysis of the thermal control of biological and artificial systems like nanoscale thermal ratchet motors.
Asunto(s)
ADN/química , Calefacción/métodos , Imagen Molecular/métodos , Miosinas/química , Pinzas Ópticas , Termografía/métodos , ADN/ultraestructura , Oro/química , Ensayo de Materiales/métodos , Nanopartículas del Metal/química , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/ultraestructura , Técnicas de Sonda Molecular , Miosinas/aislamiento & purificación , TemperaturaRESUMEN
We determined the crystal structure of the motor domain of human non-muscle myosin 2B (NM-2B) in a nucleotide-free state and at a resolution of 2.8 Å. The structure shows the motor domain with an open active site and the large cleft that divides the 50 kDa domain in a closed state. Compared to other rigor-like myosin motor domain structures, our structure shows subtle but significant conformational changes in regions important for actin binding and mechanochemical coupling. Moreover, our crystal structure helps to rationalize the impact of myosin, heavy chain 9 (MYH9)-related disease mutations Arg709Cys and Arg709His on the kinetic and functional properties of NM-2B and of the closely related non-muscle myosin 2A (NM-2A).
Asunto(s)
Miosinas/química , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación , Miosinas/genética , Miosinas/aislamiento & purificación , Estructura Terciaria de ProteínaRESUMEN
Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (â¼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) â¼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.
Asunto(s)
Miosinas/aislamiento & purificación , Miosinas/metabolismo , Estereocilios/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Calmodulina/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Chaperonas Moleculares , Datos de Secuencia Molecular , Cadenas Ligeras de Miosina/metabolismo , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/aislamiento & purificación , Subfragmentos de Miosina/metabolismo , Miosinas/genética , Pinzas Ópticas , Pliegue de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Células Sf9 , SpodopteraRESUMEN
To understand how mutations in thick filament proteins such as cardiac myosin binding protein-C or titin, cause familial hypertrophic cardiomyopathies, it is important to determine the structure of the cardiac thick filament. Techniques for the genetic manipulation of the zebrafish are well established and it has become a major model for the study of the cardiovascular system. Our goal is to develop zebrafish as an alternative system to the mammalian heart model for the study of the structure of the cardiac thick filaments and the proteins that form it. We have successfully isolated thick filaments from zebrafish cardiac muscle, using a procedure similar to those for mammalian heart, and analyzed their structure by negative-staining and electron microscopy. The isolated filaments appear well ordered with the characteristic 42.9 nm quasi-helical repeat of the myosin heads expected from x-ray diffraction. We have performed single particle image analysis on the collected electron microscopy images for the C-zone region of these filaments and obtained a three-dimensional reconstruction at 3.5 nm resolution. This reconstruction reveals structure similar to the mammalian thick filament, and demonstrates that zebrafish may provide a useful model for the study of the changes in the cardiac thick filament associated with disease processes.
Asunto(s)
Miocardio/ultraestructura , Miosinas/aislamiento & purificación , Miosinas/ultraestructura , Pez Cebra/metabolismo , Animales , Proteínas Portadoras/ultraestructura , Conectina/ultraestructura , Análisis de Fourier , Imagenología Tridimensional , Modelos MolecularesRESUMEN
Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). Structural analysis of relaxed tarantula thick filaments shows that the RLCs of the interacting free and blocked myosin heads are in different environments. This and other data suggested a phosphorylation mechanism in which Ser-35 of the free head is exposed and constitutively phosphorylated by protein kinase C, whereas the blocked head is hidden and unphosphorylated; on activation, myosin light chain kinase phosphorylates the monophosphorylated free head followed by the unphosphorylated blocked head, both at Ser-45. Our goal was to test this model of phosphorylation. Mass spectrometry of quickly frozen, intact muscles showed that only Ser-35 was phosphorylated in the relaxed state. The location of this constitutively phosphorylated Ser-35 was analyzed by immunofluorescence, using antibodies specific for unphosphorylated or phosphorylated Ser-35. In the relaxed state, myofibrils were labeled by anti-pSer-35 but not by anti-Ser-35, whereas in rigor, labeling was similar with both. This suggests that only pSer-35 is exposed in the relaxed state, while in rigor, Ser-35 is also exposed. In the interacting-head motif of relaxed filaments, only the free head RLCs are exposed, suggesting that the constitutive pSer-35 is on the free heads, consistent with the proposed mechanism.
Asunto(s)
Arácnidos , Proteínas de Artrópodos/química , Proteínas de Artrópodos/metabolismo , Miosinas/química , Miosinas/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/aislamiento & purificación , Glicerol/química , Modelos Moleculares , Datos de Secuencia Molecular , Quinasa de Cadena Ligera de Miosina/metabolismo , Miosinas/aislamiento & purificación , Fosforilación , Proteína Quinasa C/metabolismo , Serina/metabolismo , Urea/químicaRESUMEN
Chara myosin is plant myosin responsible for cytoplasmic streaming and moves actin filaments at 60 µm/s, which is the fastest of all myosins examined. The neck of the myosin molecule has usually mechanical and regulatory roles. The neck of Chara myosin is supposed to bind six light chains, but, at present, we have no knowledge about them. We found Caâºâº-calmodulin activated Chara myosin motility and its actin-activated ATPase, and actually bound with the Chara myosin heavy chain, indicating calmodulin might be one of candidates for Chara myosin light chains. Antibody against essential light chain from Physarum myosin, and antibodies against Chara calmodulin and chicken myosin light chain from lens membranes reacted with 20 kDa and 18 kDa polypeptides of Chara myosin preparation, respectively. Correspondingly, column purified Chara myosin had light chains of 20 kDa, and 18 kDa with the molar ratio of 0.7 and 2.5 to the heavy chain, respectively.
Asunto(s)
Chara/metabolismo , Miosinas/metabolismo , Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Calmodulina/aislamiento & purificación , Calmodulina/metabolismo , Chara/química , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Miosinas/química , Miosinas/aislamiento & purificación , Unión Proteica , ConejosRESUMEN
Like all eukaryotic cells, Tetrahymena thermophila contains a rich array of cytoskeletal proteins, some familiar and some novel. A detailed analysis of the structure, function, and interactions of these proteins requires procedures for purifying the individual protein components. Procedures for the purification of actin and tubulin from Tetrahymena are reviewed, followed by a description of a procedure that yields proteins from the epiplasmic layer and associated structures, including the tetrins. Finally, the challenges and opportunities for future advances are assessed.
Asunto(s)
Actinas/aislamiento & purificación , Proteínas del Citoesqueleto/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Tetrahymena thermophila/química , Tubulina (Proteína)/aislamiento & purificación , Actinas/química , Membrana Celular/química , Cilios/química , Proteínas del Citoesqueleto/química , Citoesqueleto/química , Dineínas/química , Dineínas/aislamiento & purificación , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Miosinas/química , Miosinas/aislamiento & purificación , Proteómica/métodos , Proteínas Protozoarias/química , Solubilidad , Tubulina (Proteína)/química , UltracentrifugaciónRESUMEN
Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal-troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.
Asunto(s)
Calcio/metabolismo , Músculo Esquelético/metabolismo , Enfermedades Neuromusculares/metabolismo , Troponina C/agonistas , Troponina C/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Bovinos , Humanos , Imidazoles/química , Imidazoles/uso terapéutico , Terapia Molecular Dirigida , Contracción Muscular/efectos de los fármacos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patología , Miastenia Gravis/tratamiento farmacológico , Miastenia Gravis/metabolismo , Miastenia Gravis/patología , Miosinas/aislamiento & purificación , Miosinas/metabolismo , Enfermedades Neuromusculares/tratamiento farmacológico , Enfermedades Neuromusculares/patología , Pirazinas/química , Pirazinas/uso terapéutico , Conejos , Ratas , Troponina/metabolismo , Troponina/fisiologíaRESUMEN
Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.
Asunto(s)
Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Vuelo Animal , Expresión Génica , Miosinas/genética , Miosinas/aislamiento & purificación , Transgenes/genética , Animales , Cromatografía de Afinidad/métodos , Técnicas de Inactivación de Genes , Miosinas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismoRESUMEN
A group of closely related myosins is characterized by the presence of at least one MyTH/FERM (myosin tail homology; band 4.1, ezrin, radixin, moesin) domain in their C-terminal tails. This domain interacts with a variety of binding partners, and mutations in either the MyTH4 or the FERM domain of myosin VII and myosin XV result in deafness, highlighting the functional importance of each domain. The N-terminal MyTH/FERM region of Dictyostelium myosin VII (M7) has been isolated as a first step toward gaining insight into the function of this domain and its interaction with binding partners. The M7 MyTH4/FERM domain (MF1) binds to both actin and microtubules in vitro, with dissociation constants of 13.7 and 1.7 µM, respectively. Gel filtration and UV spectroscopy reveal that MF1 exists as a monomer in solution and forms a well-folded, compact conformation with a high degree of secondary structure. These results indicate that MF1 forms an integrated structural domain that serves to couple actin filaments and microtubules in specific regions of the cytoskeleton.