RESUMEN
Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.
Asunto(s)
Arabidopsis , Calmodulina , Calmodulina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Unión ProteicaRESUMEN
Myosin essential light chains A1 and A2 are identical isoforms except for an extension of â¼40 amino acids at the N terminus of A1 that binds F-actin. The extension has no bearing on the burst hydrolysis rate (M-ATP â M-ADP-Pi) as determined by chemical quench flow (100 µM isoenzyme). Whereas actomyosin-S1A2 steady state MgATPase (low ionic strength, 20 °C) is hyperbolically dependent on concentration: Vmax 7.6 s-1, Kapp 6.4 µM (F-actin) and Vmax 10.1 s-1, Kapp 5.5 µM (native thin filaments, pCa 4), the relationship for myosin-S1A1 is bimodal; an initial rise at low concentration followed by a decline to one-third the Vmax of S1A2, indicative of more than one rate-limiting step and A1-enforced flux through the slower actomyosin-limited hydrolysis pathway. In double-mixing stopped-flow with an indicator, Ca(II)-mediated activation of Pi dissociation (regulatedAM-ADP-Pi â regulatedAM-ADP + Pi) is attenuated by A1 attachment to thin filaments (pCa 4). The maximum accelerated rates of Pi dissociation are: 81 s-1 (S1A1, Kapp 8.9 µM) versus 129 s-1 (S1A2, Kapp 58 µM). To investigate apomyosin-S1-mediated activation, thin filaments (EGTA) are premixed with a given isomyosin-S1 and double-mixing is repeated with myosin-S1A1 in the first mix. Similar maximum rates of Pi dissociation are observed, 44.5 s-1 (S1A1) and 47.1 s-1 (S1A2), which are lower than for Ca(II) activation. Overall, these results biochemically demonstrate how the longer light chain A1 can contribute to slower contraction and higher force and the shorter version A2 to faster contraction and lower force, consistent with their distribution in different types of striated muscle.
Asunto(s)
Actomiosina , Cadenas Ligeras de Miosina , Actinas/metabolismo , Actomiosina/metabolismo , Adenosina Trifosfato/metabolismo , Hidrólisis , Isoenzimas/metabolismo , Cinética , Cadenas Ligeras de Miosina/química , Subfragmentos de Miosina/metabolismo , Humanos , AnimalesRESUMEN
BACKGROUND: The interaction of N-terminal extension of the myosin A1 essential light chain (A1 ELC) with actin is receiving increasing attention as a target in utilizing synthetic A1 ELC N-terminal-derived peptides in cardiac dysfunction therapy. METHODS: To elucidate the mechanism by which these peptides regulate actin-myosin interaction, here we have investigated their effects on the myosin subfragment 1 (S1)-induced polymerization of G-actin. RESULTS: The MLCFpep and MLCSpep peptides spanning the 3-12 of A1 ELC sequences from fast and slow skeletal muscle, respectively, increased the rate of actin polymerization not only by S1(A2) but also the rate of S1(A1)-induced actin polymerization, suggesting that they did not interfere with the direct binding of A1 ELC with actin. The efficiency of actin polymerization in the presence of the N-terminal ELC peptides depended on their sequence. Substitution of aspartic acid for neutral asparagine at position 5 of MLCFpep dramatically enhanced its ability to stimulate S1-induced polymerization and enabled it to initiate polymerization of G-actin in the absence of S1. CONCLUSIONS: These and other results presented in this work suggest that the modulation of myosin motor activity by N-terminal ELC peptides is exerted through a change in actin filament conformation rather than through blocking the A1 ELC-actin interaction. GENERAL SIGNIFICANCE: The results imply the possibility of enhancing therapeutic effects of these peptides by modifications of their sequence.
Asunto(s)
Actinas , Cadenas Ligeras de Miosina , Actinas/metabolismo , Músculo Esquelético/metabolismo , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismoRESUMEN
Muscles are required for animal movement, feeding, heartbeat, and reproduction. Disruption of muscle function can lead to mobility impairments and diseases like muscular dystrophy and cardiac myopathy; therefore, research in this area has significant implications for public health. Recent work by Vaziri and colleagues has taken genetic, cell biological, and biochemical approaches to identify Protein kinase C-d (Pkcδ) as a novel regulator of the essential myosin light chain 2 (MLC2) by phosphorylation. The authors determine which residues of MLC2 are modified by Pkcδ and show that phosphorylation by Pkcδ is required for proper sarcomere assembly and function. This study underscores the importance of Drosophila melanogaster as a model system for muscle function and highlights how protein phosphorylation is a vital part of post-translational gene regulation.
Asunto(s)
Drosophila melanogaster , Cadenas Ligeras de Miosina , Animales , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Cadenas Ligeras de Miosina/química , Fosforilación , Proteína Quinasa C-delta/metabolismoRESUMEN
The normal function of heart muscle depends on its ability to contract more strongly at longer length. Increased venous filling stretches relaxed heart muscle cells, triggering a stronger contraction in the next beat- the Frank-Starling relation. Conversely, heart muscle cells are inactivated when they shorten during ejection, accelerating relaxation to facilitate refilling before the next beat. Although both effects are essential for the efficient function of the heart, the underlying mechanisms were unknown. Using bifunctional fluorescent probes on the regulatory light chain of the myosin motor we show that its N-terminal domain may be captured in the folded OFF state of the myosin dimer at the end of the working-stroke of the actin-attached motor, whilst its C-terminal domain joins the OFF state only after motor detachment from actin. We propose that sequential folding of myosin motors onto the filament backbone may be responsible for shortening-induced de-activation in the heart.
Asunto(s)
Miocardio/metabolismo , Miosinas/metabolismo , Animales , Calcio/metabolismo , Masculino , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Ratas Wistar , Sarcómeros/metabolismoRESUMEN
Phosphorylation of myosin regulatory light chain (MRLC) can regulate muscle contraction and thus affect actomyosin dissociation and meat quality. The objective of this study was to explore the mechanism by how MRLC phosphorylation regulates actomyosin dissociation and thus develop strategies for improving meat quality. Here, the phosphorylation status of MRLC was modulated by myosin light chain kinase and myosin light chain kinase inhibitor. MRLC phosphorylation at Ser17 decreased the kinetic energy and total energy of actomyosin, thus stabilized the structure, facilitating the interaction between myosin and actin; this was one possible way that MRLC phosphorylation at Ser17 negatively affects actomyosin dissociation. Moreover, MRLC phosphorylation at Ser17 was beneficial to the formation of ionic bonds, hydrogen bonds, and hydrophobic interaction between myosin and actin, and was the second possible way that MRLC phosphorylation at Ser17 negatively affects actomyosin dissociation.
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Actomiosina/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Actinas/metabolismo , Actomiosina/química , Animales , Calorimetría , Simulación de Dinámica Molecular , Cadenas Ligeras de Miosina/química , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Serina/metabolismoRESUMEN
We investigated the mechanisms associated with E22K mutation in myosin regulatory light chain (RLC), found to cause hypertrophic cardiomyopathy (HCM) in humans and mice. Specifically, we characterized the mechanical profiles of papillary muscle fibers from transgenic mice expressing human ventricular RLC wild-type (Tg-WT) or E22K mutation (Tg-E22K). Because the two mouse models expressed different amounts of transgene, the B6SJL mouse line (NTg) was used as an additional control. Mechanical experiments were carried out on Ca2+ - and ATP-activated fibers and in rigor. Sinusoidal analysis was performed to elucidate the effect of E22K on tension and stiffness during activation/rigor, tension-pCa, and myosin cross-bridge (CB) kinetics. We found significant reductions in active tension (by 54%) and stiffness (active by 40% and rigor by 54%). A decrease in the Ca2+ sensitivity of tension (by ∆pCa ~ 0.1) was observed in Tg-E22K compared with Tg-WT fibers. The apparent (=measured) rate constant of exponential process B (2πb: force generation step) was not affected by E22K, but the apparent rate constant of exponential process C (2πc: CB detachment step) was faster in Tg-E22K compared with Tg-WT fibers. Both 2πb and 2πc were smaller in NTg than in Tg-WT fibers, suggesting a kinetic difference between the human and mouse RLC. Our results of E22K-induced reduction in myofilament stiffness and tension suggest that the main effect of this mutation was to disturb the interaction of RLC with the myosin heavy chain and impose structural abnormalities in the lever arm of myosin CB. When placed in vivo, the E22K mutation is expected to result in reduced contractility and decreased cardiac output whereby leading to HCM. SUB-DISCIPLINE: Bioenergetics. DATABASE: The data that support the findings of this study are available from the corresponding authors upon reasonable request. ANIMAL PROTOCOL: BK20150353 (Soochow University). RESEARCH GOVERNANCE: School of Nursing: Hua-Gang Hu: seuboyh@163.com; Soochow University: Chen Ge chge@suda.edu.cn.
Asunto(s)
Calcio/metabolismo , Cardiomiopatía Hipertrófica/genética , Elasticidad , Mutación Missense , Miofibrillas/metabolismo , Cadenas Ligeras de Miosina/química , Adenosina Trifosfato/metabolismo , Animales , Fenómenos Biomecánicos , Cardiomiopatía Hipertrófica/metabolismo , Femenino , Masculino , Ratones , Contracción Miocárdica , Miofibrillas/química , Miofibrillas/fisiología , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismoRESUMEN
A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten's ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions.
Asunto(s)
Bencilaminas/química , Bencilaminas/metabolismo , Miosinas/metabolismo , Uracilo/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Humanos , Músculo Esquelético/metabolismo , Contracción Miocárdica/fisiología , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Miosinas/química , Fosforilación/fisiología , Uracilo/química , Uracilo/metabolismoRESUMEN
Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads1, which prevents filament formation and inactivates the motors2. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations.
Asunto(s)
Microscopía por Crioelectrón , Miosina Tipo II/química , Miosina Tipo II/ultraestructura , Animales , Activación Enzimática , Estabilidad de Enzimas , Modelos Moleculares , Músculo Liso/química , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Cadenas Ligeras de Miosina/ultraestructura , Miosina Tipo II/metabolismo , Fosforilación , Dominios Proteicos , PavosRESUMEN
Gliding, a type of motility based on an actin-myosin motor, is specific to apicomplexan parasites. Myosin A binds two light chains which further interact with glideosome associated proteins and assemble into the glideosome. The role of individual glideosome proteins is unclear due to the lack of structures of larger glideosome assemblies. Here, we investigate the role of essential light chains (ELCs) in Toxoplasma gondii and Plasmodium falciparum and present their crystal structures as part of trimeric sub-complexes. We show that although ELCs bind a conserved MyoA sequence, P. falciparum ELC adopts a distinct structure in the free and MyoA-bound state. We suggest that ELCs enhance MyoA performance by inducing secondary structure in MyoA and thus stiffen its lever arm. Structural and biophysical analysis reveals that calcium binding has no influence on the structure of ELCs. Our work represents a further step towards understanding the mechanism of gliding in Apicomplexa.
Asunto(s)
Apicomplexa , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Secuencia de Aminoácidos , Apicomplexa/metabolismo , Calcio/química , Calcio/metabolismo , Secuencia Conservada , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estabilidad Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , TermodinámicaRESUMEN
Myosin is an essential motor protein, which in muscle is comprised of two molecules each of myosin heavy-chain (MHC), the essential or alkali myosin light-chain 1 (MLC1), and the regulatory myosin light-chain 2 (MLC2). It has been shown previously that MLC2 phosphorylation at two canonical serine residues is essential for proper flight muscle function in Drosophila; however, MLC2 is also phosphorylated at additional residues for which the mechanism and functional significance is not known. We found that a hypomorphic allele of Pkcδ causes a flightless phenotype; therefore, we hypothesized that PKCδ phosphorylates MLC2. We rescued flight disability by duplication of the wild-type Pkcδ gene. Moreover, MLC2 is hypophosphorylated in Pkcδ mutant flies, but it is phosphorylated in rescued animals. Myosin isolated from Pkcδ mutant flies shows a reduced actin-activated ATPase activity, and MLC2 in these myosin preparations can be phosphorylated directly by recombinant human PKCδ. The flightless phenotype is characterized by a shortened and disorganized sarcomere phenotype that becomes apparent following eclosion. We conclude that MLC2 is a direct target of phosphorylation by PKCδ, and that this modification is necessary for flight muscle maturation and function.
Asunto(s)
Miosinas Cardíacas/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Proteína Quinasa C-delta/metabolismo , Animales , Miosinas Cardíacas/química , Miosinas Cardíacas/genética , Drosophila melanogaster , Humanos , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Fenotipo , Fosforilación , Procesamiento Proteico-Postraduccional , Sarcómeros/metabolismoRESUMEN
Peptidyl post-translational modifications (PTMs) could influence the final quality of processed meat. In this study, the peptide oxidative phenomena in Spanish dry-cured ham (Biceps femoris muscle) was evaluated at different ripening times (9, 12, 15, 18 and 24 months of processing) evidencing interactions amongst the lipid and protein oxidation, major peptidyl PTMs and the release of free amino acids (FAAs). Results showed that 12 months of processing enabled the most abundant protein-bound carbonyls, while TBARS value was significantly favored (p < 0.001) by ripening. However, FAAs were still intensively generated during overall ripening. Peptidomics and chemometrics further revealed that proteolysis mostly hampered the oxidized peptides rather than the deamidated ones during ripening. Myosin light chain (MYL1 and MYL3) showed high oxidative susceptibility owing to peptidyl methionine and proline oxidation as well as acetaldehyde adduct formation on lysine or histidine residues.
Asunto(s)
Productos de la Carne/análisis , Cadenas Ligeras de Miosina/metabolismo , Péptidos/análisis , Aminoácidos/análisis , Animales , Cromatografía Líquida de Alta Presión , Dipéptidos/análisis , Peroxidación de Lípido , Cadenas Ligeras de Miosina/química , Oxidación-Reducción , Péptidos/metabolismo , Análisis de Componente Principal , Procesamiento Proteico-Postraduccional , Porcinos , Espectrometría de Masas en TándemRESUMEN
Myosin II is the main force-generating motor during muscle contraction. Myosin II exists as different isoforms that are involved in diverse physiological functions. One outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for these distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) are known to assemble with specific MHCs, raising the intriguing possibility that light chains contribute to specialized myosin functions. Here, we asked whether different RLCs contribute to this functional diversification. To this end, we generated chimeric motors by reconstituting the MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light-chain variants. As a result of the RLC swapping, actin filament sliding velocity increased by â¼10-fold for the slow myosin and decreased by >3-fold for the fast myosin. Results from ensemble molecule solution kinetics and single-molecule optical trapping measurements provided in-depth insights into altered chemo-mechanical properties of the myosin motors that affect the sliding speed. Notably, we found that the mechanical output of both slow and fast myosins is sensitive to the RLC isoform. We therefore propose that RLCs are crucial for fine-tuning the myosin function.
Asunto(s)
Citoesqueleto de Actina/química , Cadenas Ligeras de Miosina/química , Miosina Tipo II/química , Animales , Isoenzimas/química , Pinzas Ópticas , ConejosRESUMEN
To better understand the contribution of myosin light chain (MLC) isoforms to sensory defects in Jinhua ham, dipeptidyl peptidase (DPP) activities, peptide fragments, cleavage sites and the potential of DPP to develop sensory defects of dry-cured ham were evaluated and discussed in normal and defective hams. Higher residual activities of DPP I were found in defective ham compared with normal ham; approximate 3-fold peptide fragments were identified in defective ham than in normal ham. These regions of positions 11-35 and 116-141 in MLC 1, 13-53 and 139-156 in MLC 2, and 18-50 in MLC 3 contributed to the intense generation of peptide fragments in defective ham. PLS-DA further revealed DPP I showing intense response to degrade peptides. Cleavage sites including Glu-128, Tyr-132 and Glu-133 were responsible for the intense release of dipeptides in defective ham. These cleavages could play key role in discriminating taste attributes between defective and normal hams.
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Productos de la Carne/análisis , Cadenas Ligeras de Miosina/química , Carne de Cerdo/análisis , Animales , Cadenas Ligeras de Miosina/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteolisis , PorcinosRESUMEN
Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In this study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically monophosphorylates Ser23 of human cardiac troponin I (cTnI) in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent in vitro and in situ, suggesting species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.
Asunto(s)
Contracción Miocárdica/fisiología , Miocardio/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Troponina I/metabolismo , Animales , Calcio/metabolismo , Humanos , Masculino , Miofibrillas/metabolismo , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/química , Quinasa de Cadena Ligera de Miosina/genética , Péptidos/análisis , Péptidos/química , Fosforilación , Ratas , Ratas Wistar , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Transducción de Señal , Troponina I/química , Troponina I/genéticaRESUMEN
Thick filaments from some striated muscles are regulated by phosphorylation of myosin regulatory light chains (RLCs). A tarantula thick filament quasi-atomic model achieved by cryo-electron microscopy has advanced our understanding on how this regulation occurs. In native thick filaments, an asymmetric intramolecular interaction between the actin-binding region of one myosin head ("blocked") and the converter region of the other head ("free") switches both heads off, establishing the myosin interacting-heads motif (IHM). This structural finding, together with motility assays, sequence analysis, and mass spectrometry (MS) observations have suggested a cooperative phosphorylation activation (CPA) mechanism for thick filament activation. In the CPA mechanism, some myosin free heads are phosphorylated constitutively in Ser35 by protein kinase C (PKC) and -under Ca2+ control - others (free or blocked) heads temporally on Ser45 by myosin light chain kinase (MLCK), in a way that explains both force development and post-tetanic potentiation in tarantula striated muscle. We tested this model using MS to verify if Ca2+-activation phosphorylates de novo un-phosphorylated Ser35 heads. For this purpose, we standardized an approach based on 18O isotopic ATP labeling to accurately detect by MS-MS the RLC phosphorylation under Ca2+-activation. MS spectra showed de novo18O incorporation only on Ser45 but not on Ser35. As the constitutive Ser35 phosphorylation cannot be dephosphorylated, this result suggests that the number of RLCs on free heads with constitutively phosphorylated Ser35 does remain constant on Ca2+-activation supporting that the myosin has a basal activation and force modulation or potentiation is controlled by MLCK Ser45 phosphorylation.
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Marcaje Isotópico , Miosinas/metabolismo , Isótopos de Oxígeno/metabolismo , Serina/metabolismo , Arañas/metabolismo , Secuencia de Aminoácidos , Animales , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Péptidos/química , Péptidos/metabolismo , FosforilaciónRESUMEN
Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. Here we report the first x-ray crystal structure of an insect myosin: the D melanogaster skeletal muscle myosin II embryonic isoform (EMB). Using our system for recombinant expression of myosin heavy chain (MHC) proteins in whole transgenic flies, we prepared and crystallized stable proteolytic S1-like fragments containing the entire EMB motor domain bound to an essential light chain. We solved the x-ray crystal structure by molecular replacement and refined the resulting model against diffraction data to 2.2 Å resolution. The protein is captured in two slightly different renditions of the rigor-like conformation with a citrate of crystallization at the nucleotide binding site and exhibits structural features common to myosins of diverse classes from all kingdoms of life. All atom molecular dynamics simulations on EMB in its nucleotide-free state and a derivative homology model containing 61 amino acid substitutions unique to the indirect flight muscle isoform (IFI) suggest that differences in the identity of residues within the relay and the converter that are encoded for by MHC alternative exons 9 and 11, respectively, directly contribute to increased mobility of these regions in IFI relative to EMB. This suggests the possibility that alternative folding or conformational stability within these regions contribute to the observed functional differences in Drosophila EMB and IFI myosins.
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Cadenas Pesadas de Miosina/ultraestructura , Cadenas Ligeras de Miosina/ultraestructura , Isoformas de Proteínas/ultraestructura , Miosinas del Músculo Esquelético/ultraestructura , Secuencia de Aminoácidos/genética , Animales , Cristalografía por Rayos X , Drosophila melanogaster/química , Drosophila melanogaster/ultraestructura , Simulación de Dinámica Molecular , Miofibrillas/genética , Miofibrillas/ultraestructura , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Dominios Proteicos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína , Miosinas del Músculo Esquelético/química , Miosinas del Músculo Esquelético/genéticaRESUMEN
It has recently been exhibited that Rac1 expression is increased in the bronchial tissue of a murine model with repeated antigen-challenged airway hyperresponsiveness (AHR). In the present study, the role of Rac1 in endothelin-1 (ET-1)-induced bronchial contraction and myosin light chain (MLC) phosphorylation was examined in AHR mice. Enhanced reactions in AHR mice were prevented by the Rac1 inhibitor NSC23766. These findings suggest that increased activation of Rac1 might be responsible for the enhancement of the bronchial contraction induced by ET-1 in AHR.
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Hiperreactividad Bronquial/metabolismo , Broncoconstricción , Endotelina-1/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacología , Animales , Bronquios/metabolismo , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos BALB C , Músculo Liso/metabolismo , Músculo Liso/fisiopatología , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Pirimidinas/metabolismo , Pirimidinas/farmacologíaRESUMEN
Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the ß-cardiac myosin heavy chain gene (ß-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any ß-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.
Asunto(s)
Adenosina Trifosfatasas/genética , Cardiomiopatía Hipertrófica/genética , Miosinas/genética , Miosinas Ventriculares/genética , Citoesqueleto de Actina/genética , Actinas/química , Actinas/genética , Adenosina Trifosfatasas/química , Edad de Inicio , Cardiomiopatía Hipertrófica/patología , Femenino , Humanos , Cinética , Masculino , Mutación Missense/genética , Contracción Miocárdica/genética , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Miosinas/química , Índice de Severidad de la Enfermedad , Miosinas Ventriculares/químicaRESUMEN
Rationale: Diabetes can lead to cerebral and cutaneous vascular dysfunction. However, it is still unclear how vascular function changes with the development of diabetes and what differences exist between cerebral and cutaneous vascular dysfunction. Thus, it is very important to monitor changes in cerebral and cutaneous vascular function responses in vivo and study their differences during diabetes development. Methods: With the assistance of newly developed skull and skin optical clearing techniques, we monitored the responses of sodium nitroprusside (SNP)- and acetyl choline (ACh)-induced cerebral and cutaneous vascular blood flow and blood oxygen in diabetic mice in vivo during the development of type 1 diabetes (T1D) by combining laser speckle contrast imaging with hyperspectral imaging. We then compared the differences between cerebral and cutaneous vascular responses and explored the reasons for abnormal changes induced in response to different vascular beds. Results: In the early stage of diabetes (T1D-1 week), there were abnormal changes in the cerebral vascular blood flow and blood oxygen responses to SNP and ACh as well as cutaneous vascular blood oxygen. The cutaneous vascular blood flow response also became abnormal from T1D-3 weeks. Additionally, the T1D-induced abnormal blood flow response was associated with changes in vascular myosin light chain phosphorylation and muscarinic acetylcholine receptor M3 levels, and the aberrant blood oxygen response was related to an increase in glycated hemoglobin levels. Conclusion: These results suggest that the abnormal cutaneous vascular blood oxygen response occurred earlier than the blood flow response and therefore has the potential to serve as a good assessment indicator for revealing cerebrovascular dysfunction in the early stage of diabetes.