RESUMEN
Aims: The benefit of the ß1-adrenergic receptor (ß1-AR) agonist dobutamine for treatment of acute heart failure in peripartum cardiomyopathy (PPCM) is controversial. Cardiac STAT3 expression is reduced in PPCM patients. Mice carrying a cardiomyocyte-restricted deletion of STAT3 (CKO) develop PPCM. We hypothesized that STAT3-dependent signalling networks may influence the response to ß-AR agonist treatment in PPCM patients and analysed this hypothesis in CKO mice. Methods and Results: Follow-up analyses in 27 patients with severe PPCM (left ventricular ejection fraction ≤25%) revealed that 19 of 20 patients not obtaining dobutamine improved cardiac function. All seven patients obtaining dobutamine received heart transplantation (n = 4) or left ventricular assist devices (n = 3). They displayed diminished myocardial triglyceride, pyruvate, and lactate content compared with non-failing controls. The ß-AR agonist isoproterenol (Iso) induced heart failure with high mortality in postpartum female, in non-pregnant female and in male CKO, but not in wild-type mice. Iso induced heart failure and high mortality in CKO mice by impairing fatty acid and glucose uptake, thereby generating a metabolic deficit. The latter was governed by disturbed STAT3-dependent signalling networks, microRNA-199a-5p, microRNA-7a-5p, insulin/glucose transporter-4, and neuregulin/ErbB signalling. The resulting cardiac energy depletion and oxidative stress promoted dysfunction and cardiomyocyte loss inducing irreversible heart failure, which could be attenuated by the ß1-AR blocker metoprolol or glucose-uptake-promoting drugs perhexiline and etomoxir. Conclusions: Iso impairs glucose uptake, induces energy depletion, oxidative stress, dysfunction, and death in STAT3-deficient cardiomyocytes mainly via ß1-AR stimulation. These cellular alterations may underlie the dobutamine-induced irreversible heart failure progression in PPCM patients who frequently display reduced cardiac STAT3 expression.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 1/efectos adversos , Agonistas de Receptores Adrenérgicos beta 1/toxicidad , Cardiomiopatías/inducido químicamente , Dobutamina/efectos adversos , Insuficiencia Cardíaca/tratamiento farmacológico , Trastornos Puerperales/tratamiento farmacológico , Factor de Transcripción STAT3/fisiología , Adulto , Animales , Glucemia/metabolismo , Femenino , Humanos , Isoproterenol/farmacología , Masculino , Ratones Noqueados , MicroARNs/fisiología , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Periodo Periparto , Nucleótidos de Purina/metabolismo , Distribución Aleatoria , Especies Reactivas de Oxígeno/metabolismo , Receptor ErbB-4/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/deficiencia , Disfunción Ventricular Izquierda/inducido químicamenteRESUMEN
Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.
Asunto(s)
Actinas/química , Actinas/metabolismo , Tropomiosina/química , Tropomiosina/metabolismo , Actinas/genética , Adenosina Difosfato/metabolismo , Animales , Microscopía por Crioelectrón , Magnesio/metabolismo , Ratones , Modelos Moleculares , Mutación/genética , Conformación Proteica , Conejos , Electricidad Estática , Tropomiosina/genéticaRESUMEN
Cytoplasmic ß-actin supports fundamental cellular processes in healthy and diseased cells including cell adhesion, migration, cytokinesis and maintenance of cell polarity. Mutations in ACTB, the gene encoding cytoplasmic ß-actin, lead to severe disorders with a broad range of symptoms. The two dominant heterozygous gain-of-function ß-actin mutations p.R183W and p.E364K were identified in patients with developmental malformations, deafness and juvenile-onset dystonia (p.R183W) and neutrophil dysfunction (p.E364K). Here, we report the recombinant production and functional characterization of the two mutant proteins. Arg183 is located near the nucleotide-binding pocket of actin. Our results from biochemical studies and molecular dynamics simulations show that replacement by a tryptophan residue at position 183 establishes an unusual stacking interaction with Tyr69 that perturbs nucleotide release from actin monomers and polymerization behavior by inducing a closed state conformation. The replacement of Glu364 by a lysine residue appears to act as an allosteric trigger event leading to the preferred formation of the closed state. Thus, our approach indicates that both mutations affect interdomain mobility and nucleotide interactions as a basis for the formation of disease phenotypes in patients.
Asunto(s)
Actinas/química , Actinas/genética , Mutación , Humanos , Simulación de Dinámica Molecular , Miosinas/química , Nucleótidos/metabolismo , Profilinas/química , Pliegue de Proteína , Estabilidad ProteicaRESUMEN
Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6). We found that in in vitro assays, the C-terminally-truncated TcdA1-1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1-542) or intermediate (TcdA1102-1847) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.
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Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Oligopéptidos/metabolismo , Células 3T3 , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Supervivencia Celular/efectos de los fármacos , Enterotoxinas/toxicidad , Glucosiltransferasas/metabolismo , Ratones , Oligopéptidos/toxicidad , Estructura Terciaria de ProteínaRESUMEN
Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic ß- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic ß-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by ß-actin and myosin-7A by γ-actin. Our results indicate that ß- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.
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Actinas/metabolismo , Miosinas/metabolismo , Adenosina Trifosfato/metabolismo , Citoplasma/metabolismo , Humanos , Músculo Esquelético/metabolismo , Mapas de Interacción de Proteínas , Isoformas de Proteínas/metabolismoRESUMEN
Regulation of myosin and filamentous actin interaction by tropomyosin is a central feature of contractile events in muscle and nonmuscle cells. However, little is known about molecular interactions within the complex and the trajectory of tropomyosin movement between its "open" and "closed" positions on the actin filament. Here, we report the 8 Å resolution structure of the rigor (nucleotide-free) actin-tropomyosin-myosin complex determined by cryo-electron microscopy. The pseudoatomic model of the complex, obtained from fitting crystal structures into the map, defines the large interface involving two adjacent actin monomers and one tropomyosin pseudorepeat per myosin contact. Severe forms of hereditary myopathies are linked to mutations that critically perturb this interface. Myosin binding results in a 23 Å shift of tropomyosin along actin. Complex domain motions occur in myosin, but not in actin. Based on our results, we propose a structural model for the tropomyosin-dependent modulation of myosin binding to actin.
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Actinas/química , Complejos Multiproteicos/química , Miosinas/metabolismo , Tropomiosina/química , Actinas/genética , Actinas/metabolismo , Animales , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Miosinas/química , Miosinas/genética , Conejos , Tropomiosina/genética , Tropomiosina/metabolismoRESUMEN
Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac ß-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.
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Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Cardiomiopatía Hipertrófica/metabolismo , Mutación/genética , Adenoviridae/genética , Animales , Animales Recién Nacidos , Baculoviridae/genética , Sitios de Unión , Cardiomiopatía Hipertrófica/genética , Células Cultivadas , Humanos , Immunoblotting , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miosinas/metabolismo , Ratas , Sarcómeros/fisiologíaRESUMEN
In order to determine the microRNA expression pattern in normal basal and luminal breast epithelium and to analyze the relationship of this expression pattern to different breast cancer subtypes, laser-microdissected luminal and basal cells isolated from plastic surgery tissue samples were used for comprehensive expression profiling, measuring 664 microRNAs by low-density TaqMan arrays. In a test (n = 5) and validation set (n = 9) 10 differentially expressed microRNAs were identified by TaqMan RT-qPCR. These microRNAs were studied in laser-microdissected cells of luminal A (n = 5), luminal B (n = 5), basal-like subtypes of breast cancer (n = 10), and malignant myoepithelioma of the breast (n = 10). From 116 microRNAs unequivocally expressed in normal breast epithelial cells, we identified 8 basal microRNAs (let7c, miR-125b, miR-126, miR-127-3p, miR-143, miR-145, miR-146b-5p, and miR-199a-3p), preferentially expressed in normal basal cells, exceeding luminal cells by a factor from 4 to 1000. All of these microRNAs were also found to be significantly elevated in malignant myoepithelioma but not in basal-type of breast cancer. Two members of the miR-200 family (miR-200c and miR-429) were predominantly luminal. Both microRNAs were expressed in the luminal and basal type of breast cancer in contrast to malignant myoepithelioma, which revealed significantly lower levels potentially contributing to its mesenchymal phenotype. In conclusion, normal luminal and basal mammary epithelial cells exhibit a different microRNA expression profile. Malignant myoepithelioma seems to exhibit a basal pattern of microRNA expression, whereas the so-called basal-like breast cancer is clearly different and reveals a luminal type pattern.
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Neoplasias de la Mama/genética , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Glándulas Mamarias Humanas/metabolismo , MicroARNs/metabolismo , Adolescente , Adulto , Neoplasias de la Mama/clasificación , Análisis por Conglomerados , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Especificidad de Órganos/genética , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Phalloidin and fluorescently labeled phalloidin analogs are established reagents to stabilize and mark actin filaments for the investigation of acto-myosin interactions. In the present study, we employed transient and steady-state kinetic measurements as well as in vitro motility assays to show that phalloidin perturbs the productive interaction of human non-muscle myosin-2A and -2C1 with filamentous actin. Phalloidin binding to F-actin results in faster dissociation of the complex formed with non-muscle myosin-2A and -2C1, reduced actin-activated ATP turnover, and slower velocity of actin filaments in the in vitro motility assay. In contrast, phalloidin binding to F-actin does not affect the interaction with human non-muscle myosin isoform 2B and Dictyostelium myosin-2 and myosin-5b.