RESUMO
Cardiac function requires appropriate proteins in each chamber. Atria requires slow myosin to act as reservoirs, while ventricles demand fast myosin for swift pumping. Myosins are thus under chamber-biased cis-regulation, with myosin gene expression imbalances leading to congenital heart dysfunction. To identify regulatory inputs leading to cardiac chamber-biased expression, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives preferential expression to the atria. We show that SMyHC III gene states are orchestrated by a complex Nuclear Receptor Element (cNRE) of 32 base pairs. Using transgenesis in zebrafish and mice, we demonstrate that preferential atrial expression is achieved by a combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Using comparative genomics, we show that the cNRE might have emerged from an endogenous viral element through infection of an ancestral host germline, revealing an evolutionary pathway to cardiac chamber-specific expression.
Assuntos
Átrios do Coração , Peixe-Zebra , Camundongos , Animais , Peixe-Zebra/genética , Átrios do Coração/metabolismo , Ventrículos do Coração , Miosinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
BACKGROUND: During the final stages of heart development the myocardium grows and becomes vascularized by means of paracrine factors and cell progenitors derived from the epicardium. There is evidence to suggest that retinoic acid (RA), a metabolite of vitamin A, plays an important role in epicardial-based developmental programming. However, the consequences of altered RA-signaling in coronary development have not been systematically investigated. RESULTS: We explored the developmental consequences of altered RA-signaling in late cardiogenic events that involve the epicardium. For this, we used a model of embryonic RA excess based on mouse embryos deficient in the retinaldehyde reductase DHRS3, and a complementary model of embryonic RA deficiency based on pharmacological inhibition of RA synthesis. We found that alterations in embryonic RA signaling led to a thin myocardium and aberrant coronary vessel formation and remodeling. Both excess, and deficient RA-signaling are associated with reductions in ventricular coverage and density of coronary vessels, altered vessel morphology, and impaired recruitment of epicardial-derived mural cells. Using a combined transcriptome and proteome profiling approach, we found that RA treatment of epicardial cells influenced key signaling pathways relevant for cardiac development. CONCLUSIONS: Epicardial RA-signaling plays critical roles in the development of the coronary vasculature needed to support myocardial growth. Developmental Dynamics 247:976-991, 2018. © 2018 Wiley Periodicals, Inc.
Assuntos
Vasos Coronários/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Tretinoína/farmacologia , Animais , Vasos Coronários/embriologia , Coração/crescimento & desenvolvimento , Camundongos , Pericárdio/citologia , Proteoma , TranscriptomaRESUMO
All- trans-retinoic acid (RA), a vitamin A metabolite, is an important signaling molecule required for the proper development of the heart. The epicardium is the main source of RA in the embryonic heart, yet the cardiogenic functions of epicardial-produced RA are not fully understood. Here, we investigated the roles of RA signaling in the embryonic epicardium using in vivo and in vitro models of excess or deficiency of RA. Our results suggested that RA signaling facilitates the cytoskeletal rearrangement required for the epicardial-to-mesenchymal transition of epicardial cells. In vivo treatment with an inhibitor of RA synthesis delayed the migration of epicardial-derived precursor cells (EPDCs) into the myocardium; the opposite was seen in the case of dehydrogenase/reductase superfamily (DHRS)3-deficient embryos, a mouse model of RA excess. Analysis of the behavior of epicardial cells exposed to RA receptor agonists or inhibitors of RA synthesis in vitro revealed that appropriate levels of RA are important in orchestrating the platelet-derived growth factor-induced loss of epithelial character, cytoskeletal remodeling, and migration, necessary for the infiltration of the myocardium by EPDCs. To understand the molecular mechanisms by which RA regulates epicardial cytoskeletal rearrangement, we used a whole transcriptome profiling approach, which in combination with pull-down and inhibition assays, demonstrated that the Ras homolog gene family, member A (RhoA) pathway is required for the morphologic changes induced by RA in epicardial cells. Collectively, these data demonstrate that RA regulates the cytoskeletal rearrangement of epicardial cells via a signaling cascade that involves the RhoA pathway.-Wang, S., Yu, J., Jones, J. W., Pierzchalski, K., Kane, M. A., Trainor, P. A., Xavier-Neto, J., Moise, A. R. Retinoic acid signaling promotes the cytoskeletal rearrangement of embryonic epicardial cells.
Assuntos
Citoesqueleto/metabolismo , Pericárdio/citologia , Transdução de Sinais , Tretinoína/metabolismo , Animais , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pericárdio/embriologia , Transcriptoma , Tretinoína/farmacologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5-9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes.
Assuntos
Artrogripose/virologia , Modelos Animais de Doenças , Hidrocefalia/virologia , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Artrogripose/embriologia , Artrogripose/imunologia , Artrogripose/patologia , Feminino , Humanos , Hidrocefalia/embriologia , Hidrocefalia/imunologia , Hidrocefalia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Placenta/anormalidades , Placenta/imunologia , Placenta/virologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/patologia , Teratogênicos/análise , Infecção por Zika virus/embriologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/patologiaRESUMO
Elucidating cardiac evolution has been frustrated by lack of fossils. One celebrated enigma in cardiac evolution involves the transition from a cardiac outflow tract dominated by a multi-valved conus arteriosus in basal actinopterygians, to an outflow tract commanded by the non-valved, elastic, bulbus arteriosus in higher actinopterygians. We demonstrate that cardiac preservation is possible in the extinct fish Rhacolepis buccalis from the Brazilian Cretaceous. Using X-ray synchrotron microtomography, we show that Rhacolepis fossils display hearts with a conus arteriosus containing at least five valve rows. This represents a transitional morphology between the primitive, multivalvar, conal condition and the derived, monovalvar, bulbar state of the outflow tract in modern actinopterygians. Our data rescue a long-lost cardiac phenotype (119-113 Ma) and suggest that outflow tract simplification in actinopterygians is compatible with a gradual, rather than a drastic saltation event. Overall, our results demonstrate the feasibility of studying cardiac evolution in fossils.
Assuntos
Peixes/anatomia & histologia , Fósseis , Coração/anatomia & histologia , Animais , Evolução Biológica , Microtomografia por Raio-XRESUMO
Repulsive guidance molecules (RGMs) compose a family of glycosylphosphatidylinositol (GPI)-anchored axon guidance molecules and perform several functions during neural development. New evidence has suggested possible new roles for these axon guidance molecules during skeletal muscle development, which has not been investigated thus far. In the present study, we show that RGMa, RGMb and RGMc are all induced during skeletal muscle differentiation in vitro. Immunolocalization performed on adult skeletal muscle cells revealed that RGMa, RGMb and RGMc are sarcolemmal proteins. Additionally, RGMa was found to be a sarcoplasmic protein with a surprisingly striated pattern. RGMa colocalization with known sarcoplasmic proteins suggested that this axon guidance molecule is a skeletal muscle sarcoplasmic protein. Western blot analysis revealed two RGMa fragments of 60 and 33 kDa, respectively, in adult skeletal muscle samples. RGMa phenotypes in skeletal muscle cells (C2C12 and primary myoblasts) were also investigated. RGMa overexpression produced hypertrophic cells, whereas RGMa knockdown resulted in the opposite phenotype. RGMa knockdown also blocked myotube formation in both skeletal muscle cell types. Our results are the first to show an axon guidance molecule as a skeletal muscle sarcoplasmic protein and to include RGMa in a system that regulates skeletal muscle cell size and differentiation.
Assuntos
Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Animais , Diferenciação Celular/fisiologia , Crescimento Celular , Hipertrofia/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologiaRESUMO
Transcriptional regulation controlled by thyroid hormone receptor (TR) drives events such as development, differentiation, and metabolism. TRs may act either as homodimers or as heterodimers with retinoid X receptor (RXR). Thyroid hormone T3 preferentially binds TR-RXR heterodimers, which activate transcription through coactivator recruitment. However, it is unclear whether TR-RXR heterodimers may also be responsive to the canonical RXR agonist 9-cis retinoic acid (9C) in the context of physiological gene regulation. New structural studies suggest that 9C promotes the displacement of bound coactivators from the heterodimer, modifying TR-RXR activity. To shed light on the molecular mechanisms that control TR-RXR function, we used biophysical approaches to characterize coregulator recruitment to TR-TR or to TR-RXR in the presence of T3 and/or 9C as well as cell-based assays to establish the functional significance of biophysical findings. Using cell-based and fluorescence assays with mutant and wild-type TR, we show that 9C does indeed have a function in the TR-RXR heterodimer context, in which it induces the release of corepressors. Furthermore, we show that 9C does not promote detectable conformational changes in the structure of the TR-RXR heterodimer and does not affect coactivator recruitment. Finally, our data support the view that DNA binding domain and Hinge regions are important to set up NR-coactivator binding interfaces. In summary, we showed that the RXR agonist 9C can regulate TR function through its modulation of corepressor dissociation.
Assuntos
Proteínas Correpressoras/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Receptores X de Retinoides/agonistas , Tretinoína/farmacologia , Alitretinoína , Anisotropia , Cromatografia em Gel , Dicroísmo Circular , DNA/metabolismo , Difusão Dinâmica da Luz , Fluorescência , Células HEK293 , Humanos , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores dos Hormônios Tireóideos/química , Espalhamento a Baixo Ângulo , Ativação Transcricional/genética , Triptofano/metabolismo , Ultracentrifugação , Difração de Raios XRESUMO
Retinoic acid (RA) is a terpenoid that is synthesized from vitamin A/retinol (ROL) and binds to the nuclear receptors retinoic acid receptor (RAR)/retinoid X receptor (RXR) to control multiple developmental processes in vertebrates. The available clinical and experimental data provide uncontested evidence for the pleiotropic roles of RA signaling in development of multiple embryonic structures and organs such eyes, central nervous system, gonads, lungs and heart. The development of any of these above-mentioned embryonic organ systems can be effectively utilized to showcase the many strategies utilized by RA signaling. However, it is very likely that the strategies employed to transfer RA signals during cardiac development comprise the majority of the relevant and sophisticated ways through which retinoid signals can be conveyed in a complex biological system. Here, we provide the reader with arguments indicating that RA signaling is exquisitely regulated according to specific phases of cardiac development and that RA signaling itself is one of the major regulators of the timing of cardiac morphogenesis and differentiation. We will focus on the role of signaling by RA receptors (RARs) in early phases of heart development. This article is part of a Special Issue entitled: Nuclear receptors in animal development.
Assuntos
Coração/embriologia , Receptores do Ácido Retinoico/fisiologia , Animais , Relógios Biológicos/efeitos dos fármacos , Relógios Biológicos/fisiologia , Evolução Biológica , Regulação da Expressão Gênica no Desenvolvimento , Coração/efeitos dos fármacos , Coração/crescimento & desenvolvimento , Humanos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , Tretinoína/farmacologiaRESUMO
Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr(253) in α-tubulin as a site that is phosphorylated by protein kinase C ßI (PKCßI). Thr(253) is not part of a linear PKC consensus motif. Instead, Thr(253) occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCßI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this "structurally formed" consensus motif and its importance for the protein kinase-substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.
Assuntos
Fosfotransferases/química , Motivos de Aminoácidos , Animais , Catálise , Bovinos , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas de Fluorescência Verde/química , Células HEK293 , Células HeLa , Humanos , Lisina/química , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Dobramento de Proteína , Proteína Quinase C/química , Treonina/química , Tubulina (Proteína)/químicaRESUMO
BACKGROUND: The characterization of protein binding sites is a major challenge in computational biology. Proteins interact with a wide variety of molecules and understanding of such complex interactions is essential to gain deeper knowledge of protein function. Shape complementarity is known to be important in determining protein-ligand interactions. Furthermore, these protein structural features have been shown to be useful in assisting medicinal chemists during lead discovery and optimization. RESULTS: We developed KVFinder, a highly versatile and easy-to-use tool for cavity prospection and spatial characterization. KVFinder is a geometry-based method that has an innovative customization of the search space. This feature provides the possibility of cavity segmentation, which alongside with the large set of customizable parameters, allows detailed cavity analyses. Although the main focus of KVFinder is the steered prospection of cavities, we tested it against a benchmark dataset of 198 known drug targets in order to validate our software and compare it with some of the largely accepted methods. Using the one click mode, we performed better than most of the other methods, staying behind only of hybrid prospection methods. When using just one of KVFinder's customizable features, we were able to outperform all other compared methods. KVFinder is also user friendly, as it is available as a PyMOL plugin, or command-line version. CONCLUSION: KVFinder presents novel usability features, granting full customizable and highly detailed cavity prospection on proteins, alongside with a friendly graphical interface. KVFinder is freely available on http://lnbio.cnpem.br/bioinformatics/main/software/.
Assuntos
Biologia Computacional/métodos , Proteínas/química , Software , Algoritmos , Sítios de Ligação , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Dact gene family encodes multifunctional proteins that are important modulators of Wnt and TGF-ß signaling pathways. Given that these pathways coordinate multiple steps of limb development, we investigated the expression pattern of the two chicken Dact genes (Dact1 and Dact2) from early limb bud up to stages when several tissues are differentiating. RESULTS: During early limb development (HH24-HH30) Dact1 and Dact2 were mainly expressed in the cartilaginous rudiments of the appendicular skeleton and perichondrium, presenting expression profiles related, but distinct. At later stages of development (HH31-HH35), the main sites of Dact1 and Dact2 expression were the developing synovial joints. In this context, Dact1 expression was shown to co-localize with regions enriched in the nuclear ß-catenin protein, such as developing joint capsule and interzone. In contrast, Dact2 expression was restricted to the interzone surrounding the domains of bmpR-1b expression, a TGF-ß receptor with crucial roles during digit morphogenesis. Additional sites of Dact expression were the developing tendons and digit blastemas. CONCLUSIONS: Our data indicate that Dact genes are good candidates to modulate and, possibly, integrate Wnt and TGF-ß signaling during limb development, bringing new and interesting perspectives about the roles of Dact molecules in limb birth defects and human diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Aviárias/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Membro Posterior/embriologia , Proteínas Nucleares/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Embrião de Galinha , Membro Posterior/citologia , Humanos , Membrana Sinovial/citologia , Membrana Sinovial/embriologiaRESUMO
Transgenesis refers to the molecular genetic techniques for directing specific insertions, deletions and point mutations in the genome of germ cells in order to create genetically modified organisms (GMO). Genetic modification is becoming more practicable, efficient and predictable with the development and use of a variety of cell and molecular biology tools and DNA sequencing technologies. A collection of plasmidial and viral vectors, cell-type specific promoters, positive and negative selectable markers, reporter genes, drug-inducible Cre-loxP and Flp/FRT recombinase systems are available which ensure efficient transgenesis in the mouse. The technologies for the insertion and removal of genes by homologous-directed recombination in embryonic stem cells (ES) and generation of targeted gain- and loss-of function alleles have allowed the creation of thousands of mouse models of a variety of diseases. The engineered zinc finger nucleases (ZFNs) and small hairpin RNA-expressing constructs are novel tools with useful properties for gene knockout free of ES manipulation. In this review we briefly outline the different approaches and technologies for transgenesis as well as their advantages and disadvantages. We also present an overview on how the novel integrative mouse and human genomic databases and bioinformatics approaches have been used to understand genotype-phenotype relationships of hundreds of mutated and candidate disease genes in mouse models. The updating and continued improvements of the genomic technologies will eventually help us to unraveling the biological and pathological processes in such a way that they can be translated more efficiently from mouse to human and vise-versa.
Assuntos
Técnicas de Transferência de Genes , Animais , DNA/administração & dosagem , DNA/metabolismo , Embrião de Mamíferos/metabolismo , Marcação de Genes , Vetores Genéticos/genética , Humanos , Camundongos , MicroinjeçõesRESUMO
The identification of subpharyngeal cardiac precursors has had a strong influence on the way we think about early cardiac development. From this discovery was born the concept of multiple heart fields. Early support for the concept came from gene expression, genetic retrospective fate mapping, and gene targeting studies, which collectively suggested the existence of a second heart field (SHF) on the basis of specific Islet-1 (Isl-1) expression, presence of two cardiac ancestral lineages, and compatible cardiac knockout phenotypes, respectively. A decade after the original studies, support for the SHF concept is dwindling. This is because in all bilaterian models studied, Isl expression in heart progenitors is not SHF-specific, because lineage data are best explained by alternative models including an older, truly ancestral, lineage of cardiac pioneers with unrestricted contribution to all cardiac segments and, finally, because the inflow-to-outflow segmental nature of the early vertebrate peristaltic heart has been reaffirmed with novel, less invasive, methodologies. Altogether, the paradigms derived from the discovery of subpharyngeal cardiac progenitors helped us shift from relatively simple models, which rely predominantly either on patterning, gene expression patterns or lineages, to a much more sophisticated body of knowledge in which all these parameters must be accounted. Thus, it is well possible that due consideration of the key elements contained in the inflow/outflow, pioneer/scaffold, ballooning, and SHF hypotheses may provide us with a unified framework of the early stages of cardiac development. Here, we advance into this direction by suggesting an intuitive model of early heart development based on the concept of an inflow/outflow scaffold erected by cardiac pioneers, one that is required to assemble all the subsequent cell contribution that emigrates from cardiac progenitor areas.
Assuntos
Coração/crescimento & desenvolvimento , Miocárdio/metabolismo , Evolução Biológica , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Humanos , Modelos Biológicos , Miocárdio/citologiaRESUMO
Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Celulases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cálcio/metabolismo , Celulases/química , Celulases/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/fisiologia , Temperatura Alta , Cinética , Manganês/química , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 Å(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/química , Miócitos Cardíacos/química , Miosinas/química , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Animais , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Hipertrofia/metabolismo , Camundongos , Modelos Moleculares , Miócitos Cardíacos/metabolismo , Miosinas/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.
Assuntos
Aldeído Desidrogenase/genética , Padronização Corporal/fisiologia , Evolução Molecular , Modelos Moleculares , Filogenia , Conformação Proteica , Transdução de Sinais/genética , Tretinoína/metabolismo , Animais , Sequência de Bases , Teorema de Bayes , Análise por Conglomerados , Biologia Computacional , Perfilação da Expressão Gênica , Genes Duplicados/genética , Hibridização In Situ , Funções Verossimilhança , Modelos Genéticos , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal, and differentiation. However, the function of specific PKC isoenzymes have yet to be determined. Of the PKCs expressed in undifferentiated ESCs, ßIPKC was the only isoenzyme abundantly expressed in the nuclei. To investigate the role of ßΙPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one ßIPKC-specific inhibitor peptide. We identified 13 nuclear proteins that are direct or indirect ßΙPKC substrates in undifferentiated ESCs. These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation. Inhibiting ßΙPKC had no effect on DNA synthesis in undifferentiated ESCs. However, upon differentiation, many cells seized to express ßΙPKC and ßΙPKC was frequently found in the cytoplasm. Taken together, our results suggest that ßIPKC takes part in the processes that maintain ESCs in their undifferentiated state.
Assuntos
Células-Tronco Embrionárias/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Eletroforese em Gel Bidimensional , Células-Tronco Embrionárias/citologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Peptídeos/farmacologia , Fosfoproteínas/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C beta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Transcrição GênicaRESUMO
RATIONALE: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. OBJECTIVE: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. METHODS AND RESULTS: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. CONCLUSION: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels.
Assuntos
Diferenciação Celular , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericárdio/metabolismo , Transdução de Sinais , Tretinoína/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Apoptose , Comunicação Autócrina , Diferenciação Celular/genética , Células Cultivadas , Embrião de Galinha , Técnicas de Cocultura , Vasos Coronários/embriologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Morfogênese , Miócitos Cardíacos/metabolismo , Comunicação Parácrina , Pericárdio/embriologia , Codorniz , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Técnicas de Cultura de Tecidos , Transdução Genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Introdução: A voz é o principal instrumento na vida profissional do professor, requerendo uma adaptação precisa dos órgãos da fonação. O desconhecimento da disfonia em professores de nossa região motivou esta pesquisa.Objetivos: Avaliar a frequência de disfonia em professores do Ensino Fundamentalda rede municipal em Maceió-AL e identificar sintomas associados às queixas vocais e possíveis fatores de risco ao aparecimento de alterações vocais. Metodologia: Estudo transversal abrangendo 126 docentes selecionadosaleatoriamente, avaliados a partir de entrevista, com aplicação de questionário dirigido, em 2008. Resultados: Dos 126 professores avaliados, 87,3% referiram ocorrência de disfonia na docência. Observou-se relação entre carga horária semanal e presença de disfonia (p=0,0038). Em relação ao ambiente de trabalho, poeira e ambiente seco foram as queixas mais relatadas, ambas apresentando associação significativa (p<0,04). Os sintomas de obstrução nasal, prurido, tosse e dispepsia apresentaram relação com a presença de rouquidão. Não houve associação entre disfonia e tabagismo ou tabagismopassivo (p<0,6). Conclusão: O estudo permitiu concluir que existe elevada prevalência de disfonia no grupo estudado e que o comprometimento vocal na atividade docente está relacionado aos fatores ambientais, bem como asintomas clínicos associados à rinopatia e ao refluxo gastroesofágico.
Introduction: Voice is teachers main tool and demands an accurate adaptation of the phonation organs. Teachers unawareness of dysphonia in our region lead to this survey. Objectives: Assess frequency of dysphonia in elementary public school teachers from Maceió, state of Alagoas, Brazil, and identify symptoms associated to vocal complaints and possible risk factors for changes in voice. Methodology: Transversal study with 126 teachers selected randomly, who have been assessed through interviews, and structured questionnaires in 2008. Results: From 126 teachers assessed, 87,3% reported dysphonia. A relation between weekly working hours and dysphonia (p=0,0038) was observed. In relation to work environment, dust and air dryness were the most reported complaints, both showing significant association (p<0,04). Nasal obstruction, itching, cough, and dyspepsia were the symptoms related to hoarseness. There was no association between dysphonia and smoking, either active or passive (p<0,6). Conclusion: There is a high prevalence of vocal problems in the studied group, and dysphonia in teaching activity is related to environmental factors, as well as to clinical symptoms associated to allergic rhinitis and gastroesophageal reflux.