RESUMO
Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação , Centrossomo/metabolismo , Cílios/genética , Cílios/metabolismo , Sequência Conservada , Humanos , Modelos Moleculares , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/química , Miosina Tipo V/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas RecombinantesRESUMO
Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98 th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.
Assuntos
Cerebelo/metabolismo , Miosina Tipo V/metabolismo , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Cadáver , Criança , Pré-Escolar , Eletroforese em Gel de Ágar , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Adulto JovemRESUMO
Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.
Assuntos
Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Adulto Jovem , Cerebelo/metabolismo , Miosina Tipo V/metabolismo , Fatores Etários , Cadáver , Eletroforese em Gel de Ágar , Immunoblotting , Imuno-HistoquímicaRESUMO
Myosin Va is an actin-based, processive molecular motor protein highly enriched in the nervous tissue of vertebrates. It has been associated with processes of cellular motility, which include organelle transport and neurite outgrowth. The in vivo expression of myosin Va protein in the developing nervous system of mammals has not yet been reported. We describe here the immunolocalization of myosin Va in the developing rat hippocampus. Coronal sections of the embryonic and postnatal rat hippocampus were probed with an affinity-purified, polyclonal anti-myosin Va antibody. Myosin Va was localized in the cytoplasm of granule cells in the dentate gyrus and of pyramidal cells in Ammon's horn formation. Myosin Va expression changed during development, being higher in differentiating rather than already differentiated granule and pyramidal cells. Some of these cells presented a typical migratory profile, while others resembled neurons that were in the process of differentiation. Myosin Va was also transiently expressed in fibers present in the fimbria. Myosin Va was not detected in germinative matrices of the hippocampus proper or of the dentate gyrus. In conclusion, myosin Va expression in both granule and pyramidal cells showed both position and time dependency during hippocampal development, indicating that this motor protein is under developmental regulation.
Assuntos
Animais , Feminino , Ratos , Hipocampo/embriologia , Hipocampo/metabolismo , Miosina Tipo V/análise , Giro Denteado/embriologia , Giro Denteado/metabolismo , Imuno-Histoquímica , Miosina Tipo V/metabolismo , Células Piramidais/embriologia , Células Piramidais/metabolismo , Ratos WistarRESUMO
Myosin Va is an actin-based, processive molecular motor protein highly enriched in the nervous tissue of vertebrates. It has been associated with processes of cellular motility, which include organelle transport and neurite outgrowth. The in vivo expression of myosin Va protein in the developing nervous system of mammals has not yet been reported. We describe here the immunolocalization of myosin Va in the developing rat hippocampus. Coronal sections of the embryonic and postnatal rat hippocampus were probed with an affinity-purified, polyclonal anti-myosin Va antibody. Myosin Va was localized in the cytoplasm of granule cells in the dentate gyrus and of pyramidal cells in Ammon's horn formation. Myosin Va expression changed during development, being higher in differentiating rather than already differentiated granule and pyramidal cells. Some of these cells presented a typical migratory profile, while others resembled neurons that were in the process of differentiation. Myosin Va was also transiently expressed in fibers present in the fimbria. Myosin Va was not detected in germinative matrices of the hippocampus proper or of the dentate gyrus. In conclusion, myosin Va expression in both granule and pyramidal cells showed both position and time dependency during hippocampal development, indicating that this motor protein is under developmental regulation.
Assuntos
Hipocampo/embriologia , Hipocampo/metabolismo , Miosina Tipo V/análise , Animais , Giro Denteado/embriologia , Giro Denteado/metabolismo , Feminino , Imuno-Histoquímica , Miosina Tipo V/metabolismo , Células Piramidais/embriologia , Células Piramidais/metabolismo , Ratos , Ratos WistarRESUMO
A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing.
Assuntos
Sistema Nervoso Central/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Loligo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sistema Nervoso Central/ultraestrutura , Gânglios dos Invertebrados/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/isolamento & purificação , Loligo/ultraestrutura , Peso Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/isolamento & purificação , Lobo Óptico de Animais não Mamíferos/metabolismo , Lobo Óptico de Animais não Mamíferos/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Ribonucleoproteínas Citoplasmáticas Pequenas/genética , Ribonucleoproteínas Citoplasmáticas Pequenas/metabolismo , Sinaptossomos/metabolismo , Sinaptossomos/ultraestruturaRESUMO
Brain myosin Va (MVa) is a molecular motor associated with plastic changes during development. MVa has previously been detected in the cell body and in dendrites of neuronal cells in culture, in cells of the guinea-pig cochlea, as well as in cerebellar cells. Adult Wistar rats (n=14), 250-300 g, were perfused with standard methods for immunohistochemistry, using a polyclonal, affinity-purified rabbit antibody against MVa tail domain. Anti-MVa antibody specifically stained neuronal nuclei from forebrain to cerebellar regions, and more intensely sensory nuclei. Differences in MVa immunoreactivity were detected between brain nuclei, ranging from very intense to weak staining. The analysis of MVa and glial fibrillary acidic protein staining in adjacent brain sections demonstrated a clear-cut neuronal labeling rather than an astroglial staining. The studies presented here represent a comprehensive map of MVa regional distribution in the CNS of the adult rat and may contribute to the basic understanding of its role in brain function and plasticity, particularly in relationship to phenomena that involve molecular motors, such as neurite outgrowth, organelle transport and neurotransmitter-vesicle cycling. It is important to highlight that this is a pioneer immunohistochemical study on the distribution of MVa on the whole brain of adult rats, a first step toward the understanding of its function in the CNS.
Assuntos
Química Encefálica , Encéfalo/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Animais , Western Blotting , Encéfalo/citologia , Núcleo Celular/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Masculino , Proteínas de Neurofilamentos/metabolismo , Ratos , Ratos WistarRESUMO
We show here the localisation of myosin-V in whole mount preparations of the mucous-submucous and the muscular layers of rat small intestine by using an affinity purified antibody specific to the tail domain of myosin-V. Myosin-V immunostaining was intense in the submucous and myenteric nervous plexuses, allowing the visualisation of neuronal cell bodies and fibres. Western blots of total muscle layers homogenates detected with the same antibody revealed a single band of the expected size for myosin-V. Understanding the cellular localisation and function of this class of myosin is an important challenge and the accessibility and simplicity of the enteric nervous system as compared to the central nervous system, makes the digestive tract an attractive model for studying possible functional roles of myosin-V in neurotransmission and neuroplasticity.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Sistema Nervoso Entérico/metabolismo , Miosina Tipo V , Proteínas do Tecido Nervoso/metabolismo , Animais , Western Blotting , Sistema Nervoso Entérico/citologia , Imuno-Histoquímica , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Plexo Mientérico/citologia , Plexo Mientérico/metabolismo , Ratos , Plexo Submucoso/citologia , Plexo Submucoso/metabolismoRESUMO
The lability of brain myosin-V (BM-V) to aldehyde-fixation has hindered immunohistochemical (IH) studies of this actin-based motor. We show here that BM-V immunoreactivity (IR) can be retrieved from formalin-fixed, paraffin-embedded human tissue. BM-V IR was optimally retrieved by boiling 5 microm cerebellar tissue sections in 10 mM sodium citrate buffer, pH 6, for 15 min, using a microwave oven set at 900 W and 2.45 GHz. A polyclonal, affinity purified anti-BM-V antibody, raised in rabbits against the tail domain of chicken BM-V, was shown here to recognize a single band in Western blots of human cortical homogenates. The combined use of this monospecific antibody and of the antigen retrieval (AR) method above allowed us to verify that BM-V IR is strongly expressed in human Purkinje cell bodies and dendrites, and in granule cells. The same pattern of BM-V IR expression was consistently and maximally detected in tissues stored in 10% formalin from 1 week to 2.5 months. The AR protocol for BM-V described here permits its IH study in formaldehyde-fixed tissues. It is a valuable tool to study BM-V in well fixed tissues, as occurs with the large collection of human archival tissue available.
Assuntos
Proteínas de Ligação a Calmodulina/isolamento & purificação , Cerebelo/química , Formaldeído , Micro-Ondas , Miosina Tipo V , Proteínas do Tecido Nervoso/isolamento & purificação , Inclusão em Parafina , Lobo Temporal/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Galinhas , Pré-Escolar , Feto , Humanos , Imuno-Histoquímica/métodos , Lactente , CoelhosRESUMO
PC12 cell line is a cellular model to study neurite outgrowth and neurotransmitter release mechanisms. Molecular motors may be involved in these responses and myosin V could be a candidate to mediate these effects. Overlay experiments using [(125)I]-calmodulin showed that PC12 cells possess several calmodulin-binding proteins, some of them around 190-210 kDa. Western blots using affinity purified polyclonal antibodies raised against chicken brain myosin V revealed a component of 190 kDa, a molecular mass typical of myosin V. Furthermore, Northern blots using a myosin V probe also detected a transcript of around 12 kbp. Immunofluorescence cytochemistry demonstrated the localization of myosin V throughout the cytoplasm, in the neurites, growth cone tips, and with an intense asymmetrical perinuclear labeling. Western blot analyses of PC12 cellular extracts after FGF-2 and/or dibutyryl cAMP treatment revealed variations between myosin V and myosin II expression during neuronal differentiation. These results demonstrated the presence of myosin V in PC12 cells and also suggest a role for this motor molecule in the neuronal differentiation response in PC12 cells.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Miosina Tipo V , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Animais , Western Blotting , Bucladesina/farmacologia , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/genética , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Peso Molecular , Miosinas/metabolismo , Proteínas do Tecido Nervoso/genética , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , RNA Mensageiro/análise , RNA Mensageiro/genética , RatosRESUMO
Myosin-V, an unconventional myosin, has two notable structural features: (i) a regulatory neck domain having six IQ motifs that bind calmodulin and light chains, and (ii) a structurally distinct tail domain likely responsible for its specific intracellular interactions. Myosin-V copurifies with synaptic vesicles via its tail domain, which also is a substrate for calmodulin-dependent protein kinase II. We demonstrate here that myosin-V coimmunoprecipitates with CaM-kinase II from a Triton X-100-solubilized fraction of isolated nerve terminals. The purified proteins also coimmunoprecipitate from dilute solutions and bind in overlay experiments on Western blots. The binding region on myosin-V was mapped to its proximal and medial tail domains. Autophosphorylated CaM-kinase II binds to the tail domain of myosin-V with an apparent Kd of 7.7 nM. Surprisingly, myosin-V activates CaM-kinase II activity in a Ca2+-dependent manner, without the need for additional CaM. The apparent activation constants for the autophosphorylation of CaM-kinase II were 10 and 26 nM, respectively, for myosin-V versus CaM. The maximum incorporation of 32P into CaM-kinase II activated by myosin-V was twice that for CaM, suggesting that myosin-V binding to CaM-kinase II entails alterations in kinetic and/or phosphorylation site parameters. These data suggest that myosin-V, a calmodulin-carrying myosin, binds to and delivers CaM to CaM-kinase II, a calmodulin-dependent enzyme.
Assuntos
Encéfalo/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Miosina Tipo V , Proteínas do Tecido Nervoso/metabolismo , Animais , Sítios de Ligação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Bovinos , Galinhas , Ativação Enzimática , Cinética , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteínas Qa-SNARE , Ratos , Proteínas Recombinantes/metabolismo , Sinaptossomos/metabolismoRESUMO
Myosin V isolated from chick brain (BM V) is a multimeric protein of about 640 kDa consisting of two intertwined heavy chains of 212 kDa and multiple light chains of 10 to 20 kDa. A distinctive feature of the heavy chain is an extended neck region with six consensus IQ sites for the binding of calmodulin (CaM) and myosin light chains. The actin-activated MgATPase has been shown to require >/=1 microM Ca2+ for full activity, and evidence points to a myosin-linked regulatory system where the CaM light chains participate as modulators for the Ca2+ signal. Still, the precise mechanism of Ca2+ regulation remains unknown. In the present study we have used the intrinsic tryptophan fluorescence of native BM V to monitor conformational changes of BM V induced by Ca2+, and we relate these changes to CaM dissociation from the BM V molecule. The fluorescence intensity decreases approximately 17% upon addition of sub-micromolar concentrations of Ca2+ (K0.5 = 0.038 microM). This decrease in fluorescence, which is dominated by a conformational change in the heavy chain, can be reversed by addition of 1, 2-di(2-aminoethoxy)ethane-N,N,N',N'tetraacetic acid (EGTA) followed by an excess of CaM, but not by addition of EGTA alone. Gel filtration of native BM V using HPLC shows that CaM is partially dissociated from the heavy chain in EGTA and dissociates further upon addition of sub-micromolar concentrations of Ca2+. These observations suggest that the affinity of CaM for at least one of the IQ sites on the BM V heavy chain decreases with Ca2+ and that the Ca2+ concentration required for this effect is lower than that needed to activate acto-BM V. Using a cosedimentation assay in the presence of actin, we also observe partial dissociation of CaM when Ca2+ is absent, but now the addition of Ca2+ has a biphasic effect: sub-micromolar Ca2+ concentrations lead to reassociation of CaM with the heavy chain, followed by dissociation when Ca2+ exceeds 5-10 microM. Thus, the binding of CaM to BM V is affected by both actin and Ca2+.
Assuntos
Química Encefálica , Cálcio/química , Calmodulina/química , Miosinas/química , Actinas/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Cálcio/farmacologia , Calmodulina/metabolismo , Galinhas , Fluorescência , Técnicas In Vitro , Substâncias Macromoleculares , Peso Molecular , Miosinas/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Espectrometria de FluorescênciaRESUMO
The discovery that the dilute gene encodes a class V myosin led to the hypothesis that this molecular motor is involved in melanosome transport and/or dendrite outgrowth in mammalian melanocytes. The present studies were undertaken to gain insight into the subcellular distribution of myosin-V in the melanoma cell line B16-F10, which is wild-type for the dilute gene. Immunofluorescence studies showed some degree of superimposed labeling of myosin-V with melanosomes that predominated at the cell periphery. A subcellular fraction highly enriched in melanosomes was also enriched in myosin-V based on Western blot analysis. Immunoelectron microscopy showed myosin-V labeling associated with melanosomes and other organelles. The stimulation of B16 cells with the alpha-melanocyte-stimulating hormone led to a significant increase in myosin-V expression. This is the first evidence that a cAMP signaling pathway might regulate the dilute gene expression. Immunofluorescence also showed an intense labeling of myosin-V independent of melanosomes that was observed within the dendrites and at the perinuclear region. Although the results presented herein are consistent with the hypothesis that myosin-V might act as a motor for melanosome translocation, they also suggest a broader cytoplasmic function for myosin-V, acting on other types of organelles or in cytoskeletal dynamics.
Assuntos
Genes Neoplásicos , Melanoma Experimental/patologia , Miosinas/análise , Miosinas/genética , Animais , Western Blotting , Fracionamento Celular , Imuno-Histoquímica , Hormônios Estimuladores de Melanócitos/farmacologia , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Miosinas/efeitos dos fármacos , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/ultraestruturaRESUMO
Soluble calmodulin-binding proteins from Saccharomyces carlsbergensis were analyzed in cells grown on glucose, maltose and galactose as carbon source. A large number of polypeptide chains showed affinity for calmodulin by affinity chromatography and overlay techniques. Amongst these, polypeptides of 115, 67 and 45 kDa were only detected during the second exponential phase of growth on glucose or non-fermentative carbon sources, suggesting that they might be subjected to catabolite repression. Polypeptides of 195 and 22 kDa were only observed in cells grown on maltose, whereas 88 kDa polypeptide was only observed in galactose-grown cells. Among the calmodulin -binding polypeptides, eight were phosphorylated in a Ca2+/calmodulin -dependent manner (220, 200, 175, 100, 62, 55, 31 and 16 kDa). Ca2+/calmodulin dependent [gamma-32P] incorporation was dramatically decreased in yeast cells submitted to a heat treatment.
Assuntos
Proteínas de Ligação a Calmodulina/isolamento & purificação , Galactose/metabolismo , Maltose/metabolismo , Saccharomyces/metabolismo , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Temperatura Alta , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
In this review we focus on the biochemical and structural properties of the myosin-V class of unconventional myosins as an example of the diversity of molecular motors within the myosin superfamily. A member of this class was first identified as a novel calmodulin-binding protein in mammalian brain (Larson RE, Pitta DE and Ferro JA (1988). Brazilian Journal of Medical and Biological Research, 21: 213-217). To date, the myosin-V class is represented by two molecules from yeast, one from nematodes, several from vertebrates (chickens, rats, mice and humans) and possibly one from plants. The domain structure of these myosins features a highly conserved head containing the ATP-hydrolysis and actin-binding sites, an extended neck composed of six tandem IQ-motifs which are sites for calmodulin binding and a large tail which has coiled-coil segments intercalated with globular regions of as yet unknown function. Biochemical studies on purified myosin-V from vertebrate brains and the description of myosin-V mutants in yeast and mice have made myosin-V one of the best characterized, unconventional myosin classes at the present time, surpassed only by the well-studied myosin-I class.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a Calmodulina/química , Miosina Tipo V , Proteínas do Tecido Nervoso/química , Sequência de Aminoácidos , Amoeba , Animais , Sítios de Ligação , Aves , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/ultraestrutura , Drosophila , Humanos , Mamíferos , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Nematoides , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/ultraestrutura , Plantas , Ratos , Análise de Sequência de DNA , LevedurasAssuntos
Animais , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a Calmodulina/química , Proteínas do Tecido Nervoso/química , Sítios de Ligação , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/ultraestrutura , Estrutura Molecular , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/ultraestrutura , Análise de Sequência de DNARESUMO
With the aim of further understanding the structure/function relationships in the membrane-damaging activity of the Lys49 phospholipase A2 (Lys49-PLA2) sub-family, we used PCR (polymerase chain reaction) on total venom gland cDNAs from Bothrops jararacussu with degenerate oligodeoxyribonucleotides encoding the N- and C-termini of myotoxin II, a Lys49-PLA2 from Bothrops asper. A 350-bp cDNA coding for bothropstoxin I (BtxtxI) was amplified. Sequencing of the amplified fragment shows that BtxtxI has a Lys49, and comparison with the known structure of myotoxin II showed that the amino acids involved in the formation of a novel dimeric structure in this protein were also conserved.
Assuntos
Bothrops/genética , Venenos de Crotalídeos/genética , Fosfolipases A , Sequência de Aminoácidos , Animais , Sequência de Bases , Venenos de Crotalídeos/farmacologia , DNA Complementar/genética , Fosfolipases A2 do Grupo II , Membranas/efeitos dos fármacos , Dados de Sequência Molecular , Músculos/efeitos dos fármacos , Neurotoxinas/genética , Fosfolipases A2 , Reação em Cadeia da Polimerase , Proteínas de Répteis , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The subcellular localization in brain of an unconventional, calmodulin-binding myosin (myosin-V) found in neurons, astrocytes and other secretory cells of vertebrates has been investigated by probing Western blots of synaptic fractions from rat cerebral cortex with affinity-purified polyclonal antibodies against myosin-V. Myosin-V was detected in intact synaptosomes and in lysed synaptosomes associated with a particulate fraction. Our data suggest a role for brain myosin-V in membrane-cytoskeleton function in the synaptic region.
Assuntos
Proteínas de Ligação a Calmodulina/análise , Córtex Cerebral/química , Miosina Tipo V , Proteínas do Tecido Nervoso/análise , Sinaptossomos/química , Animais , RatosRESUMO
the subcellular localization in brain of an unconventional, calmodulin-binding myosin (myosin-V) found in neurons, astrocytes and other secretory cells of vertebrates has been investigated by probing Western blots of synaptic fractions from rat cerebral cortex with affinity-purified polyclonal antibodies against myosin-V. Myosin-V was detected in intact synaptosomes and in lysed synaptosomes associated with a particulate fration. Our data suggest a role for brain myosin-V in membrane-cytoskeleton function in the synaptic region
Assuntos
Animais , Ratos , Córtex Cerebral/química , Proteínas do Tecido Nervoso/química , Sinaptossomos/química , Western BlottingRESUMO
1. Myosin-V from vertebrate brain is a novel molecular motor with a myosin-like head domain, a calmodulin-binding neck region and a unique tail domain of unknown function. Previous studies showed brain myosin-V to be a phosphoprotein substrate for Ca2+/calmodulin-dependent protein kinase associated with actomyosin. In the present study we describe the preparation of a specific actin-cytoskeletal fraction which is enriched in brain myosin-V. 2. We show that Ca2+/calmodulin-dependent protein kinase activity is also associated with this preparation and phosphorylates brain myosin-V. 3. Calpain, a Ca(2+)-dependent protease, generates a M(r) 80,000 fragment from the COOH terminal region of brain myosin-V containing most or all of the phosphorylation sites. 4. These results suggest that the unique tail domain of this novel myosin is subject to Ca2+ control via phosphorylation by kinase activity associated with the actin cytoskeleton.