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1.
Proc Natl Acad Sci U S A ; 116(38): 18943-18950, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31484776

RESUMO

Rapid advances in genomic technologies have led to a wealth of diverse data, from which novel discoveries can be gleaned through the application of robust statistical and computational methods. Here, we describe GeneFishing, a semisupervised computational approach to reconstruct context-specific portraits of biological processes by leveraging gene-gene coexpression information. GeneFishing incorporates multiple high-dimensional statistical ideas, including dimensionality reduction, clustering, subsampling, and results aggregation, to produce robust results. To illustrate the power of our method, we applied it using 21 genes involved in cholesterol metabolism as "bait" to "fish out" (or identify) genes not previously identified as being connected to cholesterol metabolism. Using simulation and real datasets, we found that the results obtained through GeneFishing were more interesting for our study than those provided by related gene prioritization methods. In particular, application of GeneFishing to the GTEx liver RNA sequencing (RNAseq) data not only reidentified many known cholesterol-related genes, but also pointed to glyoxalase I (GLO1) as a gene implicated in cholesterol metabolism. In a follow-up experiment, we found that GLO1 knockdown in human hepatoma cell lines increased levels of cellular cholesterol ester, validating a role for GLO1 in cholesterol metabolism. In addition, we performed pantissue analysis by applying GeneFishing on various tissues and identified many potential tissue-specific cholesterol metabolism-related genes. GeneFishing appears to be a powerful tool for identifying related components of complex biological systems and may be used across a wide range of applications.


Assuntos
Fenômenos Biológicos/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Genômica/métodos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Bases de Dados Genéticas , Humanos , Lactoilglutationa Liase/genética , Metabolismo dos Lipídeos/genética , Especificidade de Órgãos/genética , Reprodutibilidade dos Testes , Fluxo de Trabalho
2.
Circ Cardiovasc Genet ; 9(3): 223-30, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27071970

RESUMO

BACKGROUND: Numerous genetic contributors to cardiovascular disease risk have been identified through genome-wide association studies; however, identifying the molecular mechanism underlying these associations is not straightforward. The Justification for the Use of Statins in Primary Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER) trial of rosuvastatin users identified a sub-genome-wide association of rs6924995, a single-nucleotide polymorphism ≈10 kb downstream of myosin regulatory light chain interacting protein (MYLIP, aka IDOL and inducible degrader of low-density lipoprotein receptor [LDLR]), with LDL cholesterol statin response. Interestingly, although this signal was initially attributed to MYLIP, rs6924995 lies within RP1-13D10.2, an uncharacterized long noncoding RNA. METHODS AND RESULTS: Using simvastatin and sham incubated lymphoblastoid cell lines from participants of the Cholesterol and Pharmacogenetics (CAP) simvastatin clinical trial, we found that statin-induced change in RP1-13D10.2 levels differed between cell lines from the tails of the white and black low-density lipoprotein cholesterol response distributions, whereas no difference in MYLIP was observed. RP1-13D10.2 overexpression in Huh7 and HepG2 increased LDLR transcript levels, increased LDL uptake, and decreased media levels of apolipoprotein B. In addition, we found a trend of slight differences in the effects of RP1-13D10.2 overexpression on LDLR transcript levels between hepatoma cells transfected with the rs6924995 A versus G allele and a suggestion of an association between rs6924995 and RP1-10D13.2 expression levels in the CAP lymphoblastoid cell lines. Finally, RP1-13D10.2 expression levels seem to be sterol regulated, consistent with its potential role as a novel lipid regulator. CONCLUSIONS: RP1-13D10.2 is a long noncoding RNA that regulates LDLR and may contribute to low-density lipoprotein cholesterol response to statin treatment. These findings highlight the potential role of noncoding RNAs as determinants of interindividual variation in drug response.


Assuntos
LDL-Colesterol/metabolismo , Dislipidemias/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , RNA Longo não Codificante/genética , Sinvastatina/farmacologia , Adulto , Idoso , Apolipoproteína B-100/metabolismo , Biomarcadores/sangue , Ensaios Clínicos como Assunto , Dislipidemias/sangue , Dislipidemias/diagnóstico , Dislipidemias/genética , Feminino , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Regulação para Cima
3.
Bone ; 81: 97-103, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26151122

RESUMO

Osteoblasts perceive and respond to changes in their pericellular environment, including biophysical signals and oxygen availability, to elicit an anabolic or catabolic response. Parathyroid hormone (PTH) affects each arm of skeletal remodeling, with net anabolic or catabolic effects dependent upon duration of exposure. Similarly, the capacity of osteoblastic cells to perceive pericellular oxygen has a profound effect on skeletal mass and architecture, as mice expressing stable hypoxia-inducible factor (HIF)-1α and -2α demonstrate age-dependent increases in bone volume per tissue volume and osteoblast number. Further, HIF levels and signaling can be influenced in an oxygen-independent manner. Because the cellular mechanisms involved in PTH regulation of the skeleton remain vague, we sought whether PTH could influence HIF-1α expression and HIF-α-driven luciferase activity independently of altered oxygen availability. Using UMR106.01 mature osteoblasts, we observed that 100nM hPTH(1-34) decreased HIF-1α and HIF-responsive luciferase activity in a process involving heat shock protein 90 (Hsp90) and cyclic AMP but not intracellular calcium. Altering activity of the small GTPase RhoA and its effector kinase ROCK altered HIF-α-driven luciferase activity in the absence and presence of PTH. Taken together, these data introduce PTH as a regulator of oxygen-independent HIF-1α levels through a mechanism involving cyclic AMP, Hsp90, and the cytoskeleton.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Osteoblastos/metabolismo , Osteogênese/fisiologia , Hormônio Paratireóideo/metabolismo , Transdução de Sinais/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Western Blotting , Linhagem Celular , Humanos , Mutagênese Sítio-Dirigida , Ratos , Transfecção
4.
Nature ; 512(7515): 449-52, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25164756

RESUMO

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.


Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Animais , Linhagem Celular , Centrômero/genética , Centrômero/metabolismo , Cromatina/química , Montagem e Desmontagem da Cromatina/genética , Replicação do DNA/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Anotação de Sequência Molecular , Lâmina Nuclear/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , Especificidade da Espécie
5.
Curr Biol ; 22(4): 320-5, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22264607

RESUMO

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo III/metabolismo , Actinas/metabolismo , Motivos de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Humanos , Camundongos , Proteínas dos Microfilamentos/química , Microscopia Confocal , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Ligação Proteica , Transporte Proteico , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Ratos , Estereocílios/metabolismo , Transfecção
6.
J Neurochem ; 119(4): 772-84, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21895655

RESUMO

As class III unconventional myosins are motor proteins with an N-terminal kinase domain, it seems likely they play a role in both signaling and actin based transport. A growing body of evidence indicates that the motor functions of human class IIIA myosin, which has been implicated in progressive hearing loss, are modulated by intermolecular autophosphorylation. However, the phosphorylation sites have not been identified. We studied the kinase activity and phosphorylation sites of mouse class III myosins, mMyo3A and 3B, which are highly similar to their human orthologs. We demonstrate that the kinase domains of mMyo3A and 3B are active kinases, and that they have similar, if not identical, substrate specificities. We show that the kinase domains of these proteins autophosphorylate, and that they can phosphorylate sites within their myosin and tail domains. Using liquid chromatography-mass spectrometry, we identified phosphorylated sites in the kinase, myosin motor and tail domains of both mMyo3A and 3B. Most of the phosphorylated sites we identified and their consensus phosphorylation motifs are highly conserved among vertebrate class III myosins, including human class III myosins. Our findings are a major step toward understanding how the functions of class III myosins are regulated by phosphorylation.


Assuntos
Miosina Tipo III/química , Miosina Tipo III/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Aminoácidos , Animais , Humanos , Espectrometria de Massas , Camundongos , Miosina Tipo III/classificação , Miosina Tipo III/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Especificidade por Substrato
7.
Science ; 330(6012): 1775-87, 2010 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-21177976

RESUMO

We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor-binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor-binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.


Assuntos
Caenorhabditis elegans/genética , Cromossomos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma Helmíntico , Anotação de Sequência Molecular , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Cromossomos/genética , Cromossomos/metabolismo , Cromossomos/ultraestrutura , Biologia Computacional/métodos , Sequência Conservada , Evolução Molecular , Redes Reguladoras de Genes , Genes de Helmintos , Genômica/métodos , Histonas/metabolismo , Modelos Genéticos , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Exp Eye Res ; 89(2): 224-37, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19332056

RESUMO

Class III myosins are important for the function and survival of photoreceptors and ciliary hair cells. Although vertebrates possess two class III myosin genes, myo3A and myo3B, recent studies have focused on Myo3A because mutations in the human gene are implicated in progressive hearing loss. Myo3B may compensate for defects in Myo3A, yet little is known about its distribution and function. This study focuses on Myo3B expression in the mouse retina. We cloned two variants of myo3B from mouse retina and determined that they are expressed early in retinal development. In this study we show for the first time in a mammal that both Myo3B and Myo3A proteins are present in inner segments of all photoreceptors. Myo3B is also present in outer segments of S opsin-immunoreactive cones but not M opsin dominant cones. Myo3B is also detected in rare cells of the inner nuclear layer and some ganglion cells. Myo3B may have diverse roles in retinal neurons. In photoreceptor inner segments Myo3B is positioned appropriately to prevent photoreceptor loss of function caused by Myo3A defects.


Assuntos
Proteínas do Olho/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo III/metabolismo , Retina/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Soros Imunes , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/imunologia , Miosina Tipo III/genética , Miosina Tipo III/imunologia , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Ganglionares da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Distribuição Tecidual
9.
Nat Cell Biol ; 11(4): 443-50, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19287378

RESUMO

Two proteins implicated in inherited deafness, myosin IIIa, a plus-end-directed motor, and espin, an actin-bundling protein containing the actin-monomer-binding motif WH2, have been shown to influence the length of mechanosensory stereocilia. Here we report that espin 1, an ankyrin repeat-containing isoform of espin, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that combined overexpression of these proteins causes greater elongation of stereocilia, compared with overexpression of either myosin IIIa alone or espin 1 alone. When these two proteins were co-expressed in the fibroblast-like COS-7 cell line they induced a tenfold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role of myosin IIIa and espin 1 in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.


Assuntos
Citoesqueleto de Actina/metabolismo , Cílios/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miosina Tipo III/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Repetição de Anquirina , Células COS , Chlorocebus aethiops , Cílios/ultraestrutura , Camundongos , Proteínas dos Microfilamentos/química , Ligação Proteica , Transporte Proteico , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Ratos , Transfecção
10.
Biochemistry ; 47(8): 2485-96, 2008 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-18229949

RESUMO

Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells.


Assuntos
Movimento , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo III/química , Miosina Tipo III/fisiologia , Fosfotransferases , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Células HeLa , Humanos , Hidrólise , Cinética , Modelos Biológicos , Movimento/fisiologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo III/genética , Miosina Tipo III/metabolismo , Neurônios Aferentes/metabolismo , Fosfotransferases/metabolismo , Fosfotransferases/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Coelhos , Spodoptera , Transfecção
11.
J Biol Chem ; 282(1): 216-31, 2007 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-17074769

RESUMO

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k(cat) = 0.77 +/- 0.08 s(-1)), and high actin concentrations are required to fully activate the ATPase rate (K(ATPase) = 34 +/- 11 microm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K(actin) approximately 7 microm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K(1)k(+2) = 0.020 and 0.001 microm(-1).s(-1), respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s(-1)) than the k(cat), whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s(-1)). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K(d) approximately 5 microm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.


Assuntos
Miosina Tipo III/química , Actinas/química , Motivos de Aminoácidos , Relação Dose-Resposta a Droga , Humanos , Cinética , Luz , Modelos Químicos , Miosinas/química , Fosfatos/química , Fosforilação , Conformação Proteica , Estrutura Terciária de Proteína , Espalhamento de Radiação , Espectrometria de Fluorescência
12.
J Neurosci ; 26(40): 10243-52, 2006 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-17021180

RESUMO

Class III myosins are motor proteins that contain an N-terminal kinase domain and a C-terminal actin-binding domain. We show that myosin IIIa, which has been implicated in nonsyndromic progressive hearing loss, is localized at stereocilia tips. Myosin IIIa progressively accumulates during stereocilia maturation in a thimble-like pattern around the stereocilia tip, distinct from the cap-like localization of myosin XVa and the shaft localization of myosin Ic. Overexpression of deletion mutants for functional domains of green fluorescent protein (GFP)-myosin IIIa shows that the motor domain, but not the actin-binding tail domain, is required for stereocilia tip localization. Deletion of the kinase domain produces stereocilia elongation and bulging of the stereocilia tips. The thimble-like localization and the influence myosin IIIa has on stereocilia shape reveal a previously unrecognized molecular compartment at the distal end of stereocilia, the site of actin polymerization as well as operation of the mechanoelectrical transduction apparatus.


Assuntos
Orelha Interna/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cadeias Pesadas de Miosina/biossíntese , Miosina Tipo III/biossíntese , Animais , Anuros , Bass , Células COS , Células Cultivadas , Galinhas , Chlorocebus aethiops , Cílios/genética , Cílios/metabolismo , Cobaias , Humanos , Camundongos , Cadeias Pesadas de Miosina/genética , Miosina Tipo III/genética , Ratos , Fatores de Tempo
13.
Mol Biol Cell ; 14(10): 4173-80, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14517327

RESUMO

Bass Myo3A, a class III myosin, was expressed in HeLa cells as a GFP fusion in order to study its cellular localization. GFP-Myo3A localized to the cytoplasm and to the tips of F-actin bundles in filopodia, a localization that is consistent with the observed concentration toward the distal ends of F-actin bundles in photoreceptor cells. A mutation in the motor active site resulted in a loss of filopodia localization, suggesting that Myo3A motor activity is required for filopodial tip localization. Deletion analyses showed that the NH2-terminal kinase domain is not required but the CO2H-terminal 22 amino acids of the Myo3A tail are required for filopodial localization. Expression of this tail fragment alone produced fluorescence associated with F-actin throughout the cytoplasm and filopodia and a recombinant tail fragment bound to F-actin in vitro. An actin-binding motif was identified within this tail fragment, and a mutation within this motif abolished both filopodia localization by Myo3A and F-actin binding by the tail fragment alone. Calmodulin localized to filopodial tips when coexpressed with Myo3A but not in the absence of Myo3A, an observation consistent with the previous proposal that class III myosins bind calmodulin and thereby localize it in certain cell types.


Assuntos
Actinas/metabolismo , Calmodulina/metabolismo , Miosina Tipo III/metabolismo , Pseudópodes/metabolismo , Sequência de Aminoácidos , Análise Mutacional de DNA , Primers do DNA/genética , Imunofluorescência , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Miosina Tipo III/genética , Ligação Proteica , Estrutura Terciária de Proteína , Pseudópodes/genética , Proteínas Recombinantes de Fusão/metabolismo
14.
Mol Biol Cell ; 14(3): 1058-73, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12631723

RESUMO

The striped bass has two retina-expressed class III myosin genes, each composed of a kinase, motor, and tail domain. We report the cloning, sequence analysis, and expression patterns of the long (Myo3A) and short (Myo3B) class III myosins, as well as cellular localization and biochemical characterization of the long isoform, Myo3A. Myo3A (209 kDa) is expressed in the retina, brain, testis, and sacculus, and Myo3B (155 kDa) is expressed in the retina, intestine, and testis. The tails of these two isoforms contain two highly conserved domains, 3THDI and 3THDII. Whereas Myo3B has three IQ motifs, Myo3A has nine IQ motifs, four in its neck and five in its tail domain. Myo3A localizes to actin filament bundles of photoreceptors and is concentrated in the calycal processes. An anti-Myo3A antibody decorates the actin cytoskeleton of rod inner/outer segments, and this labeling is reduced by the presence of ATP. The ATP-sensitive actin association is a feature characteristic of myosin motors. The numerous IQ motifs may play a structural or signaling role in the Myo3A, and its localization to calycal processes indicates that this myosin mediates a local function at this site in vertebrate photoreceptors.


Assuntos
Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sáculo e Utrículo/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Bass/metabolismo , Calmodulina/metabolismo , Citoesqueleto/metabolismo , Humanos , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/classificação , Cadeias Pesadas de Miosina/genética , Miosina Tipo III , Filogenia , Isoformas de Proteínas/genética , Retina/química , Retina/citologia , Células Fotorreceptoras Retinianas Cones/química , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/química , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Sáculo e Utrículo/química , Alinhamento de Sequência , Distribuição Tecidual , Tubulina (Proteína)/metabolismo
15.
Genomics ; 79(5): 621-4, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11991710

RESUMO

Class III myosins are actin-based motors with amino-terminal kinase domains. Expression of these motors is highly enhanced in retinal photoreceptors. As mutations in the gene encoding NINAC, a Drosophila melanogaster class III myosin, cause retinal degeneration, human homologs of this gene are potential candidates for human retinal disease. We have recently reported the cloning of MYO3A, a human myosin III expressed predominantly in the retina and retinal pigmented epithelium [1]. The map locus of MYO3A is close to, but does not overlap, that of human Usher's 1F [2]. Here we introduce a shorter class III myosin isoform, MYO3B, which is expressed in the retina, kidney, and testis. We describe the cDNA sequence, genomic organization, and splice variants of MYO3B expressed in the human retina. A product of 36 exons, MYO3B has several splice variants containing either one or two calmodulin binding (IQ) motifs in the neck domain and one of three predominant tail variations: a short tail ending just past the second IQ motif, or two alternatively spliced longer tails. MYO3B maps to 2q31.1-q31.2, a region that overlaps the locus for a Bardet-Biedl syndrome (BBS5) linked to markers at 2q31 [3].


Assuntos
Síndrome de Bardet-Biedl/genética , Predisposição Genética para Doença/genética , Cadeias Pesadas de Miosina/genética , Retina/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Linhagem Celular , Éxons , Feminino , Expressão Gênica , Genes/genética , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Miosina Tipo III , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos
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