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1.
Genes Dev ; 28(10): 1068-84, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24788092

RESUMO

The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Processamento Alternativo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Isoformas de Proteínas , Fatores de Processamento de RNA , Spliceossomos
2.
J Immunol ; 193(2): 860-70, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24935926

RESUMO

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRß activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRß, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.


Assuntos
Artrite Experimental/imunologia , Citocinas/imunologia , Inflamação/imunologia , Receptores Imunológicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Artrite Experimental/metabolismo , Artrite Experimental/prevenção & controle , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Células Cultivadas , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Células HEK293 , Membro Posterior/efeitos dos fármacos , Membro Posterior/imunologia , Membro Posterior/patologia , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , Inflamação/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , Osteoartrite/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/genética , Transcriptoma/imunologia
3.
Blood ; 121(7): e25-33, 2013 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-23255559

RESUMO

Despite significant progresses, cell-based assays still have major limitations part to because of their plate format. Here, we present a wall-less plate technology based on unique liquid dynamics named DropArray that takes advantage of hydrophobic and hydrophilic surface properties. Liquid velocities within the DropArray plate were quantified through fluid dynamics simulation and complete retention of suspension cells experimentally demonstrated within the range of simulated shear stresses. Subsequently, we compared the DropArray technology with conventional microtiter plates in a cell-based protein-binding assay. Although the wall-less plate produced similar results with adherent cells, the advantage of the DropArray technology was absolutely clear when semiadherent or suspension cells were used in this multistep experimental procedure. The technology also was evaluated for the cell viability assay and generated similar results to conventional plate format while enabling significant reduction in toxic reagent use. Finally, we developed a DropArray cell-based assay to evaluate a bispecific antibody designed to engage cytotoxic T cells and trigger tumor cell killing. This assay enables for the first time the visualization and quantification of the specific killing events and represents a very powerful tool to further investigate functional aspects of the cancer immunotherapy.


Assuntos
Técnicas Citológicas/métodos , Animais , Anticorpos Biespecíficos , Linfócitos B/imunologia , Células COS , Linhagem Celular , Sobrevivência Celular , Chlorocebus aethiops , Técnicas Citológicas/instrumentação , Testes Imunológicos de Citotoxicidade/instrumentação , Testes Imunológicos de Citotoxicidade/métodos , Células HEK293 , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunoterapia , Células K562 , Ativação Linfocitária , Neoplasias/imunologia , Neoplasias/terapia , Ligação Proteica , Linfócitos T Citotóxicos/imunologia , Células U937
4.
J Biol Chem ; 287(19): 15837-50, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22396535

RESUMO

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.


Assuntos
Evolução Molecular , Glicoproteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Receptores Imunológicos/metabolismo , Antígeno 12E7 , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Arginina/química , Arginina/genética , Arginina/metabolismo , Sítios de Ligação/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Colectinas/química , Colectinas/genética , Colectinas/metabolismo , Sequência Conservada/genética , Células HEK293 , Humanos , Ligantes , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Depuradores/química , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Homologia de Sequência de Aminoácidos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Células Vero
5.
J Cell Biochem ; 113(7): 2397-405, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22573556

RESUMO

Sensitive detection of protein interactions is a critical step toward understanding complex cellular processes. As an alternative to fluorescence-based detection, Renilla reniformis luciferase conjugated to quantum dots results in self-illuminating bioluminescence resonance energy transfer quantum dot (BRET-Qdot) nanoprobes that emit red to near-infrared bioluminescence light. Here, we report the development of an ultrasensitive technology based on BRET-Qdot conjugates modified with streptavidin ([BRET-Qdot]-SA) to detect cell-surface protein interactions. Transfected COS7 cells expressing human cell-surface proteins were interrogated with a human Fc tagged protein of interest. Specific protein interactions were detected using a biotinylated anti-human Fc region specific antibody followed by incubation with [BRET-Qdot]-SA. The luciferase substrate coelenterazine activated bioluminescence light emission was detected with an ultra-fast and -sensitive imager. Protein interactions barely detectable by the fluorescence-based approach were readily quantified using this technology. The results demonstrate the successful application and the flexibility of the BRET-Qdot-based imaging technology to the ultrasensitive investigation of cell-surface proteins and protein-protein interactions.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Proteínas de Membrana/metabolismo , Mapeamento de Interação de Proteínas/métodos , Células 3T3 , Animais , Células COS , Chlorocebus aethiops , Humanos , Imidazóis/farmacologia , Luciferases de Renilla , Camundongos , Nanopartículas , Pirazinas/farmacologia , Pontos Quânticos , Receptores Fc/imunologia , Estreptavidina/química
6.
Mol Biol Cell ; 15(12): 5295-305, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15456898

RESUMO

Most cells on earth exist in a quiescent state. In yeast, quiescence is induced by carbon starvation, and exit occurs when a carbon source becomes available. To understand how cells survive in, and exit from this state, mRNA abundance was examined using oligonucleotide-based microarrays and quantitative reverse transcription-polymerase chain reaction. Cells in stationary-phase cultures exhibited a coordinated response within 5-10 min of refeeding. Levels of >1800 mRNAs increased dramatically (>or=64-fold), and a smaller group of stationary-phase mRNAs decreased in abundance. Motif analysis of sequences upstream of genes clustered by VxInsight identified an overrepresentation of Rap1p and BUF (RPA) binding sites in genes whose mRNA levels rapidly increased during exit. Examination of 95 strains carrying deletions in stationary-phase genes induced identified 32 genes essential for survival in stationary-phase at 37 degrees C. Analysis of these genes suggests that mitochondrial function is critical for entry into stationary-phase and that posttranslational modifications and protection from oxidative stress become important later. The phylogenetic conservation of stationary-phase genes, and our findings that two-thirds of the essential stationary-phase genes have human homologues and of these, many have human homologues that are disease related, demonstrate that yeast is a bona fide model system for studying the quiescent state of eukaryotic cells.


Assuntos
Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/genética , Genes Essenciais/genética , Genes Fúngicos/genética , Genômica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Sequência de Bases , Ciclo Celular , Evolução Molecular , Genoma Fúngico , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência/genética , Fatores de Tempo , Transcrição Gênica/genética
7.
J Microbiol Methods ; 65(2): 357-60, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16198434

RESUMO

Here we describe an automated, pressure-driven, sampling device for harvesting 10 to 30 ml samples, in replicate, with intervals as short as 10 s. Correlation between biological replicate time courses measured by microarrays was extremely high. The sampler enables sampling at intervals within the range of many important biological processes.


Assuntos
Técnicas Microbiológicas/instrumentação , Leveduras , Automação , Meios de Cultura , Desenho de Equipamento , Análise de Sequência com Séries de Oligonucleotídeos , RNA Fúngico/análise , RNA Fúngico/isolamento & purificação , Reprodutibilidade dos Testes , Leveduras/genética , Leveduras/crescimento & desenvolvimento , Leveduras/isolamento & purificação , Leveduras/metabolismo
8.
PLoS One ; 9(1): e84823, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24400119

RESUMO

The growth and guidance of many axons in the developing nervous system require Netrin-mediated activation of Deleted in Colorectal Cancer (DCC) and other still unknown signaling cues. Commissural axon guidance defects are more severe in DCC mutant mice than Netrin-1 mutant mice, suggesting additional DCC activating signals besides Netrin-1 are involved in proper axon growth. Here we report that interaction screens on extracellular protein microarrays representing over 1,000 proteins uniquely identified Cerebellin 4 (CBLN4), a member of the C1q-tumor necrosis factor (TNF) family, and Netrin-1 as extracellular DCC-binding partners. Immunofluorescence and radio-ligand binding studies demonstrate that Netrin-1 competes with CBLN4 binding at an overlapping site within the membrane-proximal fibronectin domains (FN) 4-6 of DCC and binds with ∼5-fold higher affinity. CBLN4 also binds to the DCC homolog, Neogenin-1 (NEO1), but with a lower affinity compared to DCC. CBLN4-null mice did not show a defect in commissural axons of the developing spinal cord but did display a transient increase in the number of wandering axons in the brachial plexus, consistent with a role in axon guidance. Overall, the data solidifies CBLN4 as a bona fide DCC ligand and strengthens its implication in axon guidance.


Assuntos
Receptores de Superfície Celular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Axônios/metabolismo , Proteínas de Transporte , Receptor DCC , Desenvolvimento Embrionário/genética , Humanos , Cinética , Ligantes , Camundongos , Mutação , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Netrina-1 , Neurogênese/genética , Neurônios/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Precursores de Proteínas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
9.
PLoS One ; 9(7): e102695, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25029443

RESUMO

Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, ß-catenin independent pathway and suppress a canonical Wnt/ß-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Evolução Molecular , Modelos Moleculares , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Catálise , Células HEK293 , Humanos , Immunoblotting , Luciferases , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de Proteína , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/química , Alinhamento de Sequência , Especificidade da Espécie
10.
Cell Cycle ; 9(13): 2522-8, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20581461

RESUMO

While directed cellular migration facilitates the coordinated movement of cells during development and tissue repair, the precise mechanisms regulating the interplay between the extracellular environment, the actin cytoskeleton, and the overlying plasma membrane remain inadequately understood. The BAR domain family of lipid binding, actin cytoskeletal regulators are gaining greater appreciation for their role in these critical processes. BAR domain proteins are involved as both positive and negative regulators of endocytosis, membrane plasticity, and directional cell migration. This review focuses on the functional relationship between different classes of BAR domain proteins and their role in guiding cell migration through regulation of the endocytic machinery. Competition for key signaling substrates by positive and negative BAR domain endocytic regulators appears to mediate control of directional cell migration, and may have wider applicability to other trafficking functions associated with development and carcinogenesis.


Assuntos
Movimento Celular , Estrutura Terciária de Proteína , Animais , Endocitose , Humanos , Modelos Biológicos
11.
J Cell Biol ; 189(2): 353-67, 2010 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-20385776

RESUMO

Although directed cellular migration facilitates the coordinated movement of cells during development and repair, the mechanisms regulating such migration remain poorly understood. Missing-in-metastasis (MIM) is a defining member of the inverse Bin/Amphiphysin/Rvs domain (I-BAR) subfamily of lipid binding, cytoskeletal regulators whose levels are altered in a number of cancers. Here, we provide the first genetic evidence that an I-BAR protein regulates directed cell migration in vivo. Drosophila MIM (dmim) is involved in Drosophila border cell migration, with loss of dmim function resulting in a lack of directional movement by the border cell cluster. In vivo endocytosis assays combined with genetic analyses demonstrate that the dmim product regulates directed cell movement by inhibiting endocytosis and antagonizing the activities of the CD2-associated protein/cortactin complex in these cells. These studies demonstrate that DMIM antagonizes pro-endocytic components to facilitate polarity and localized guidance cue sensing during directional cell migration.


Assuntos
Movimento Celular/fisiologia , Proteínas de Drosophila/metabolismo , Endocitose/fisiologia , Proteínas dos Microfilamentos/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Animais Geneticamente Modificados , Cortactina/genética , Cortactina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/fisiologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células Germinativas/citologia , Células Germinativas/fisiologia , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Mutação , Ligação Proteica
12.
Genome Biol ; 7(2): R9, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16507144

RESUMO

BACKGROUND: As carbon sources are exhausted, Saccharomyces cerevisiae cells exhibit reduced metabolic activity and cultures enter the stationary phase. We asked whether cells in stationary phase cultures respond to additional stress at the level of transcript abundance. RESULTS: Microarrays were used to quantify changes in transcript abundance in cells from stationary phase cultures in response to stress. More than 800 mRNAs increased in abundance by one minute after oxidative stress. A significant number of these mRNAs encode proteins involved in stress responses. We tested whether mRNA increases were due to new transcription, rapid poly-adenylation of message (which would not be detected by microarrays), or potential release of mature mRNA present in the cell but resistant to extraction during RNA isolation. Examination of the response to oxidative stress in an RNA polymerase II mutant, rpb1-1, suggested that new transcription was not required. Quantitative RT-PCR analysis of a subset of these transcripts further suggested that the transcripts present in isolated total RNA from stationary phase cultures were polyadenylated. In contrast, over 2,000 transcripts increased after protease treatment of cell-free lysates from stationary phase but not exponentially growing cultures. Different subsets of transcripts were released by oxidative stress and temperature upshift, suggesting that mRNA release is stress-specific. CONCLUSIONS: Cells in stationary phase cultures contain a large number of extraction-resistant mRNAs in a protease-labile, rapidly releasable form. The transcript release appears to be stress-specific. We hypothesize that these transcripts are associated with P-bodies.


Assuntos
Estresse Oxidativo/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sistema Livre de Células , Endopeptidase K/farmacologia , Regulação Fúngica da Expressão Gênica , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , RNA Polimerase II/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/efeitos dos fármacos
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