RESUMO
With the advent of high-throughput sequencing of immunoglobulin genes (Ig-Seq), the understanding of antibody repertoires and their dynamics among individuals and populations has become an exciting area of research. There is an increasing number of computational tools that aid in every step of the immune repertoire characterization. However, since not all tools function identically, every pipeline has its unique rationale and capabilities, creating a rich blend of useful features that may appear intimidating for newcomer laboratories with the desire to plunge into immune repertoire analysis to expand and improve their research; hence, all pipeline strengths and differences may not seem evident. In this review we provide a practical and organized list of the current set of computational tools, focusing on their most attractive features and differences in order to carry out the characterization of antibody repertoires so that the reader better decides a strategic approach for the experimental design, and computational pathways for the analyses of immune repertoires.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Software , Recombinação V(D)J , Animais , Humanos , ImunoglobulinasRESUMO
This data contains information related to the research article entitled "Osteopontin splice variants and polymorphisms in Cancer Progression and Prognosis" [1]. Here, we describe an in silico analysis of transcription factors that could have altered binding to their DNA target sequence as a result of SNPs in the osteopontin gene promoter. We concentrated on SNPs associated with cancer risk and development. The analysis was performed with PROMO v3.0.2 software which incorporates TRANSFACT v6.4 of. We also present a figure depicting the putative transcription factor binding according to genotype.
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Osteopontin (OPN) is an extracellular matrix protein that is overexpressed in various cancers and promotes oncogenic features including cell proliferation, survival, migration, and angiogenesis, among others. OPN can participate in the regulation of the tumor microenvironment, affecting both cancer and neighboring cells. Here, we review the roles of OPN splice variants (a, b, c) in cancer development, progression, and prognosis, and also discuss the identities of isoforms 4 and 5. We also discussed how single-nucleotide polymorphisms (SNPs) of the OPN gene are an additional factor influencing the level of OPN in individuals, modulating the risks of cancer development and outcome.
Assuntos
Neoplasias/genética , Neoplasias/patologia , Osteopontina/genética , Polimorfismo Genético/genética , Splicing de RNA/genética , Sequência de Aminoácidos , Sequência de Bases , Progressão da Doença , Humanos , PrognósticoRESUMO
TDP-43 is a nuclear protein involved in many aspects of RNA metabolism. To ensure cellular viability, its expression levels within cells must be tightly regulated. We have previously demonstrated that TDP-43 autoregulation occurs through the activation of a normally silent intron in its 3'-UTR sequence that results in the use of alternative polyadenylation sites. In this work, we analyse which is the dominant event in autoregulation: the recognition of the splice sites of 3'-UTR intron 7 or the intrinsic quality of the alternative polyadenylation sites. A panel of minigene constructs was tested for autoregulation functionality, protein production and subcellular messenger RNA localization. Our data clearly indicate that constitutive spliceosome complex formation across intron 7 does not lead to high protein production but, on the contrary, to lower TDP-43 messenger RNA and protein levels. This is due to altered nucleocytoplasmic distribution of the RNA that is mostly retained in the nucleus and degraded. This study provides a novel in-depth characterization of how RNA binding proteins can autoregulate their own levels within cells, an essential regulatory process in maintaining cellular viability.
Assuntos
Proteínas de Ligação a DNA/genética , Poliadenilação , Splicing de RNA , Proteínas de Ligação a RNA/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Homeostase , Humanos , Íntrons , Sítios de Splice de RNA , Transporte de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Spliceossomos/metabolismoRESUMO
Using C9 cells stably expressing LPA1 receptors fused to the enhanced green fluorescent protein, it was observed that activation of protein kinase C induced a rapid and strong increase in the phosphorylation state of these receptors. Overnight incubation with phorbol esters markedly decreased the amount of conventional (α, ßI, ßII and γ) and novel (δ) but not atypical (ζ) immunodetected PKC isoforms, this treatment blocks the action of protein kinase on receptor function and phosphorylation. Bis-indolylmaleimide I a general, non-subtype selective protein kinase C inhibitor, and Gö 6976, selective for the isoforms α and ß, were also able to block LPA1 receptor desensitization and phosphorylation; hispidin, isoform ß-selective blocker partially avoided receptor desensitization. Expression of dominant-negative protein kinase C α or ß II mutants and knocking down the expression of these kinase isozymes markedly decreased phorbol ester-induced LPA1 receptor phosphorylation without avoiding receptor desensitization. This effect was blocked by bis-indolyl-maleimide and Gö 6976, suggesting that these genetic interventions were not completely effective. It was also observed that protein kinase C α and ß II isozymes co-immunoprecipitate with LPA1 receptors and that such an association was further increased by cell treatments with phorbol esters or lysophosphatidic acid. Our data suggest that conventional protein kinase C α and ß isozymes modulate LPA1 receptor phosphorylation state. Receptor desensitization appears to be a more complex process that might involve additional elements.
Assuntos
Proteína Quinase C/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Linhagem Celular , Isoenzimas/metabolismo , Lisofosfolipídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
TDP-43 is a critical RNA-binding factor associated with pre-mRNA splicing in mammals. Its expression is tightly autoregulated, with loss of this regulation implicated in human neuropathology. We demonstrate that TDP-43 overexpression in humans and mice activates a 3' untranslated region (UTR) intron, resulting in excision of the proximal polyA site (PAS) pA(1). This activates a cryptic PAS that prevents TDP-43 expression through a nuclear retention mechanism. Superimposed on this process, overexpression of TDP-43 blocks recognition of pA(1) by competing with CstF-64 for PAS binding. Overall, we uncover complex interplay between transcription, splicing, and 3' end processing to effect autoregulation of TDP-43.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Poli A/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Transcrição Gênica , Processamento Alternativo , Animais , Sequência de Bases , Linhagem Celular , Fator Estimulador de Clivagem/química , Fator Estimulador de Clivagem/metabolismo , Proteínas de Ligação a DNA/genética , Homeostase , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Sítios de Splice de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismoRESUMO
The overview of TDP 43 functions immediately disclose a number of open questions regarding its pathological role. The formation of TDP-43 aggregates is one of the major distinguishing features of TDP-43 proteinopathies, especially in patients affected by Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar degeneration (FTLD). At the moment, however, very little is known regarding the biological processes that underlie TDP-43 aggregation and, most importantly, its potential consequences on cellular metabolism. For these reasons, it is particularly important to further investigate this process in order to gain a better understanding of the pathology and to develop novel therapeutic effectors. In this report, we focus on a series of missense mutations associated with disease in the 342-366 region of this protein to examine their ability to affect RNA splicing regulation and to induce aggregate formation. In particular, aggregate formation was assessed in a novel system capable of inducing TDP-43 aggregation in experimental cell lines and primary neuronal cultures. The results of this analysis showed that the presence of two of these missense mutations in the 342-366 region (G348V and N352S) could differentially affect the levels and appearance of TDP-43 aggregation with respect to the wild-type protein. This article is part of a Special Issue entitled RNA-Binding Proteins.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Fluorescência Verde/farmacologia , Mutação/fisiologia , Western Blotting , Fracionamento Celular , Ensaio de Desvio de Mobilidade Eletroforética , Éxons/genética , Células HeLa , Humanos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Mutação/genética , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Neuropeptídeos/química , Plasmídeos/genética , Splicing de RNA , RNA Interferente Pequeno/genética , Proteinopatias TDP-43 , Técnicas de Cultura de Tecidos , TransfecçãoRESUMO
BACKGROUND: Lysophosphatidic acid (LPA) is a local mediator that exerts its actions through G protein coupled receptors. Knowledge on the regulation of such receptors is scarce to date. Here we show that bidirectional cross-talk exits between LPA(1) and EGF receptors. METHODS: C9 cells expressing LPA(1) receptor fussed to the enhanced green fluorescent protein were used. We studied intracellular calcium concentration, Akt/PKB phosphorylation, LPA(1) and EGF receptor phosphorylation. RESULTS: EGF diminished LPA-mediated intracellular calcium response and induced LPA(1) receptor phosphorylation, which was sensitive to protein kinase C inhibitors. Angiotensin II and LPA induced EGF receptor transactivation as evidenced by Akt/PKB phosphorylation through metalloproteinase-catalyzed membrane shedding of heparin-binding EGF and autocrine/paracrine activation of EGF receptors. This process was found to be of major importance in angiotensin II-induced LPA(1) receptor phosphorylation. Attempts to define a role for EGF receptor transactivation in homologous LPA(1) receptor desensitization and phosphorylation suggested that G protein-coupled receptor kinases are the major players in this process, overshadowing other events. CONCLUSIONS: EGF receptors and LPA(1) receptors are engaged in an intense liaison, in that EGF receptors are capable of modulating LPA(1) receptor function through phosphorylation cascades. EGF transactivation plays a dual role: it mediates some LPA actions, and it modulates LPA(1) receptor function in inhibitory fashion. GENERAL SIGNIFICANCE: EGF and LPA receptors coexist in many cell types and play key roles in maintaining the delicate equilibrium that we call health and in the pathogenesis of many diseases. The intense cross-talk described here has important physiological and pathophysiological implications.
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Angiotensina II/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Western Blotting , Linhagem Celular , Imunoprecipitação , Fosforilação , RatosRESUMO
TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3' UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43-hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3' UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis.
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Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Sequência de Bases , Northern Blotting , Linhagem Celular , Proteínas de Ligação a DNA/genética , Biblioteca Gênica , Humanos , Immunoblotting , Imunoprecipitação , Dados de Sequência Molecular , Plasmídeos/genética , Reação em Cadeia da Polimerase , Interferência de RNA , Análise de Sequência de DNARESUMO
17beta-Estradiol induced LPA(1) receptor desensitization in C9 cells stably expressing LPA(1) receptors and transiently expressing estrogen receptor alpha. Such desensitization was evidenced by a reduction in lysophosphatidic acid-mediated Ca(2+)mobilization and it was associated to receptor phosphorylation and internalization. These effects of 17beta-estradiol were rapid (taking place over 5 min) and were blocked by the estrogen receptor antagonist ICI 182780. Similarly, inhibitors of phosphoinositide 3-kinase (wortmannin and LY294002) and of protein kinase C (staurosporine and Gö 6976) blocked 17beta-estradiol-induced LPA(1) receptor desensitization and phosphorylation. Confocal microscopy evidenced LPA(1) receptor internalization in response to 17beta-estradiol treatment. Association between LPA(1) receptors and protein kinase C alpha was suggested by co-immunoprecipitation assays. Protein kinase C alpha was associated with LPA(1) receptors in the absence of stimulus and such association further increased in a dynamic fashion in response to 17beta-estradiol. The results demonstrated that in C9 cells estrogens modulate LPA(1) action through estrogen receptor alpha with the participation of protein kinase C alpha and phosphoinositide 3-kinase.
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Estrogênios/farmacologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Endocitose/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Humanos , Imunoprecipitação , Lisofosfolipídeos/farmacologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/efeitos dos fármacos , TransfecçãoRESUMO
The effect of insulin-like growth factor-I (IGF-I) on human alpha(1B)-adrenoceptor function, phosphorylation state and cellular location was studied. Rat-1 fibroblasts were transfected with a plasmid construction containing enhanced green fluorescent protein joined to the carboxyl terminus of the human alpha(1B)-adrenoceptor. Receptors were identified by radioligand binding and photoaffinity labeling, and were immunoprecipitated with an antiserum generated against the enhanced green fluorescent protein. The receptor was functional, as evidenced by noradrenaline action on intracellular calcium and inositol phosphate production. IGF-I had no significant effect by itself on these parameters but markedly reduced the effects of noradrenaline. IGF-I induced alpha(1B)-adrenoceptor phosphorylation, which was markedly reduced by the following agents: pertussis toxin, a metalloproteinase inhibitor, diphtheria toxin mutant CRM 197, an epidermal growth factor (EGF) receptor intrinsic kinase activity inhibitor, and by phosphoinositide 3-kinase and protein kinase C inhibitors. IGF-I action appears to involve activation of a pertussis toxin-sensitive G protein, shedding of heparin-binding EGF and autocrine activation of EGF receptors. G protein subunits and phosphotyrosine residues stimulate phosphoinositide 3-kinase activity leading to activation of protein kinase C, which in turn phosphorylates alpha(1B)-adrenoceptors. Confocal fluorescent microscopy showed that alpha(1B)-adrenoceptors fussed to the green fluorescent protein were located in plasma membrane and intracellular vesicles in the basal state. IGF-I induced receptor redistribution favoring the intracellular location; this effect was blocked by hypertonic sucrose and concanavalin A. Our data show that IGF-I induces alpha(1B)-adrenoceptor desensitization associated to receptor phosphorylation and internalization.
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Proteínas de Ligação ao GTP/metabolismo , Fator de Crescimento Insulin-Like I/fisiologia , Receptores Adrenérgicos alfa 1/metabolismo , Animais , Linhagem Celular , Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Microscopia Confocal , Toxina Pertussis/farmacologia , Fosforilação , Marcadores de Fotoafinidade , Ensaio Radioligante , RatosRESUMO
beta-Estradiol induced alpha1b-adrenergic receptor desensitization in U373 MG cells stably expressing alpha1b-adrenoceptors, as evidenced by a reduction in the adrenergic-mediated Ca2+ mobilization; desensitization was associated with receptor phosphorylation and internalization. These effects of beta-estradiol were rapid (taking place during 15 min) and were blocked by the estrogen receptor antagonist ICI 182,780 (faslodex). Likewise, inhibitors of phosphoinositide 3-kinase [wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002)] and of protein kinase C [staurosporine, 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (Ro31-8220), and rottlerin] blocked the desensitization and phosphorylation of alpha1b-adrenoceptors induced by estradiol. The formation of a complex was suggested by coimmunoprecipitation assays. The regulatory and catalytic subunits of phosphoinositide 3-kinase (p85 and p110) and protein kinase C delta were associated with alpha1b-adrenoceptors in the absence of stimulus, and such association further increased in a dynamic fashion in response to beta-estradiol. In cells cotransfected with the estrogen receptor alpha and alpha1b-adrenoceptors, beta-estradiol induced phosphorylation, desensitization and internalization of the adrenergic receptors; pretreatment with ICI 182,780 inhibited these effects. Our data support the idea that estrogens modulate alpha1b-adrenergic action through estrogen receptor alpha.
Assuntos
Estrogênios/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Androstadienos/farmacologia , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Endocitose/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Fulvestranto , Humanos , Microscopia Confocal , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Receptor Cross-Talk/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/genética , Estaurosporina/farmacologia , Transfecção , WortmaninaRESUMO
In C9 cells, LPA (lysophosphatidic acid) induced inositol phosphate production, increased intracellular calcium concentration and inhibited adenylate cyclase activity. These responses were abolished in cells challenged with active phorbol esters. Action of phorbol esters was blocked by inhibitors of PKC (protein kinase C) and by its down-regulation. LPA1 receptor phosphorylation was observed in response to phorbol esters. The effect was rapid (t1/2 approximately 1 min), intense (2-fold) and sustained (at least 60 min). PKC inhibitors markedly decreased the LPA1 receptor phosphorylation induced by phorbol esters. LPA1 receptor tagged with the green fluorescent protein internalized in response to PKC activation. In addition, LPA and angiotensin II were also capable of inducing LPA1 receptor phosphorylation, showing that LPA1 receptor can be subjected to homologous and heterologous desensitization.