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2.
Nat Commun ; 11(1): 3133, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561764

RESUMO

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.


Assuntos
Carbono-Nitrogênio Ligases/genética , Proteínas de Escherichia coli/genética , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Proteínas Repressoras/genética , Sarcômeros/metabolismo , Animais , Animais Recém-Nascidos , Biotinilação/genética , Feminino , Técnicas de Introdução de Genes , Masculino , Redes e Vias Metabólicas , Camundongos Transgênicos , Modelos Animais , Miocárdio/citologia , Miocárdio/metabolismo , Estudo de Prova de Conceito , Mapas de Interação de Proteínas/fisiologia , Proteínas Quinases/genética , Proteostase/fisiologia , Músculo Quadríceps/citologia , Músculo Quadríceps/metabolismo , Sarcômeros/genética , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade
3.
Cancers (Basel) ; 12(6)2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32471029

RESUMO

Osteosarcoma (OS) is a primary malignant bone tumor and OS metastases are mostly found in the lung. The limited understanding of the biology of metastatic processes in OS limits the ability for effective treatment. Alterations to the metabolome and its transformation during metastasis aids the understanding of the mechanism and provides information on treatment and prognosis. The current study intended to identify metabolic alterations during OS progression by using a targeted gas chromatography mass spectrometry approach. Using a female OS cell line model, malignant and metastatic cells increased their energy metabolism compared to benign OS cells. The metastatic cell line showed a faster metabolic flux compared to the malignant cell line, leading to reduced metabolite pools. However, inhibiting both glycolysis and glutaminolysis resulted in a reduced proliferation. In contrast, malignant but non-metastatic OS cells showed a resistance to glycolytic inhibition but a strong dependency on glutamine as an energy source. Our in vivo metabolic approach hinted at a potential sex-dependent metabolic alteration in OS patients with lung metastases (LM), although this will require validation with larger sample sizes. In line with the in vitro results, we found that female LM patients showed a decreased central carbon metabolism compared to metastases from male patients.

4.
Nat Commun ; 11(1): 179, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924766

RESUMO

Hereditary autoinflammatory diseases are caused by gene mutations of the innate immune pathway, e.g. nucleotide receptor protein 3 (NLRP3). Here, we report a four-generation family with cold-induced urticarial rash, arthralgia, chills, headache and malaise associated with an autosomal-dominant inheritance. Genetic studies identify a substitution mutation in gene F12 (T859A, resulting in p.W268R) which encodes coagulation factor XII (FXII). Functional analysis reveals enhanced autocatalytic cleavage of the mutated protein and spontaneous FXII activation in patient plasma and in supernatant of transfected HEK293 cells expressing recombinant W268R-mutated proteins. Furthermore, we observe reduced plasma prekallikrein, cleaved high molecular weight kininogen and elevated plasma bradykinin. Neutrophils are identified as a local source of FXII. Interleukin-1ß (IL-1ß) is upregulated in lesional skin and mononuclear donor cells exposed to recombinant mutant proteins. Treatment with icatibant (bradykinin-B2-antagonist) or anakinra (interleukin-1-antagonist) reduces disease activity in patients. In conclusion, our findings provide a link between contact system activation and cytokine-mediated inflammation.


Assuntos
Temperatura Baixa/efeitos adversos , Fator XII/metabolismo , Doenças Hereditárias Autoinflamatórias/metabolismo , Adulto , Coagulação Sanguínea , Bradicinina/análogos & derivados , Bradicinina/sangue , Bradicinina/uso terapêutico , Fator XII/genética , Feminino , Células HEK293 , Doenças Hereditárias Autoinflamatórias/genética , Doenças Hereditárias Autoinflamatórias/patologia , Humanos , Mediadores da Inflamação , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/metabolismo , Cininogênio de Alto Peso Molecular/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neutrófilos , Linhagem , Fenótipo , Calicreína Plasmática/metabolismo , Proteínas Recombinantes , Pele/patologia
5.
Cell ; 178(1): 242-260.e29, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31155234

RESUMO

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.


Assuntos
Miocárdio/metabolismo , Biossíntese de Proteínas , Adolescente , Adulto , Idoso , Animais , Códon/genética , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , RNA Circular/genética , RNA Circular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ribossomos/genética , Ribossomos/metabolismo , Adulto Jovem
6.
iScience ; 13: 351-370, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30884312

RESUMO

CCAAT enhancer-binding protein beta (C/EBPß) is a pioneer transcription factor that specifies cell differentiation. C/EBPß is intrinsically unstructured, a molecular feature common to many proteins involved in signal processing and epigenetics. The structure of C/EBPß differs depending on alternative translation initiation and multiple post-translational modifications (PTM). Mutation of distinct PTM sites in C/EBPß alters protein interactions and cell differentiation, suggesting that a C/EBPß PTM indexing code determines epigenetic outcomes. Herein, we systematically explored the interactome of C/EBPß using an array technique based on spot-synthesized C/EBPß-derived linear tiling peptides with and without PTM, combined with mass spectrometric proteomic analysis of protein interactions. We identified interaction footprints of ∼1,300 proteins in nuclear extracts, many with chromatin modifying, chromatin remodeling, and RNA processing functions. The results suggest that C/EBPß acts as a multi-tasking molecular switchboard, integrating signal-dependent modifications and structural plasticity to orchestrate interactions with numerous protein complexes directing cell fate and function.

7.
Sci Rep ; 9(1): 3183, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30816308

RESUMO

Phosphatase and tensin homolog (PTEN) signalling might influence neuronal survival after brain ischemia. However, the influence of the less studied longer variant termed PTEN-L (or PTENα) has not been studied to date. Therefore, we examined the translational variant PTEN-L in the context of neuronal survival. We identified PTEN-L by proteomics in murine neuronal cultures and brain lysates and established a novel model to analyse PTEN or PTEN-L variants independently in vitro while avoiding overexpression. We found that PTEN-L, unlike PTEN, localises predominantly in the cytosol and translocates to the nucleus 10-20 minutes after glutamate stress. Genomic ablation of PTEN and PTEN-L increased neuronal susceptibility to oxygen-glucose deprivation. This effect was rescued by expression of either PTEN-L indicating that both PTEN isoforms might contribute to a neuroprotective response. However, in direct comparison, PTEN-L replaced neurons were protected against ischemic-like stress compared to neurons expressing PTEN. Neurons expressing strictly nuclear PTEN-L NLS showed increased vulnerability, indicating that nuclear PTEN-L alone is not sufficient in protecting against stress. We identified mutually exclusive binding partners of PTEN-L or PTEN in cytosolic or nuclear fractions, which were regulated after ischemic-like stress. GRB2-associated-binding protein 2, which is known to interact with phosphoinositol-3-kinase, was enriched specifically with PTEN-L in the cytosol in proximity to the plasma membrane and their interaction was lost after glutamate exposure. The present study revealed that PTEN and PTEN-L have distinct functions in response to stress and might be involved in different mechanisms of neuroprotection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Isquemia Encefálica/genética , Encéfalo/metabolismo , PTEN Fosfo-Hidrolase/genética , Acidente Vascular Cerebral/genética , Animais , Encéfalo/patologia , Isquemia Encefálica/patologia , Núcleo Celular/genética , Modelos Animais de Doenças , Proteína Adaptadora GRB2/genética , Regulação da Expressão Gênica/genética , Glucose/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Neuroproteção/genética , Oxigênio/metabolismo , Isoformas de Proteínas/genética , Proteômica/métodos , Transdução de Sinais/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia
8.
Acta Biomater ; 86: 171-184, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30616076

RESUMO

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.


Assuntos
Colágeno Tipo I/farmacologia , Consolidação da Fratura/efeitos dos fármacos , Osteotomia , Tecidos Suporte/química , Animais , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/patologia , Calcificação Fisiológica/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/ultraestrutura , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fator de Necrose Tumoral alfa/metabolismo , Microtomografia por Raio-X
9.
Cell ; 175(1): 239-253.e17, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30197081

RESUMO

Many disease-causing missense mutations affect intrinsically disordered regions (IDRs) of proteins, but the molecular mechanism of their pathogenicity is enigmatic. Here, we employ a peptide-based proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1, and CACNA1H) lead to an increased clathrin binding. All three mutations create dileucine motifs known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), the glucose transporter causative of GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. Mutant GLUT1 interacts with adaptor proteins (APs) in vitro, and knocking down AP-2 reverts the cellular mislocalization and restores glucose transport. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in structurally disordered cytosolic domains of transmembrane proteins. Thus, several mutations in disordered regions appear to cause "dileucineopathies."


Assuntos
Transportador de Glucose Tipo 1/fisiologia , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/fisiologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/fisiologia , Erros Inatos do Metabolismo dos Carboidratos , Clatrina/metabolismo , Citoplasma/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Proteínas Intrinsicamente Desordenadas/metabolismo , Leucina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte de Monossacarídeos/deficiência , Mutação/genética , Peptídeos , Ligação Proteica , Proteômica/métodos
10.
Mol Biol Cell ; 29(22): 2674-2686, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30156465

RESUMO

Lamellipodia are flat membrane protrusions formed during mesenchymal motion. Polymerization at the leading edge assembles the actin filament network and generates protrusion force. How this force is supported by the network and how the assembly rate is shared between protrusion and network retrograde flow determines the protrusion rate. We use mathematical modeling to understand experiments changing the F-actin density in lamellipodia of B16-F1 melanoma cells by modulation of Arp2/3 complex activity or knockout of the formins FMNL2 and FMNL3. Cells respond to a reduction of density with a decrease of protrusion velocity, an increase in the ratio of force to filament number, but constant network assembly rate. The relation between protrusion force and tension gradient in the F-actin network and the density dependency of friction, elasticity, and viscosity of the network explain the experimental observations. The formins act as filament nucleators and elongators with differential rates. Modulation of their activity suggests an effect on network assembly rate. Contrary to these expectations, the effect of changes in elongator composition is much weaker than the consequences of the density change. We conclude that the force acting on the leading edge membrane is the force required to drive F-actin network retrograde flow.


Assuntos
Citoesqueleto de Actina/metabolismo , Movimento Celular , Extensões da Superfície Celular/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Simulação por Computador , Melanoma Experimental/patologia , Camundongos , Modelos Biológicos , Pseudópodes/metabolismo
12.
Nat Genet ; 50(3): 349-354, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29403011

RESUMO

Primary aldosteronism, a common cause of severe hypertension 1 , features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II) 2 and 80 additional probands with unsolved early-onset primary aldosteronism. Eight probands had novel heterozygous variants in CLCN2, including two de novo mutations and four independent occurrences of a mutation encoding an identical p.Arg172Gln substitution; all relatives with early-onset primary aldosteronism carried the CLCN2 variant found in the proband. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. Channel opening depolarizes glomerulosa cells and induces expression of aldosterone synthase, the rate-limiting enzyme for aldosterone biosynthesis. Mutant channels show gain of function, with higher open probabilities at the glomerulosa resting potential. These findings for the first time demonstrate a role of anion channels in glomerulosa membrane potential determination, aldosterone production and hypertension. They establish the cause of a substantial fraction of early-onset primary aldosteronism.


Assuntos
Canais de Cloreto/genética , Hiperaldosteronismo/genética , Mutação , Adolescente , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Adulto , Sequência de Aminoácidos , Criança , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Humanos , Hiperaldosteronismo/patologia , Lactente , Masculino , Linhagem , Adulto Jovem
13.
Nucleic Acids Res ; 45(2): 938-950, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-27604873

RESUMO

Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.


Assuntos
Evolução Molecular , Inativação Gênica , MicroRNAs/genética , Interferência de RNA , Animais , Linhagem Celular , Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética
14.
Cell ; 160(4): 759-770, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25679765

RESUMO

Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.


Assuntos
Comunicação Autócrina , Neurônios/metabolismo , Dor/metabolismo , Receptores de GABA-B/metabolismo , Canais de Cátion TRPV/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Células Cultivadas , Retroalimentação , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
15.
Elife ; 32014 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-25392983

RESUMO

CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. In this study, we report that the CIDE-N protein Drep-2 is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Memória , Sinapses/metabolismo , Animais , Apoptose , Condicionamento Psicológico , Proteína do X Frágil de Retardo Mental/metabolismo , Espectrometria de Massas , Corpos Pedunculados/metabolismo , Mutação , Neurônios/citologia , Neurônios/metabolismo , Fenótipo , Densidade Pós-Sináptica/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Olfato
16.
J Clin Invest ; 124(8): 3419-30, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24960161

RESUMO

Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3' and 5' splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure.


Assuntos
Processamento Alternativo , Miocárdio/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Estudos de Coortes , Éxons , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Mutação , Miócitos Cardíacos/metabolismo , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , Sítios de Splice de RNA , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Seleção Genética , Spliceossomos/metabolismo
17.
EMBO J ; 33(16): 1751-66, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-24957527

RESUMO

The oocyte-to-embryo transition (OET) is thought to be mainly driven by post-transcriptional gene regulation. However, expression of both RNAs and proteins during the OET has not been comprehensively assayed. Furthermore, specific molecular mechanisms that regulate gene expression during OET are largely unknown. Here, we quantify and analyze transcriptome-wide, expression of mRNAs and thousands of proteins in Caenorhabditis elegans oocytes, 1-cell, and 2-cell embryos. This represents a first comprehensive gene expression atlas during the OET in animals. We discovered a first wave of degradation in which thousands of mRNAs are cleared shortly after fertilization. Sequence analysis revealed a statistically highly significant presence of a polyC motif in the 3' untranslated regions of most of these degraded mRNAs. Transgenic reporter assays demonstrated that this polyC motif is required and sufficient for mRNA degradation after fertilization. We show that orthologs of human polyC-binding protein specifically bind this motif. Our data suggest a mechanism in which the polyC motif and binding partners direct degradation of maternal mRNAs. Our data also indicate that endogenous siRNAs but not miRNAs promote mRNA clearance during the OET.


Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Oócitos/fisiologia , Estabilidade de RNA , Regiões 3' não Traduzidas , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Feminino , Fertilização/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs , Poli C , Proteoma/metabolismo , RNA Mensageiro Estocado/metabolismo , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
18.
Cell Rep ; 6(3): 565-77, 2014 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-24462290

RESUMO

Spatiotemporal control of gene expression is crucial for development and subject to evolutionary changes. Although proteins are the final product of most genes, the developmental proteome of an animal has not yet been comprehensively defined, and the correlation between mRNA and protein abundance during development is largely unknown. Here, we globally measured and compared protein and mRNA expression changes during the life cycle of the nematodes C. elegans and C. briggsae, separated by ~30 million years of evolution. We observed that developmental mRNA and protein changes were highly conserved to a surprisingly similar degree but were poorly correlated within a species, suggesting important and widespread posttranscriptional regulation. Posttranscriptional control was particularly well conserved if mRNA fold changes were buffered on the protein level, indicating a predominant repressive function. Finally, among divergently expressed genes, we identified insulin signaling, a pathway involved in lifespan determination, as a putative target of adaptive evolution.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Evolução Biológica , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/metabolismo , Análise por Conglomerados , Embrião não Mamífero/metabolismo , Éxons/genética , Perfilação da Expressão Gênica , Marcação por Isótopo , Larva/genética , Larva/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Transcrição Genética
19.
PLoS One ; 7(6): e40000, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22768194

RESUMO

Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease.


Assuntos
Ensaios Enzimáticos/métodos , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Peixe-Zebra/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Embrião não Mamífero/enzimologia , Isoenzimas/química , Dados de Sequência Molecular , Proteínas Mutantes/química , Fosforilação , Proteína Quinase C/química , Especificidade por Substrato , Compostos de Sulfidrila/metabolismo , Peixe-Zebra/embriologia
20.
Methods Mol Biol ; 893: 175-99, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22665302

RESUMO

Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex biological samples. Improved instrumentation, quantification strategies and data analysis tools now enable protein analysis on a genome-wide scale. Particularly, quantification based on stable isotope labeling with amino acids (SILAC) has emerged as a robust, reliable and simple method for accurate large-scale protein quantification. The spectrum of applications ranges from bacteria and eukaryotic cell culture systems to multicellular organisms. Here, we provide a step-by-step protocol on how to plan and perform large-scale quantitative proteome analysis using SILAC, from sample preparation to final data analysis.


Assuntos
Proteoma/metabolismo , Aminoácidos , Animais , Cromatografia de Fase Reversa , Interpretação Estatística de Dados , Eletroforese em Gel de Poliacrilamida , Humanos , Marcação por Isótopo , Mapeamento de Peptídeos , Proteólise , Proteoma/química , Proteoma/isolamento & purificação , Projetos de Pesquisa , Software , Espectrometria de Massas em Tandem , Tripsina/química
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