Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Plant Physiol ; 192(4): 3049-3068, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37073492

RESUMO

NLP7 (NIN-LIKE-PROTEIN 7) is the major transcriptional factor responsible for the primary nitrate response (PNR), but the role of its homolog, NLP6, in nitrogen signaling and the interplay between NLP6 and NLP7 remain to be elucidated. In this study, we show that, like NLP7, nuclear localization of NLP6 via a nuclear retention mechanism is nitrate dependent, but nucleocytosolic shuttling of both NLP6 and NLP7 is independent of each other. Compared with single mutants, the nlp6nlp7 double mutant displays a synergistic growth retardation phenotype in response to nitrate. The transcriptome analysis of the PNR showed that NLP6 and NLP7 govern ∼50% of nitrate-induced genes, with cluster analysis highlighting 2 distinct patterns. In the A1 cluster, NLP7 plays the major role, whereas in the A2 cluster, NLP6 and NLP7 are partially functionally redundant. Interestingly, comparing the growth phenotype and PNR under high- and low-nitrate conditions demonstrated that NLP6 and NLP7 exert a more dominant role in the response to high nitrate. Apart from nitrate signaling, NLP6 and NLP7 also participated in high ammonium conditions. Growth phenotypes and transcriptome data revealed that NLP6 and NLP7 are completely functionally redundant and may act as repressors in response to ammonium. Other NLP family members also participated in the PNR, with NLP2 and NLP7 acting as broader regulators and NLP4, -5, -6, and -8 regulating PNR in a gene-dependent manner. Thus, our findings indicate that multiple modes of interplay exist between NLP6 and NLP7 that differ depending on nitrogen sources and gene clusters.


Assuntos
Compostos de Amônio , Proteínas de Arabidopsis , Arabidopsis , Nitratos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição/metabolismo , Nitrogênio/metabolismo , Compostos de Amônio/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo
2.
Mol Cell ; 61(2): 305-14, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26799765

RESUMO

The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.


Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/metabolismo , Humanos , Mamíferos , Espectrometria de Massas , Dados de Sequência Molecular , Biblioteca de Peptídeos , Fosforilação , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequências Repetitivas de Aminoácidos , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
3.
PLoS Genet ; 11(10): e1005574, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26485514

RESUMO

Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.


Assuntos
Proteínas de Transporte/genética , Dineínas/genética , Larva/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Células Fotorreceptoras de Vertebrados , Retina/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Animais , Transporte Biológico/genética , Cílios/genética , Células HEK293 , Humanos , Larva/crescimento & desenvolvimento , Neurogênese/genética , Proteômica , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
4.
Mol Cell Proteomics ; 13(5): 1382-91, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24563533

RESUMO

Analyzing the molecular architecture of native multiprotein complexes via biochemical methods has so far been difficult and error prone. Protein complex isolation by affinity purification can define the protein repertoire of a given complex, yet, it remains difficult to gain knowledge of its substructure or modular composition. Here, we introduce SDS concentration gradient induced decomposition of protein complexes coupled to quantitative mass spectrometry and in silico elution profile distance analysis. By applying this new method to a cellular transport module, the IFT/lebercilin complex, we demonstrate its ability to determine modular composition as well as sensitively detect known and novel complex components. We show that the IFT/lebercilin complex can be separated into at least five submodules, the IFT complex A, the IFT complex B, the 14-3-3 protein complex and the CTLH complex, as well as the dynein light chain complex. Furthermore, we identify the protein TULP3 as a potential new member of the IFT complex A and showed that several proteins, classified as IFT complex B-associated, are integral parts of this complex. To further demonstrate EPASIS general applicability, we analyzed the modular substructure of two additional complexes, that of B-RAF and of 14-3-3-ε. The results show, that EPASIS provides a robust as well as sensitive strategy to dissect the substructure of large multiprotein complexes in a highly time- as well as cost-effective manner.


Assuntos
Espectrometria de Massas/métodos , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/isolamento & purificação , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas/economia , Proteínas/metabolismo , Proteômica , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/isolamento & purificação , Dodecilsulfato de Sódio
5.
Plant J ; 57(3): 426-35, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18826430

RESUMO

Nitrate is an essential nutrient, and is involved in many adaptive responses of plants, such as localized proliferation of roots, flowering or stomatal movements. How such nitrate-specific mechanisms are regulated at the molecular level is poorly understood. Although the Arabidopsis ANR1 transcription factor appears to control stimulation of lateral root elongation in response to nitrate, no regulators of nitrate assimilation have so far been identified in higher plants. Legume-specific symbiotic nitrogen fixation is under the control of the putative transcription factor, NIN, in Lotus japonicus. Recently, the algal homologue NIT2 was found to regulate nitrate assimilation. Here we report that Arabidopsis thaliana NIN-like protein 7 (NLP7) knockout mutants constitutively show several features of nitrogen-starved plants, and that they are tolerant to drought stress. We show that nlp7 mutants are impaired in transduction of the nitrate signal, and that the NLP7 expression pattern is consistent with a function of NLP7 in the sensing of nitrogen. Translational fusions with GFP showed a nuclear localization for the NLP7 putative transcription factor. We propose NLP7 as an important element of the nitrate signal transduction pathway and as a new regulatory protein specific for nitrogen assimilation in non-nodulating plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Nitratos/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Secas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mutação , Nitrogênio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA de Plantas/genética , Estresse Fisiológico , Fatores de Transcrição/genética
6.
PLoS One ; 14(5): e0216705, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31095607

RESUMO

The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse, zebrafish and nematode) and techniques. For example, we show that OSCP1, which has previously been implicated in two distinct non-ciliary processes, causes ciliogenic and ciliopathy-associated tissue phenotypes when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 956 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.


Assuntos
Cílios/genética , Genômica , Animais , Teorema de Bayes , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Anotação de Sequência Molecular , Fenótipo , Reprodutibilidade dos Testes , Células Receptoras Sensoriais/metabolismo , Peixe-Zebra/genética
7.
Cell Signal ; 28(9): 1432-1439, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27345148

RESUMO

The BRAF proto-oncogene serine/threonine-protein kinase, known as BRAF, belongs to the RAF kinase family. It regulates the MAPK/ERK signalling pathway affecting several cellular processes such as growth, survival, differentiation, and cellular transformation. BRAF is mutated in ~8% of all human cancers with the V600E mutation constituting ~90% of mutations. Here, we have used quantitative mass spectrometry to map and compare phosphorylation site patterns between BRAF and BRAF V600E. We identified sites that are shared as well as several quantitative differences in phosphorylation abundance. The highest difference is phosphorylation of S614 in the activation loop which is ~5fold enhanced in BRAF V600E. Mutation of S614 increases the kinase activity of both BRAF and BRAF V600E and the transforming ability of BRAF V600E. The phosphorylation of S614 is mitogen inducible and the result of autophosphorylation. These data suggest that phosphorylation at this site is inhibitory, and part of the physiological shut-down mechanism of BRAF signalling.


Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Fosfosserina/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação/genética , Fosforilação , Ligação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas B-raf/química , Ratos
8.
Nat Commun ; 7: 11491, 2016 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-27173435

RESUMO

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.


Assuntos
Cílios/metabolismo , Ciliopatias/genética , Nanismo/genética , Hipotonia Muscular/genética , Mapas de Interação de Proteínas , Proteínas/metabolismo , Coluna Vertebral/anormalidades , Transporte Biológico/fisiologia , Cromatografia de Afinidade/métodos , Ciliopatias/patologia , Ciliopatias/terapia , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Nanismo/patologia , Nanismo/terapia , Fibroblastos , Células HEK293 , Humanos , Espectrometria de Massas , Terapia de Alvo Molecular/métodos , Hipotonia Muscular/patologia , Hipotonia Muscular/terapia , Mapeamento de Interação de Proteínas/métodos , Proteínas/genética , Proteínas/isolamento & purificação , Proteômica/métodos , Coluna Vertebral/patologia , Análise de Sistemas
9.
Methods Mol Biol ; 1188: 177-90, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25059612

RESUMO

Pull-downs based on tag fusion proteins as well as immunoprecipitations (IP) are widely used methods to analyze protein interactions. Selectivity and specificity of both methods are compromised by nonspecific binding to the capture agent or carrier beads thereby generating false positives. Here, we provide a method combining stable isotope labeling of amino acids in cell culture (SILAC) with affinity purification, coupled to quantitative tandem mass spectrometry. It permits the analysis of protein interactions with high sensitivity, while being able to discriminate contaminants and nonspecific binders. Besides pruning out contaminants, high-resolution MS data combined with quantitative proteomics software allow the comparative analysis of protein interaction patterns of different protein variants, for example mutated versus normal protein variant or of regulatory changes in a given protein complex due to different states of activity.


Assuntos
Aminoácidos/química , Marcação por Isótopo/métodos , Proteínas/química , Proteínas/metabolismo , Proteômica/métodos , Métodos Analíticos de Preparação de Amostras , Morte Celular , Células HEK293 , Humanos , Espectrometria de Massas , Proteínas/isolamento & purificação
10.
J Clin Invest ; 121(6): 2169-80, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21606596

RESUMO

The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.


Assuntos
Proteínas do Olho/fisiologia , Amaurose Congênita de Leber/fisiopatologia , Proteínas Associadas aos Microtúbulos/fisiologia , Cílio Conector dos Fotorreceptores/fisiologia , Transporte Proteico/fisiologia , Animais , Arrestinas/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Proteínas do Olho/genética , Humanos , Amaurose Congênita de Leber/genética , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Complexos Multiproteicos , Opsinas/metabolismo , Mapeamento de Interação de Proteínas , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/fisiologia , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/patologia , Visão Ocular/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA