RESUMO
Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Sfmbt. The spacer region of Pho, separated from the DNA-binding domain by a long flexible linker, forms a tight complex with the four malignant brain tumor (4MBT) domain of Sfmbt. The highly conserved spacer region of the human Pho ortholog YY1 binds three of the four human 4MBT domain proteins in an analogous manner but with lower affinity. Comparison of the Drosophila Pho:Sfmbt and human YY1:MBTD1 complex structures provides a molecular explanation for the lower affinity of YY1 for human 4MBT domain proteins. Structure-guided mutations that disrupt the interaction between Pho and Sfmbt abolish formation of a ternary Sfmbt:Pho:DNA complex in vitro and repression of developmental regulator genes in Drosophila. PRE tethering of Sfmbt by Pho is therefore essential for Polycomb repression in Drosophila. Our results support a model where DNA tethering of Sfmbt by Pho and multivalent interactions of Sfmbt with histone modifications and other PcG proteins create a hub for PcG protein complex assembly at PREs.
Assuntos
Proteínas de Drosophila/química , Drosophila melanogaster , Regulação da Expressão Gênica no Desenvolvimento , Modelos Moleculares , Proteínas do Grupo Polycomb/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Dados de Sequência Molecular , Mutação/genética , Proteínas do Grupo Polycomb/química , Proteínas do Grupo Polycomb/genética , Ligação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Fator de Transcrição YY1/química , Fator de Transcrição YY1/metabolismoRESUMO
Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscriptional regulatory mechanism responding to stress and inflammation. This mechanism is triggered by external stimuli that activate p38 MAPK. In turn, p38 MAPK upmodulates canonical 3' end processing components and phosphorylates the RNA-binding proteins FBP2 and FBP3, which inhibit 3' end processing of mRNAs, such as prothrombin mRNA, that bear a defined upstream sequence element (USE) in their 3'UTRs. Upon phosphorylation, FBP2 and FBP3 dissociate from the USE, making it accessible to proteins that stimulate 3' end processing. We provide in vivo evidence suggesting the importance of this mechanism in inflammatory hypercoagulation and tumor invasion. Regulated 3' end processing thus emerges as a key mechanism of gene regulation with broad biological and medical implications.
Assuntos
Regulação Enzimológica da Expressão Gênica , Protrombina/metabolismo , Processamento de Terminações 3' de RNA , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Invasividade Neoplásica , Polímeros/metabolismo , Ligação Proteica , Protrombina/genética , RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. The method can be easily implemented on any LC-MS/MS system without modification to hardware or software and at no additional cost.
Assuntos
Dimetil Sulfóxido/química , Peptídeos/análise , Proteômica/métodos , Solventes/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Peptídeos/química , Proteômica/normas , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/normas , Espectrometria de Massas em Tandem/normasRESUMO
Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1-4). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/fisiologia , Alelos , Animais , Biocatálise , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inativação Gênica , Genes Homeobox/genética , Genes de Insetos/genética , Teste de Complementação Genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Nucleossomos/química , Nucleossomos/metabolismo , Complexo Repressor Polycomb 1 , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/genéticaRESUMO
RBM5/Luca-15/H37 is a gene frequently inactivated in lung cancers and overexpressed in breast tumors. Its protein product has been detected in prespliceosomal complexes and modulates cell proliferation and Fas-mediated apoptosis. We report that RBM5 is a component of complexes involved in 3' splice site recognition and regulates alternative splicing of apoptosis-related genes, including the Fas receptor, switching between isoforms with antagonistic functions in programmed cell death. In contrast with classical mechanisms of splicing regulation, RBM5 does not affect early events of splice site recognition that lead to Fas exon 6 definition. Instead, RBM5 inhibits the transition between prespliceosomal complexes assembled around exon 6 to mature spliceosomes assembled on the flanking introns and promotes sequence-specific pairing of the distal splice sites. An OCRE domain important for RBM5 function contacts components of the U4/5/6 tri-snRNP, consistent with the idea that RBM5 modulates splice site pairing after prespliceosome assembly and exon definition.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sítios de Splice de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Receptor fas/genética , Processamento Alternativo , Apoptose/genética , Sequência de Bases , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Éxons , Genes Supressores de Tumor , Células HeLa , Humanos , Técnicas In Vitro , Íntrons , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AF , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Electrospray ionization is today the most widely used ionization technique in chemical and bio-chemical analysis. Interfaced with a mass spectrometer it allows to investigate the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which enables even the investigation of large non-covalent protein complexes. Its high ionization efficiency profoundly changed bio-molecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion. This review article focusses mainly on the exploration of the underlying ionization mechanism. Some ionization characteristics are discussed which are related to this mechanism. Typical spectra of peptides, proteins and non-covalent complexes are shown and the quantitative character of spectra is highlighted. Finally the possibilities and limitations in measuring the association constant of bivalent non-covalent complexes are described.
RESUMO
Electrospray ionization is today the most widely used ionization technique in chemical and biochemical analysis. Interfaced with a mass spectrometer it allows the investigation of the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which thus enables even the investigation of large noncovalent protein complexes. Its high ionization efficiency profoundly changed biomolecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion. This review article focuses mainly on the exploration of the underlying ionization mechanism. Some ionization characteristics are discussed that are related to this mechanism. Typical spectra of peptides, proteins, and noncovalent complexes are shown and the quantitative character of spectra is highlighted. Finally the possibilities and limitations in measuring the association constant of bivalent noncovalent complexes are described.
Assuntos
Espectrometria de Massas por Ionização por Electrospray/métodos , Modelos Químicos , Fragmentos de Peptídeos/química , Ligação Proteica , Proteínas/químicaRESUMO
Asymmetric distribution of cellular components underlies many biological processes, and the localization of mRNAs within domains of the cytoplasm is one important mechanism of establishing and maintaining cellular asymmetry. mRNA localization often involves assembly of large ribonucleoproteins (RNPs) in the cytoplasm. Using an RNA affinity chromatography approach, we investigated localization RNP formation on the vegetal localization element (VLE) of the mRNA encoding Vg1, a Xenopus TGF-beta family member. We identified 40LoVe, an hnRNP D family protein, as a specific VLE binding protein from Xenopus oocytes. Interaction of 40LoVe with the VLE strictly correlates with the ability of the RNA to localize, and antibodies against 40LoVe inhibit vegetal localization in vivo in oocytes. Our results associate an hnRNP D protein with mRNA localization and have implications for several functions mediated by this important protein family.
Assuntos
Glicoproteínas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Oogênese/fisiologia , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade/métodos , Glicoproteínas/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/classificação , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas/classificação , Ribonucleoproteínas Nucleares Heterogêneas/genética , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/metabolismo , Filogenia , Ligação Proteica , Alinhamento de Sequência , Fator de Crescimento Transformador beta/genética , Proteínas de Xenopus/classificação , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Eukaryotic mRNAs possess a poly(A) tail that enhances translation via the (7)mGpppN cap structure or internal ribosome entry sequences (IRESs). Here we address the question of how cellular IRESs recruit the ribosome and how recruitment is augmented by the poly(A) tail. We show that the poly(A) tail enhances 48S complex assembly by the c-myc IRES. Remarkably, this process is independent of the poly(A) binding protein (PABP). Purification of native 48S initiation complexes assembled on c-myc IRES mRNAs and quantitative label-free analysis by liquid chromatography and mass spectrometry directly identify eIFs 2, 3, 4A, 4B, 4GI, and 5 as components of the c-myc IRES 48S initiation complex. Our results demonstrate for the first time that the poly(A) tail augments the initiation step of cellular IRES-driven translation and implicate a distinct subset of translation initiation factors in this process. The mechanistic distinctions from cap-dependent translation may allow specific translational control of the c-myc mRNA and possibly other cellular mRNAs that initiate translation via IRESs.
Assuntos
Iniciação Traducional da Cadeia Peptídica , Poli A/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Sistema Livre de Células , Células HeLa , Humanos , Capuzes de RNA/metabolismoRESUMO
Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kbeta, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinasebeta (PI3Kbeta), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.
Assuntos
Endocitose , Fosfatidilinositóis/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/metabolismo , Catálise , Compartimento Celular , Cromatografia de Afinidade , Regulação para Baixo/genética , Ativação Enzimática , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Transporte Proteico , Soro , Transferrina/metabolismo , Proteínas rab5 de Ligação ao GTP/isolamento & purificaçãoRESUMO
In metazoa, the nuclear envelope breaks down and reforms during each cell cycle. Nuclear pore complexes (NPCs), which serve as channels for transport between the nucleus and cytoplasm, assemble into the reforming nuclear envelope in a sequential process involving association of a subset of NPC proteins, nucleoporins, with chromatin followed by the formation of a closed nuclear envelope fenestrated by NPCs. How chromatin recruitment of nucleoporins and NPC assembly are regulated is unknown. Here we demonstrate that RanGTP production is required to dissociate nucleoporins Nup107, Nup153 and Nup358 from Importin beta, to target them to chromatin and to induce association between separate NPC subcomplexes. Additionally, either an excess of RanGTP or removal of Importin beta induces formation of NPC-containing membrane structures--annulate lamellae--both in vitro in the absence of chromatin and in vivo. Annulate lamellae formation is strongly and specifically inhibited by an excess of Importin beta. The data demonstrate that RanGTP triggers distinct steps of NPC assembly, and suggest a mechanism for the spatial restriction of NPC assembly to the surface of chromatin.
Assuntos
Cromatina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Extratos Celulares , Feminino , Masculino , Mutação , Oócitos , Fosforilação , Transporte Proteico , Interferência de RNA , Espermatozoides , Xenopus laevis , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/genéticaRESUMO
Proteomics has enabled the direct investigation of biological material, at first through the analysis of individual proteins, then of lysates from cell cultures, and finally of extracts from tissues and biopsies from entire organisms. Its latest manifestation - quantitative proteomics - allows deeper insight into biological systems. This article reviews the different methods used to extract quantitative information from mass spectra. It follows the technical developments aimed toward global proteomics, the attempt to characterize every expressed protein in a cell by at least one peptide. When applications of the technology are discussed, the focus is placed on yeast biology. In particular, differential quantitative proteomics, the comparison between an experiment and its control, is very discriminating for proteins involved in the process being studied. When trying to understand biological processes on a molecular level, differential quantitative proteomics tends to give a clearer picture than global transcription analyses. As a result, MS has become an even more indispensable tool for biochemically motivated biological research.
Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Saccharomyces cerevisiae/química , HumanosRESUMO
BACKGROUND: GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. RESULTS: Here we identify a complex that is required for the conversion of aster-like to spindle-like structures. The complex consists of two characterized MAPs (TPX2, XMAP215), a plus end-directed motor (Eg5), a mitotic kinase (Aurora A), and HURP, a protein associated with hepatocellular carcinoma. Formation and function of the complex is dependent on Aurora A activity. HURP protein was further characterized and shown to bind microtubules and affect their organization both in vitro and in vivo. In egg extract, anti-HURP antibodies disrupt the formation of both Ran-dependent and chromatin and centrosome-induced spindles. HURP is also required for the proper formation and function of mitotic spindles in HeLa cells. CONCLUSIONS: HURP is a new and essential component of the mitotic apparatus. HURP acts as part of a multicomponent complex that affects the growth or stability of spindle MTs and is required for spindle MT organization.
Assuntos
Proteínas de Neoplasias/fisiologia , Fuso Acromático/metabolismo , Animais , Aurora Quinases , Segregação de Cromossomos , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Complexos Multiproteicos , Proteínas de Neoplasias/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Xenopus , Proteínas de Xenopus/fisiologia , Proteína ran de Ligação ao GTP/metabolismoRESUMO
In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export. The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p. Whether a complex with similar functions assembles in metazoa has not yet been established. Here we report that Drosophila melanogaster THO consists of THO2, HPR1 and three proteins, THOC5-THOC7, which have no orthologs in budding yeast. Gene expression profiling in cells depleted of THO components revealed that <20% of the transcriptome was regulated by THO. Nonetheless, export of heat-shock mRNAs under heat stress was strictly dependent on THO function. Notably, 8% of upregulated genes encode proteins involved in DNA repair. Thus, although THO function seems to be conserved, the vast majority of mRNAs are transcribed and exported independently of THO in D. melanogaster.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , RNA Mensageiro/genética , Fatores de Transcrição , Transporte Ativo do Núcleo Celular , Animais , Reparo do DNA , Perfilação da Expressão Gênica , Genoma , Ligação ProteicaRESUMO
Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) alpha, Impbeta, and Imp7. While Impalpha and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impbeta specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.
Assuntos
Microtúbulos/fisiologia , Oócitos/fisiologia , Xenopus laevis/fisiologia , Proteína ran de Ligação ao GTP/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Meiose , Microtúbulos/ultraestrutura , Oócitos/citologia , Transporte Proteico , Interferência de RNA , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Proteína ran de Ligação ao GTP/genéticaRESUMO
Neurofibromin is the protein product of the tumor suppressor gene NF1, alterations of which are responsible for the pathogenesis of the common disorder Neurofibromatosis type I (NF1). The only well-characterized function of neurofibromin is its RasGAP activity, contained in the central GAP related domain (GRD). By solving the crystal structure of a 31 kDa fragment at the C-terminal end of the GRD we have recently identified a novel bipartite lipid-binding module composed of a Sec14 homologous and a previously undetected pleckstrin homology (PH)-like domain. Using lipid exchange assays along with mass spectrometry we show here that the Sec14-like portion binds to 1-(3-sn-phosphatidyl)-sn-glycerol (PtdGro), (3-sn-phosphatidyl)-ethanolamine (PtdEtn) and -choline (PtdCho) and to a minor extent to (3-sn-phosphatidyl)-l-serine (PtdSer) and 1-(3-sn-phosphatidyl)-d-myo-inositol (PtdIns). Phosphorylated PtdIns (PtdInsPs) are not detected as binders in the mass spectrometry assay, but their soluble inositol-phosphate headgroups and related compounds can inhibit the lipid exchange reaction. We also present here the crystal structure of this module with the Sec14 portion bound to a cellular glycerophospholipid ligand. Our structure has model character for the substrate-bound form of yeast Sec14p, of which only detergent bound structures are available so far. To assess potential regulation of the lipid exchange reaction in detail, we present a novel strategy using nanospray mass spectrometry. Ion intensities of initial phospholipids and exchanged deuterated analogues bound by the protein module allow the quantitative analysis of differences in the exchange activity under various conditions.
Assuntos
Glicerofosfolipídeos/metabolismo , Espectrometria de Massas/métodos , Neurofibromina 1/metabolismo , Estrutura Terciária de Proteína , Cristalização , Cristalografia por Raios X , Lipídeos , Neurofibromina 1/genética , Neurofibromina 1/isolamento & purificação , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.
Assuntos
Endocitose/fisiologia , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas rab5 de Ligação ao GTP/fisiologia , Adenoviridae/genética , Androstadienos/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Clonagem Molecular , Cães , Regulação para Baixo , Endossomos/metabolismo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/química , Humanos , Fosfatos de Inositol/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipossomos/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Vídeo , Movimento , Células NIH 3T3 , Pinocitose , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Proteínas Recombinantes/química , Fatores de Tempo , WortmaninaRESUMO
The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi) to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.
Assuntos
Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Proteínas/isolamento & purificação , Proteoma/química , Proteômica/métodos , Animais , Células Cultivadas , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Células Eucarióticas/química , Células Eucarióticas/fisiologia , Substâncias Macromoleculares , Complexos Multiproteicos , Proteínas/genética , Proteoma/genética , Interferência de RNA , RNA de Cadeia Dupla/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
We present a method for the systematic identification of picogram quantities of new lipids in total extracts of tissues and fluids. It relies on the modularity of lipid structures and applies all-ions fragmentation LC-MS/MS and Arcadiate software to recognize individual modules originating from the same lipid precursor of known or assumed structure. In this way it alleviates the need to recognize and fragment very low abundant precursors of novel molecules in complex lipid extracts. In a single analysis of rat kidney extract the method identified 58 known and discovered 74 novel endogenous endocannabinoids and endocannabinoid-related molecules, including a novel class of N-acylaspartates that inhibit Hedgehog signaling while having no impact on endocannabinoid receptors.
Assuntos
Cromatografia Líquida/métodos , Lipídeos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Endocanabinoides/análise , Rim/química , Ratos , SoftwareRESUMO
We cloned Xenopus laevis CRISP, XCRISP, a homologue of the mammalian family of cysteine-rich secretory proteins (CRISPs), which has been previously identified as a Wnt3a/noggin responsive gene in an expression screen [Mech. Dev. 87 (1999) 21]. We detected XCRISP expression exclusively in the hatching gland. XCRISP enters the secretory pathway and accumulates on the surface of presumptive hatching gland cells. Overexpression studies of XCRISP and XCRISP-mutants show that XCRISP induces premature hatching of embryos preceded by degradation of the vitelline envelope. A deletion mutant that lacks a 35 amino acid domain even accelerates hatching, while further deletion of the carboxy-terminus reverses these effects. From our studies, we conclude that XCRISP is sufficient to induce degradation of vitelline envelopes and that this activity maps to the most C-terminal amino acids, while the adjacent domain regulates XCRISP activity.