RESUMEN
The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.
Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Nucléolo Celular/efectos de los fármacos , Ciclopentanos/farmacología , Genes p53/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirimidinas/farmacología , Ubiquitinas/antagonistas & inhibidores , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans , Línea Celular/efectos de los fármacos , Humanos , Células MCF-7/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Ratones Mutantes , Proteína NEDD8 , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/genética , Ribosomas/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus. This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus. Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p-Nup159p-Nsp1p complex. In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome. Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export. Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport. Thus, the Nup82p-Nup159p-Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Proteínas de Unión al Calcio , Proteínas de Complejo Poro Nuclear/genética , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Citoplasma/química , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hibridación in Situ , Microscopía Electrónica , Mutagénesis/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Ribosómico/análisis , Ribonucleoproteínas/análisis , Ribonucleoproteínas/metabolismo , Ribosomas/química , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructuraRESUMEN
Eukaryotic ribosome maturation depends on a set of well ordered processing steps. Here we describe the functional characterization of yeast Nog2p (Ynr053cp), a highly conserved nuclear protein. Nog2p contains a putative GTP-binding site, which is essential in vivo. Kinetic and steady-state measurements of the levels of pre-rRNAs in Nog2p-depleted cells showed a defect in 5.8S and 25S maturation and a concomitant increase in the levels of both 27SB(S) and 7S(S) precursors. We found Nog2p physically associated with large pre-60S complexes highly enriched in the 27SB and 7S rRNA precursors. These complexes contained, besides a subset of ribosomal proteins, at least two additional factors, Nog1p, another putative GTP-binding protein, and Rlp24p (Ylr009wp), which belongs to the Rpl24e family of archaeal and eukaryotic ribosomal proteins. In the absence of Nog2p, the pre-60S ribosomal complexes left the nucleolus, but were retained in the nucleoplasm. These results suggest that transient, possibly GTP-dependent association of Nog2p with the pre-ribosomes might trigger late rRNA maturation steps in ribosomal large subunit biogenesis.
Asunto(s)
GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Transporte Activo de Núcleo Celular , Empalme Alternativo , Secuencia de Aminoácidos , Sitios de Unión , Northern Blotting , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , ADN Complementario/metabolismo , GTP Fosfohidrolasas/química , Proteínas de Unión al GTP/metabolismo , Genotipo , Glucosa/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Hibridación in Situ , Cinética , Proteínas Luminiscentes/metabolismo , Espectrometría de Masas , Microscopía Electrónica , Microscopía Fluorescente , Modelos Genéticos , Datos de Secuencia Molecular , Plásmidos/metabolismo , Polirribosomas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Numerous non-ribosomal trans-acting factors involved in pre-ribosomal RNA processing have been characterized, but none of them is specifically required for the last cytoplasmic steps of 18S rRNA maturation. Here we demonstrate that Rio1p/Rrp10p is such a factor. Previous studies showed that the RIO1 gene is essential for cell viability and conserved from archaebacteria to man. We isolated a RIO1 mutant in a screen for mutations synthetically lethal with a mutant allele of GAR1, an essential gene required for 18S rRNA production and rRNA pseudouridylation. We show that RIO1 encodes a cytoplasmic non-ribosomal protein, and that depletion of Rio1p blocks 18S rRNA production leading to 20S pre-rRNA accumulation. In situ hybridization reveals that, in Rio1p depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. This strongly suggests that Rio1p is involved in the cytoplasmic cleavage of 20S pre-rRNA at site D, producing mature 18S rRNA. Thus, Rio1p has been renamed Rrp10p (ribosomal RNA processing #10). Rio1p/Rrp10p is the first non-ribosomal factor characterized specifically required for 20S pre-rRNA processing.
Asunto(s)
Proteínas Fúngicas/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Hongos/biosíntesis , ARN Ribosómico 18S/biosíntesis , Ribonucleoproteínas Nucleolares Pequeñas , Proteínas de Saccharomyces cerevisiae , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Proteínas Nucleares/genética , Biosíntesis de Proteínas , Ribosomas/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation. High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus. rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization. Although morphologically different, the mininucleoli observed in cells transcribing rDNA with RNA polymerase II also contain a fibrillar subregion of analogous function, in addition to a dense core of unknown nature. Upon repression of rDNA transcription in this strain or in an RNA polymerase I thermosensitive mutant, the nucleolar structure falls apart (in a reversible manner), and nucleolar constituents partially relocate to the nucleoplasm, indicating that rRNA is a primary determinant for the assembly of the nucleolus.
Asunto(s)
Proteínas Bacterianas , Nucléolo Celular/ultraestructura , Mutación , Región Organizadora del Nucléolo/ultraestructura , Ribonucleoproteínas Nucleolares Pequeñas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/ultraestructura , Sitios de Unión , ADN de Hongos , ADN Ribosómico , Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Región Organizadora del Nucléolo/metabolismo , Región Organizadora del Nucléolo/fisiología , Plásmidos , Procesamiento Postranscripcional del ARN , ARN de Hongos , ARN Ribosómico , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia niTN-5B1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells. Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)(2)(Mannose)(3)Fucose, although hybrid and complex N-glycosylations were also detected. In High-Five cells, the peptide was found to be modified with a wide variety of hybrid and complex sugars in addition to paucomanosidic oligosaccharides. Most glycans had one or two fucose residues bound through alpha1,3 and alpha1,6 linkages to the innermost GlcNAc. On the basis of these results and on the structure of an eight-cysteine domain from fibrillin-1, we present a model of glycosylated CR3 and discuss the role of glycosylation in eight-cysteine domain protein-protein interactions.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cisteína/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Polisacáridos/genética , Polisacáridos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Conformación de Carbohidratos , Proteínas Portadoras/química , Línea Celular , Cromatografía Líquida de Alta Presión , Secuencia Conservada , Cisteína/química , Cisteína/genética , Glicosilación , Humanos , Proteínas de Unión a TGF-beta Latente , Datos de Secuencia Molecular , Oligosacáridos/química , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Polisacáridos/química , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Spodoptera/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Recent molecular genetics studies in humans and mice showed that transforming growth factor-beta (TGF-beta) is involved in vasculogenesis and maintenance of blood vessel integrity. These results confirm earlier in vitro studies demonstrating generation of active TGF-beta when endothelial cells are cocultured with smooth muscle cells or pericytes. TGF-beta is secreted as a latent, inactive complex and becomes active only when released. Latent TGF-beta binds covalently to proteins (LTBP) that target it to the extracellular matrix. Thus, the latency of TGF-beta is essential to the regulation of the bioavailability and activity of this cytokine. The development of methods for measuring activation of latent TGF-beta in cell cultures and identification of the proteins contained in the latent TGF-beta complex have shed new light on the mechanism of activation of latent TGF-beta possibly involved in vasculogenesis, angiogenesis, and other processes.
Asunto(s)
Neovascularización Fisiológica , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Técnicas de Cocultivo , Endotelio Vascular/metabolismo , Humanos , Ratones , Músculo Liso Vascular/metabolismoRESUMEN
BACKGROUND: Many cytokines regulate processes involved in the pathogenesis of proliferative vitreoretinopathy. Transforming growth factor-beta (TGF-beta) is an example of a pluripotent growth factor that regulates cell proliferation, extracellular matrix (ECM) deposition, cell migration, and differentiation--all biological activities involved in the formation and progression of proliferative vitreoretinopathies. METHODS: A review of experimental results that demonstrate how vascular cells generate biologically active TGF-beta is presented. Most cell types--including endothelial cells and pericytes, which form the retinal microvasculature--express TGF-beta as a large latent TGF-beta complex. Mature TGF-beta, the biologically active form, must be generated from the large latent complex before it can signal by binding to its high affinity cell surface receptors. RESULTS: A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that co-culturing bovine smooth muscle cells or pericytes and endothelial cells generates active TGF-beta. CONCLUSION: The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation. Studying TGF-beta generation by co-cultures of pericytes and endothelial cells can provide us with insights into how disruption of latent TGF-beta activation may lead to unregulated endothelial proliferation, ECM deposition, and cellular infiltration, as observed clinically in neovascular- and fibrotic-related pathologies.
Asunto(s)
Endotelio Vascular/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Vasos Retinianos/metabolismo , Animales , Técnicas de Cultivo de Célula , División Celular , Movimiento Celular , Endotelio Vascular/patología , Humanos , Músculo Liso Vascular/patología , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Vasos Retinianos/patología , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/patologíaRESUMEN
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor which is synthesized and secreted by many differentiated cells in response to various stimuli including hypoxia and growth factor exposure. Alternative splicing of vascular endothelial growth factor mRNA results in three distinct molecular forms: V189 and V165 or V121 which lack the exons 6 or 6 and 7, respectively. To clarify the functions of the 24-amino acid insertion, the biological activity of V165 was compared with that exerted by purified recombinant V189 and a synthetic peptide designed on the sequence encoded by exon 6 (Ex6P). V189 and Ex6P, but not V165, induced cell proliferation on corneal endothelial cells cultured in vitro. These effects were due to the release of fibroblast growth factor 2 (FGF2) stored in the extracellular matrix but not to direct interactions with FGF receptors since V189 was inefficient on heparan sulfate-deficient cells expressing constitutively FGF-R1. Moreover corneas incubated ex vivo with Ex6P solubilized 10-fold more FGF2 than a isocationic peptide containing a scrambled sequence. Ex6P elicited an angiogenic response in a corneal pocket assay which was totally inhibited by addition of anti-FGF2 IgG. Moreover the angiogenic response to V189, but not to V165, was inhibited by FGF2 immunoneutralization. These findings demonstrate that the presence of the exon 6-encoded sequence confers VEGF with the ability to exert its biological effects through FGF2 signaling pathways.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Endotelio Vascular/metabolismo , Exones , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Linfocinas/genética , Secuencia de Aminoácidos , Animales , Células CHO , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Córnea/citología , Córnea/metabolismo , Cricetinae , Endotelio Vascular/citología , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Células del Estroma/citología , Células del Estroma/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Transforming growth factor-beta are cytokines with a wide range of biological effects. They play a pathologic role in inflammatory and fibrosing diseases such as nephrosclerosis. TGF-beta s are secreted in a latent form due to noncovalent association with latency associated peptide (LAP), which is a homodimer formed from the propeptide region of TGF-beta. LAP is disulfide linked to another protein, latent TGF-beta binding protein (LTBP). LTBP has features in common with extracellular matrix proteins, and targets latent TGF-beta to the matrix. Activation of latent TGF-beta can be accomplished in vitro by denaturing treatments, plasmin digestion, ionizing radiation and interaction with thrombospondin. The mechanisms by which latent TGF-beta is activated physiologically are not well understood. Results to date suggest an important role for proteases, particularly plasmin, although other mechanisms probably exist. A general model of activation is proposed in which latent TGF-beta is released from the extracellular matrix by proteases, localized to cell surfaces, and activated by cell-associated plasmin.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Factor de Crecimiento Transformador beta/fisiología , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Endotelio Vascular/fisiología , Fibrinolisina/fisiología , Humanos , Proteínas de Unión a TGF-beta Latente , Factor de Crecimiento Transformador beta/químicaRESUMEN
Transforming growth factor-beta (TGF-beta) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-beta, the TGF-beta propeptide, and the latent TGF-beta binding protein (LTBP). To interact with its cell surface receptors, TGF-beta must be released from the latent complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-beta. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S-transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in cross-linking and formation of TGF-beta by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-beta generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (deltaN293) or 441 (deltaN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that deltaN293 LTBP-1S was matrix associated via a transglutaminase-dependent reaction, whereas deltaN441 LTBP-1S was not. This suggests that residues 294-441 are critical to the transglutaminase reactivity of LTBP-1S.
Asunto(s)
Proteínas Portadoras/metabolismo , Matriz Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Factor de Crecimiento Transformador beta/metabolismo , Transglutaminasas/farmacología , Animales , Células CHO , Cadaverina/análogos & derivados , Cadaverina/farmacología , Proteínas Portadoras/genética , Células Cultivadas , Cricetinae , Reactivos de Enlaces Cruzados , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de Unión a TGF-beta Latente , Proteínas Recombinantes de Fusión , Porcinos , Transglutaminasas/antagonistas & inhibidoresRESUMEN
Transforming growth factor (TGF-) beta is secreted as a latent complex in which the mature growth factor remains associated with its propeptide. In order to elicit a biological response, the cytokine must be released from the latent complex, a process termed latent TGF-beta activation or TGF-beta formation. Although latent TGF-beta activation is a critical step in the regulation of its activity, little is known about the molecular mechanisms that lead to the production of active TGF-beta. In this article, we present an overview of the data available on this topic, and we propose a tentative model for the mechanism of TGF-beta formation based upon the observations with different cell systems and on recent findings on the structure of the latent TGF-beta complex.
Asunto(s)
Células Madre/química , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismo , AnimalesRESUMEN
The plasminogen/plasminogen activator system is widely used in extracellular proteolysis. In this review the involvement of this system in tumour invasion, cell migration, growth factor presentation and inhibition of angiogenesis are discussed.
Asunto(s)
Sustancias de Crecimiento/metabolismo , Activadores Plasminogénicos/metabolismo , Plasminógeno/metabolismo , Proteínas/metabolismo , Animales , Humanos , Hidrólisis , Invasividad Neoplásica , Estimulación Química , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Most cultured cell types secrete small latent transforming growth factor-beta (TGF-beta) as a disulfide-bonded complex with a member of the latent TGF-beta binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-beta1. Coexpression in Sf9 cells of small latent TGF-beta1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-beta1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-beta, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-beta1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-beta.
Asunto(s)
Proteínas Portadoras/química , Cisteína/química , Péptidos y Proteínas de Señalización Intracelular , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células CHO , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Cricetinae , Humanos , Proteínas de Unión a TGF-beta Latente , Mutagénesis , Nucleopoliedrovirus/genética , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , SpodopteraRESUMEN
bFGF endocytosis in BHK cells was examined by electron microscopy using a conjugate of recombinant human bFGF and digoxigenin (bFGF-DIG). This probe keeps the biological activity of non-labeled bFGF and can be readily detected with anti-digoxigenin antibodies (Gleizes et al., Anal. Biochem. 219, 360-367 (1994)). Time-course studies of bFGF-DIG endocytosis were performed by incubating BHK cells at 4 degrees C in the presence of first 20 ng/ml bFGF-DIG and then antidigoxigenin antibodies adsorbed onto 10-nm gold particles. A semi-quantitative study revealed that caveolae were the main endocytic pathway of bFGF-DIG in these cells, whereas clathrin-coated pits were scarcely labeled. After occurring in caveolae, bFGF-DIG was sequentially detected in tubulovesicular early endosomes, multivesicular late endosomes, and lysosomes. Under the same conditions, low density lipoprotein (LDL)-gold was seen entering the cell exclusively through clathrin-coated pits. However, LDL-gold and bFGF-DIG were colocalized, at least in part, in common endosomal structures. Pretreatment of the cells with phosphatidylinositol-phospholipase C reduced the proportion of membrane-bound bFGF-DIG in caveolae, but did not inhibit bFGF-DIG presence in caveolae. These data suggest that bFGF enters into BHK cells through caveolae and is then shuttled into a degradative pathway similar to that of LDL.
Asunto(s)
Membrana Celular/metabolismo , Endocitosis/fisiología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Animales , Línea Celular , Membrana Celular/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Cricetinae , Endocitosis/efectos de los fármacos , Endotelio Vascular , Factor 2 de Crecimiento de Fibroblastos/ultraestructura , Riñón , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/ultraestructura , Microscopía Electrónica , Fosfatidilinositol Diacilglicerol-Liasa , Hidrolasas Diéster Fosfóricas/farmacología , Proteínas Recombinantes , Factores de TiempoRESUMEN
Most cell types express transforming growth factor-beta (TGF-beta) as a large latent TGF-beta complex that must be converted to an active form before TGF-beta can interact with cell surface TGF-beta receptors. This conversion involves the release of mature TGF-beta from the complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide, latency associated peptide. A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that coculturing bovine endothelial and smooth muscle cells generates active TGF-beta. Coculture activation of the large latent TGF-beta complex occurs through a plasmin-dependent mechanism that requires concentration of reactants on the cell surface and/or extracellular matrix. The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation.
Asunto(s)
Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismo , Animales , Bovinos , Técnicas de Cocultivo , Endotelio/metabolismo , Fibrinolisina/metabolismo , Músculo Liso/metabolismoRESUMEN
Biochemical studies have shown that basic fibroblast growth factor (bFGF or FGF-2) is internalized by two pathways, after binding to either FGF tyrosine kinase receptors or to heparan sulfate proteoglycans (HSPG). To get insights on the HSPG-mediated pathway, we have examined by electron microscopy the intracellular route of bFGF-HRP, a monovalent conjugate of bFGF and horseradish peroxidase which was found to bind to HSPG only and was detectable by electron microscopy. bFGF-HRP association to adult bovine aortic endothelial (ABAE) cells or baby hamster kidney (BHK) cells was inhibited by a high molar excess of native bFGF, a 2 M NaCl wash at neutral pH, heparin and heparan sulfate, but not by chondroitin 4-sulfate or chondroitin 6-sulfate. bFGF-HRP was not able to displace [125I]bFGF from its high-affinity binding sites, and the dissociation constant of its binding to ABAE cells was estimated at 3 nM. Time-course experiments were performed to follow bFGF-HRP endocytosis in ABAE cells. bFGF-HRP was found to enter the cell after binding to the plasma membrane or extracellular matrix. On the cell surface, the probe accumulated in noncoated flask-shaped invaginations and in caveolae rather than in clathrin-coated pits. Immediately after endocytosis, bFGF-HRP was detected in pleiomorphic tubulovesicular and tubulocisternal early endosomes. Multivesicular bodies contained diaminobenzidine (DAB) precipitate after 5 to 15 min, but lysosomes were not labeled before 1 h, indicating a delayed transfer from late endosomes to lysosomes. Labeling was never detected in the nucleus, even after intensification of the DAB reaction product by silver-gold enhancement. Similar endocytic pathways and intracellular locations were observed in other endothelial and non-endothelial cell types. These results suggest that bFGF associated to HSPG can enter the cell via several pathways and follows mainly a degradative route.
Asunto(s)
Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Proteoglicanos/metabolismo , Animales , Bovinos , Línea Celular , Endocitosis/fisiología , Endotelio Vascular/citología , Endotelio Vascular/ultraestructura , Factor 2 de Crecimiento de Fibroblastos/química , Peroxidasa de Rábano Silvestre/química , Microscopía ElectrónicaRESUMEN
Digoxigenin, a 391-Da plant sterol, was conjugated to recombinant bFGF with the aim of detecting it with high specificity and sensitivity in cultured eukaryotic cells using antibodies against digoxigenin. The conjugate, bFGF-DIG, displayed a mitogenic activity on endothelial cells equivalent to that of nonlabeled bFGF. Binding of the probe on the cell surface was assessed by ELISA on cells, which allowed discrimination between low- and high-affinity bFGF binding sites. Using a chemiluminescent system, chemical cross-linking of bFGF-DIG with FGF receptors was analyzed directly on Western blots of cell extracts with anti-digoxigenin antibodies. The labeling pattern was identical to that reported with iodinated bFGF, showing that bFGF-DIG bound to the same receptors. The time course of intracellular degradation of internalized bFGF-DIG was also followed by immunodetection on Western blots: the low speed of the catabolic process and the size of the degradation products were comparable to those previously described with iodinated bFGF. In parallel, bFGF-DIG was readily detected by immunofluorescence in cultured cells, and was shown to be an interesting probe to determine bFGF endocytosis pathways by electron microscopy. bFGF-DIG appeared as a multifunctional nonradioactive probe suitable for combined biochemical and cytological studies of bFGF.