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1.
BMC Genomics ; 19(1): 592, 2018 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-30086708

RESUMEN

BACKGROUND: Anisakis simplex sensu stricto and Anisakis pegreffii are sibling species of nematodes parasitic on marine mammals. Zoonotic human infection with third stage infective larvae causes anisakiasis, a debilitating and potentially fatal disease. These 2 species show evidence of hybridisation in geographical areas where they are sympatric. How the species and their hybrids differ is still poorly understood. RESULTS: Third stage larvae of Anisakis simplex s.s., Anisakis pegreffii and hybrids were sampled from Merluccius merluccius (Teleosti) hosts captured in waters of the FAO 27 geographical area. Specimens of each species and hybrids were distinguished with a diagnostic genetic marker (ITS). RNA was extracted from pools of 10 individuals of each taxon. Transcriptomes were generated using Illumina RNA-Seq, and assembled de novo. A joint assembly (here called merged transcriptome) of all 3 samples was also generated. The inferred transcript sets were functionally annotated and compared globally and also on subsets of secreted proteins and putative allergen families. While intermediary metabolism appeared to be typical for nematodes in the 3 evaluated taxa, their transcriptomes present strong levels of differential expression and enrichment, mainly of transcripts related to metabolic pathways and gene ontologies associated to energy metabolism and other pathways, with significant presence of excreted/secreted proteins, most of them allergens. The allergome of the 2 species and their hybrids has also been thoroughly studied; at least 74 different allergen families were identified in the transcriptomes. CONCLUSIONS: A. simplex s.s., A. pegreffi and their hybrids differ in gene expression patterns in the L3 stage. Strong parent-of-origin effects were observed: A. pegreffi alleles dominate in the expression patterns of hybrids albeit the latter, and A. pegreffii also display significant differences indicating that hybrids are intermediate biological entities among their parental species, and thus of outstanding interest in the study of speciation in nematodes. Analyses of differential expression based on genes coding for secreted proteins suggests that co-infections presents different repertoires of released protein to the host environment. Both species and their hybrids, share more allergen genes than previously thought and are likely to induce overlapping disease responses.


Asunto(s)
Anisakis/genética , Gadiformes/parasitología , Perfilación de la Expresión Génica/métodos , Proteínas del Helminto/genética , Alérgenos/genética , Animales , Anisakis/aislamiento & purificación , Anisakis/patogenicidad , Cruzamiento , Metabolismo Energético , Enfermedades de los Peces/parasitología , Regulación de la Expresión Génica , Larva/genética , Larva/patogenicidad , Anotación de Secuencia Molecular , Análisis de Secuencia de ARN/métodos , Factores de Virulencia/genética
2.
J Proteome Res ; 12(1): 112-22, 2013 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-23234512

RESUMEN

The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed.


Asunto(s)
Cromosomas Humanos Par 16 , Bases de Datos de Proteínas , Proteínas , Proteoma/análisis , Línea Celular , Cromosomas Humanos Par 16/genética , Cromosomas Humanos Par 16/metabolismo , Expresión Génica , Genoma Humano , Humanos , Espectrometría de Masas , Proteínas/clasificación , Proteínas/genética , Proteínas/metabolismo , Transcriptoma
4.
J Proteomics ; 71(1): 11-8, 2008 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-18541470

RESUMEN

Plans for the European Proteomics Association (EuPA) were conceived and established during 2004 and 2005, and culminated in the formal inception of the organisation during the 4th HUPO World Congress held in Munich in 2005. The mission from the outset has been three-tiered and is to: i) strengthen the national Proteomics organizations in their efforts; ii) to co-ordinate and provide educational programs, and iii) to advance the networking of scientists through meetings, workshops and student exchange. Linked to the mission were objectives to emphasise the benefits and contributions of Proteomics to biological and industrial researchers, the general public and science policy makers in Europe. In addition, the EuPA set out to promote scientific exchange for all applications and technology development related to Proteomics, and coordinate joint activities of national Proteomics societies at the European level. To achieve these tasks an organisational structure was conceived whereby four Activity Committees (Conferences/Communications, Education, EuPA-HUPO-Interactions and Funding) were implemented and a General Council consisting of all member countries. The remarkable rise and progress the EuPA has achieved in this small time frame is reported here.


Asunto(s)
Proteómica , Sociedades Científicas/organización & administración , Europa (Continente) , Historia del Siglo XXI , Proteómica/educación , Proteómica/organización & administración , Sociedades Científicas/historia , Sociedades Científicas/tendencias
5.
J Neurosci Res ; 86(8): 1871-83, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18241054

RESUMEN

The antigen recognized by the monoclonal antibody 3CB2 (3CB2-Ag and 3CB2 mAb) is expressed by radial glia and astrocytes in the developing and adult vertebrate central nervous system (CNS) of vertebrates as well as in neural stem cells. Here we identified the 3CB2-Ag as vimentin by proteomic analysis of human glial cell line U-87 extracts (derived from a malignant astrocytoma). Indeed, the 3CB2 mAb recognized three vimentin isoforms in glial cell lines. In the human retina, 3CB2-Ag was expressed in Müller cells, astrocytes, some blood vessels, and cells in the horizontal cell layer, as determined by immunoprecipitation and immunofluorescence. Three populations of astrocytes were distinguishable by double-labeling immunohistochemistry: vimentin+/GFAP+, vimentin-/GFAP+, and vimentin+/GFAP-. Hence, we conclude that 1) the 3CB2-Ag is vimentin; 2) vimentin isoforms are differentially expressed in normal and transformed astrocytes; 3) human retinal astrocytes display molecular heterogeneity; and 4) the 3CB2 mAb is a valuable tool to study vimentin expression and its function in the human retina.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Regulación de la Expresión Génica/fisiología , Retina/metabolismo , Vimentina/biosíntesis , Adolescente , Adulto , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Astrocitos/metabolismo , Astrocitoma/metabolismo , Astrocitoma/patología , Línea Celular Transformada , Línea Celular Tumoral , Humanos , Persona de Mediana Edad , Neuroglía/metabolismo , Neuroglía/patología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Ratas , Retina/inmunología , Vimentina/genética , Vimentina/inmunología
6.
Biochemistry ; 42(29): 8879-84, 2003 Jul 29.
Artículo en Inglés | MEDLINE | ID: mdl-12873149

RESUMEN

A synthetic peptide patterned after the sequence of the inactivating ball domain of the Shaker B K(+) channel, the ShB peptide, fully restores fast inactivation in the deletion Shaker BDelta6-46 K(+) channel, which lacks the constitutive ball domains. On the contrary, a similar peptide in which tyrosine 8 is substituted by the secondary structure-disrupting d-tyrosine stereoisomer does not. This suggests that the stereoisomeric substitution prevents the peptide from adopting a structured conformation when bound to the channel during inactivation. Moreover, characteristic in vitro features of the wild-type ShB peptide such as the marked propensity to adopt an intramolecular beta-hairpin structure when challenged by anionic phospholipid vesicles, a model target mimicking features of the inactivation site in the channel protein, or to insert into their hydrophobic bilayers, are lost in the d-tyrosine-containing peptide, whose behavior is practically identical to that of noninactivating peptide mutants. In the absence of high resolution crystallographic data on the inactivated channel/peptide complex, these latter findings suggest that the structured conformation required for the peptide to promote channel inactivation, as referred to above, is likely to be beta-hairpin.


Asunto(s)
Péptidos/química , Tirosina/química , Secuencia de Aminoácidos , Animales , Células CHO , Rastreo Diferencial de Calorimetría , Cricetinae , Cristalografía por Rayos X , Péptidos y Proteínas de Señalización Intracelular , Membrana Dobles de Lípidos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Homología de Secuencia de Aminoácido , Estereoisomerismo , Temperatura
7.
Biochemistry ; 41(40): 12263-9, 2002 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-12356329

RESUMEN

A synthetic peptide patterned after the sequence of the inactivating "ball" domain of the Shaker B K(+) channel restores fast (N-type) inactivation in mutant deletion channels lacking their constitutive ball domains, as well as in K(+) channels that do not normally inactivate. We now report on the effect of phosphorylation at a single tyrosine in position 8 of the inactivating peptide both on its ability to restore fast channel inactivation in deletion mutant channels and on the conformation adopted by the phosphorylated peptide when challenged by anionic lipid vesicles, a model target mimicking features of the inactivation site in the channel protein. We find that the inactivating peptide phosphorylated at Y8 behaves functionally as well as structurally as the noninactivating mutant carrying the mutation L7E. Moreover, it is observed that the inactivating peptide can be phosphorylated by the Src tyrosine kinase either as a free peptide in solution or when forming part of the membrane-bound protein channel as the constitutive inactivating domain. These findings suggest that tyrosine phosphorylation-dephosphorylation of this inactivating ball domain could be of physiological relevance to rapidly interconvert fast-inactivating channels into delayed rectifiers and vice versa.


Asunto(s)
Péptidos/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Liposomas/metabolismo , Datos de Secuencia Molecular , Oocitos , Fosforilación , Relación Estructura-Actividad , Xenopus , Familia-src Quinasas/metabolismo
8.
J Proteome Res ; 1(5): 421-7, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12645913

RESUMEN

Total protein variation as revealed by two-dimensional electrophoresis (2D-E) was studied in 18 isolates from populations of Meloidogyne arenaria (six isolates), Meloidogyne incognita (10 isolates), and Meloidogyne javanica (one isolate) plus an unclassified isolate. Gels (80 x 60 x 0.75 mm) were silverstained and digitized in order to compare their protein patterns. Optical density and position of protein patterns were measured using statistical cluster analysis and computer-assisted image analysis software. Only those protein stains or positions that were clearly defined (i.e., without background) were considered. The number of positions in gels ranged from 86 to 203. Each of these positions had 95 clearly expressed proteins that were present in at least two replicates for each isolate. Spot position was considered a taxonomical character with two different states: presence (1) and absence (0). Accordingly, genetic distance was estimated among isolates and species, and a phylogenetic tree was constructed following the cladistic approach based on maximum parsimony analysis. Isolates of M. arenaria--M. javanica--Meloidogyne sp. and of M. incognita formed two separate monophyletic groups. Both groups were clearly defined on the basis of two sets of protein positions that can be considered as diagnostic characters. An attempt to identify these proteins by mass spectrometry was made. Group diagnostic proteins for M. incognita and M. arenaria (and for other proteins common to all isolates) were distinguished by protonated mass signals in the MALDI fingerprinting spectrum.


Asunto(s)
Proteínas/análisis , Animales , Bases de Datos como Asunto , Electroforesis en Gel Bidimensional , Espectrometría de Masas , Filogenia , Proteínas/química , Proteoma , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tylenchoidea
9.
EMBO Rep ; 2(10): 945-51, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11571266

RESUMEN

Tumor necrosis factor (TNF) ligand family members are synthesized as transmembrane proteins, and cleavage of the membrane-anchored proteins from the cell surface is frequently observed. The TNF-related ligands APRIL and BLyS and their cognate receptors BCMA/TACI form a two ligand/two receptor system that has been shown to participate in B- and T-cell stimulation. In contrast to BLyS, which is known to be cleaved from the cell surface, we found that APRIL is processed intracellularly by furin convertase. Blockage of protein transport from the endoplasmic reticulum to the Golgi apparatus by Brefeldin A treatment abrogated APRIL processing, whereas monensin, an inhibitor of post-Golgi transport, did not interfere with cleavage of APRIL, but blocked secretion of processed APRIL. Thus, APRIL shows a unique maturation pathway among the TNF ligand family members, as it not detectable as a membrane-anchored protein at the cell surface, but is processed in the Golgi apparatus prior to its secretion.


Asunto(s)
Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Subtilisinas/metabolismo , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos Monoclonales/metabolismo , Transporte Biológico , Western Blotting , Brefeldino A/farmacología , División Celular , Línea Celular , Retículo Endoplásmico/metabolismo , Furina , Aparato de Golgi/genética , Humanos , Células Jurkat , Ligandos , Microscopía Fluorescente , Mutación , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Subtilisinas/química , Factores de Tiempo , Transfección , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral
10.
Proc Natl Acad Sci U S A ; 98(20): 11515-20, 2001 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-11562487

RESUMEN

The concept for cellular immunotherapy of solid tumors relies heavily on the capacity of class I MHC-restricted cytotoxic T lymphocytes (CTLs) to eliminate tumor cells. However, tumors often have managed to escape from the cytolytic machinery of these effector cells. Therefore, it is very important to chart the mechanisms through which this escape can occur. Target-cell killing by CTLs involves the induction of apoptosis by two major mechanisms: through death receptors and the perforin/granzyme B (GrB) pathway. Whereas tumors previously were shown to exhibit mechanisms for blocking the death receptor pathway, we now demonstrate that they also can resist CTL-mediated killing through interference with the perforin/GrB pathway. This escape mechanism involves expression of the serine protease inhibitor PI-9/SPI-6, which inactivates the apoptotic effector molecule GrB. Expression of PI-9 was observed in a variety of human and murine tumors. Moreover, we show that, indeed, expression results in the resistance of tumor cells to CTL-mediated killing both in vitro and in vivo. Our data reveal that PI-9/SPI-6 is an important parameter determining the success of T cell-based immunotherapeutic modalities against cancer.


Asunto(s)
Glicoproteínas de Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Animales , Neoplasias de la Mama/inmunología , Neoplasias del Colon/inmunología , Cartilla de ADN , Femenino , Citometría de Flujo/métodos , Granzimas , Humanos , Proteínas de Insectos , Melanoma/inmunología , Ratones , Perforina , Proteínas Citotóxicas Formadoras de Poros , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/inmunología
11.
FEBS Lett ; 503(2-3): 135-41, 2001 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-11513870

RESUMEN

Dynein is a minus end-directed microtubule motor that serves multiple cellular functions. We have performed a fine mapping of the 8 kDa dynein light chain (LC8) binding sites throughout the development of a library of consecutive synthetic dodecapeptides covering the amino acid sequences of the various proteins known to interact with this dynein member according to the yeast two hybrid system. Two different consensus sequences were identified: GIQVD present in nNOS, in DNA cytosine methyl transferase and also in GKAP, where it is present twice in the protein sequence. The other LC8 binding motif is KSTQT, present in Bim, dynein heavy chain, Kid-1, protein 4 and also in swallow. Interestingly, this KSTQT motif is also present in several viruses known to associate with microtubules during retrograde transport from the plasma membrane to the nucleus during viral infection.


Asunto(s)
Dineínas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dineínas Citoplasmáticas , Dineínas/química , Dineínas/genética , Humanos , Técnicas In Vitro , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Mapeo Peptídico , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo
12.
Proc Natl Acad Sci U S A ; 98(17): 9859-64, 2001 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-11493675

RESUMEN

Preparations of purified full-length fusion (F) protein of human respiratory syncytial virus (HRSV) expressed in recombinant vaccinia-F infected cells, or of an anchorless mutant (F(TM(-))) lacking the C-terminal 50 amino acids secreted from vaccinia-F(TM(-))-infected cells contain a minor polypeptide that is an intermediate product of proteolytic processing of the F protein precursor F0. N-terminal sequencing of the intermediate demonstrated that it is generated by cleavage at a furin-motif, residues 106-109 of the F sequence. By contrast, the F1 N terminus derives from cleavage at residue 137 of F0 which is also C-terminal to a furin recognition site at residues 131-136. Site-directed mutagenesis indicates that processing of F0 protein involves independent cleavage at both sites. Both cleavages are required for the F protein to be active in membrane fusion as judged by syncytia formation, and they allow changes in F structure from cone- to lollipop-shaped spikes and the formation of rosettes by anchorless F.


Asunto(s)
Fusión de Membrana/fisiología , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Virus Sincitiales Respiratorios/fisiología , Proteínas Virales de Fusión/fisiología , Proteínas Virales/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Efecto Citopatogénico Viral , Endopeptidasas/metabolismo , Células Gigantes , Humanos , Riñón , Mesocricetus , Microscopía Electrónica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Precursores de Proteínas/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/fisiología , Virus Sincitiales Respiratorios/genética , Relación Estructura-Actividad , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/ultraestructura , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/ultraestructura
13.
J Biol Chem ; 276(40): 37491-500, 2001 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-11448964

RESUMEN

Matrix metalloproteinases are thought to play an important role in endothelial cell migration and matrix remodeling. We have used an in vitro wound healing migration model and newly generated anti-membrane type 1-matrix metalloproteinase (MT1-MMP) monoclonal antibodies (mAbs) to characterize the role of MT1-MMP during this process. First, the expression and shedding of MT1-MMP are up-regulated upon induction of migration in endothelial cells, as demonstrated by flow cytometry and Western blot analysis. Furthermore, MT1-MMP is concentrated at discrete areas in migrating endothelial cells, in contrast to the diffuse pattern observed in confluent cells. Interestingly, migration of endothelial cells results in the stimulation of MT1-MMP activity, as shown by its ability to process pro-MMP-2 and to degrade fibrinogen assessed by zymography. Moreover, MT1-MMP-mediated gelatin degradation is enriched at migration sites. mAbs generated against the MT1-MMP catalytic domain are shown to inhibit MT1-MMP enzymatic activity and to impair both phorbol 12-myristate 13-acetate-induced endothelial migration and invasion of collagen and fibrin gels. Furthermore, a reduction in the formation of capillary tubes in Matrigel is also observed when endothelial cells are pretreated with the blocking anti-MT1-MMP mAbs. Altogether, these data demonstrate that MT1-MMP plays an important role during endothelial cell migration, and its activity can modulate endothelial migration, invasion, and formation of capillary tubes during the angiogenic response.


Asunto(s)
Movimiento Celular/fisiología , Endotelio Vascular/enzimología , Matriz Extracelular/fisiología , Metaloendopeptidasas/metabolismo , Anticuerpos Monoclonales/farmacología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Colágeno/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Activación Enzimática , Matriz Extracelular/efectos de los fármacos , Fibrina/fisiología , Fibrinógeno/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/inmunología , Regulación hacia Arriba
14.
FEBS Lett ; 501(1): 79-83, 2001 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-11457460

RESUMEN

Apoptotic protease activating factor-1 (Apaf-1) is an adaptor molecule essential for caspase-9 activation. Subcellular analysis of Apaf-1 in NIH-3T3 fibroblasts and the immature murine B cell lymphoma WEHI-231 indicates that Apaf-1 is localized in the Golgi apparatus and cytoplasm. Bcl-2 overexpression in WEHI-231 cells disrupts Apaf-1 localization in Golgi, causing a perinuclear Apaf-1 redistribution. Bcl-2 overexpression in NIH-3T3 fibroblasts however does not cause similar Apaf-1 redistribution, suggesting that cell type factors are involved in the redistribution process. The ability of Bcl-2 to modify Apaf-1 subcellular localization is not explained by direct interaction between Apaf-1 and Bcl-2. These data may help to clarify the anti-apoptotic Bcl-2 function.


Asunto(s)
Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células 3T3 , Animales , Factor Apoptótico 1 Activador de Proteasas , Células COS , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes bcl-2/genética , Aparato de Golgi/metabolismo , Humanos , Ratones , Especificidad de Órganos , Unión Proteica , Transporte de Proteínas , Proteínas/química , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transfección , Células Tumorales Cultivadas
15.
J Immunol ; 166(12): 7345-52, 2001 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-11390485

RESUMEN

The diverse forms of protein phosphatase 1 (PP1) in vivo result from the association of the catalytic subunit with different regulatory subunits. We recently have described that PP1alpha is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. With the yeast two-hybrid system, GST fusion proteins, indirect immunofluorescence, and coimmunoprecipitation, we found that Bcl-2 interacts with PP1alpha and Bad. In contrast, Bad did not interact with 14-3-3 protein. Bcl-2 depletion decreased phosphatase activity and association of PP1alpha to Bad. Bcl-2 contains the RIVAF motif, analogous to the well characterized R/KXV/IXF consensus motif shared by most PP1-interacting proteins. This sequence is involved in the binding of Bcl-2 to PP1alpha. Disruption of Bcl-2/PP1alpha association strongly decreased Bcl-2 and Bad-associated phosphatase activity and formation of the trimolecular complex. These results suggest that Bcl-2 targets PP1alpha to Bad.


Asunto(s)
Proteínas Portadoras/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis/inmunología , Unión Competitiva/genética , Unión Competitiva/inmunología , Proteínas Portadoras/genética , Línea Celular , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , Pruebas de Precipitina , Unión Proteica/genética , Unión Proteica/inmunología , Proteína Fosfatasa 1 , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Linfocitos T/citología , Linfocitos T/enzimología , Técnicas del Sistema de Dos Híbridos , Proteína Letal Asociada a bcl
16.
J Biol Chem ; 276(30): 28372-9, 2001 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-11373289

RESUMEN

CCL20/MIP-3alpha is a beta-chemokine expressed in the thymus, skin, and intestinal epithelial cells that exclusively binds and activates the CCR6 receptor in both mice and humans. The strict receptor binding specificity of CCL20 is exceptional; other chemokines and their receptors bind promiscuously with multiple partners. Toward determining the structural basis for the selective receptor specificity of CCL20, we have determined its three-dimensional structure by 1H NMR spectroscopy. CCL20 exhibits the same monomeric structure previously described for other chemokines: a three-stranded beta-sheet and an overlying alpha-helix. The CCL20 receptor selectivity could arise from the rigid conformation of the N-terminal DCCL motif as well as the groove between the N-loop and the beta2-beta3 hairpin, which is significantly narrower in CCL20 than in other chemokines. Similar structural features are seen in human beta-defensin 2, a small nonchemokine polypeptide reported to selectively bind and activate CCR6, which stresses their importance for the specific binding of both CCL20 and beta-defensin 2 to CCR6. CCL20's structure will be useful to design tools aimed to modulate its important biological functions.


Asunto(s)
Quimiocinas CC/química , Quimiocinas CC/metabolismo , Células Dendríticas/metabolismo , Linfocitos/metabolismo , Proteínas Inflamatorias de Macrófagos/química , Proteínas Inflamatorias de Macrófagos/metabolismo , Estrés Oxidativo , Receptores de Quimiocina/metabolismo , Secuencias de Aminoácidos , Animales , Quimiocina CCL20 , Factores Quimiotácticos/metabolismo , Dimerización , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Modelos Químicos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Receptores CCR6 , beta-Defensinas/química
17.
Lab Invest ; 81(3): 409-18, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11310833

RESUMEN

Using new human CXCR3 chemokine receptor-specific monoclonal antibodies, we studied human CXCR3 tissue distribution in lymphoid and nonlymphoid organs, as well as in inflammatory conditions, including rheumatoid arthritis, Hashimoto's thyroiditis, and dermal vasculitis. CXCR3 was expressed by certain dendritic cell subsets, specifically myeloid-derived CD11c positive cells, not only in those present in normal lymphoid organs, but also in germinal centers generated in inflammatory conditions. CXCR3 expression was also detected in some lymphocyte subsets such as intraepithelial lymphocytes of secondary lymphoid organs and infiltrating lymphocytes in inflammatory conditions. In addition, CXCR3 was constitutively expressed by endothelial cells (EC) of vessels of medium and large caliber but not in small vessels from different organs. Finally, enhanced CXCR3 expression was found in EC and in infiltrating lymphocytes with an activated phenotype in inflammatory diseases. The CXCR3 chemokine receptor may play a role in the regulation of leukocyte migration to inflammatory sites.


Asunto(s)
Células Dendríticas/química , Células Dendríticas/inmunología , Endotelio Vascular/química , Activación de Linfocitos/inmunología , Receptores de Quimiocina/análisis , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Células Cultivadas , Quimiotaxis de Leucocito/inmunología , Endotelio Vascular/inmunología , Humanos , Riñón/citología , Linfocitos/química , Linfocitos/inmunología , Tejido Linfoide/química , Tejido Linfoide/inmunología , Tejido Linfoide/patología , Ratones , Receptores CXCR3 , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Sinovitis/inmunología , Sinovitis/patología , Tiroiditis Autoinmune/inmunología , Tiroiditis Autoinmune/patología , Transfección , Vasculitis/inmunología , Vasculitis/patología
18.
J Biol Chem ; 276(25): 23009-17, 2001 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-11294831

RESUMEN

The MAL proteolipid, an integral protein present in glycolipid- and cholesterol-enriched membrane (GEM) rafts, is an element of the machinery necessary for apical sorting in polarized epithelial Madin-Darby canine kidney cells. MAL was the first member identified of an extended family of proteins that have significant overall sequence identity. In this study we have used a newly generated monoclonal antibody to investigate an unedited member of this family, named BENE, which was found to be expressed in endothelial-like ECV304 cells and normal human endothelium. Human BENE was characterized as a proteolipid protein predominantly present in GEM rafts in ECV304 cells. Coimmunoprecipitation experiments revealed that BENE interacted with caveolin-1. Confocal immunofluorescence and electron microscopic analyses indicated that BENE mainly accumulated into intracellular vesicular/tubular structures that partially colocalize with internal caveolin-1. In response to cell surface cholesterol oxidation, BENE redistributed to the dilated vesicular structures that concentrate most of the caveolin-1 originally on the cell surface. After cessation of cholesterol oxidation, a detectable fraction of the BENE molecules migrated to the plasmalemma accompanying caveolin-1 and then returned progressively to its steady state distribution. Together, these features highlight the BENE proteolipid as being an element of the machinery for raft-mediated trafficking in endothelial cells.


Asunto(s)
Proteínas Portadoras/metabolismo , Caveolinas/metabolismo , Endotelio/metabolismo , Proteínas de la Membrana/metabolismo , Proteolípidos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Bovinos , Caveolina 1 , Línea Celular , Colesterol/metabolismo , Endotelio/citología , Endotelio/ultraestructura , Regulación de la Expresión Génica , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito , Oxidación-Reducción , Proteolípidos/química , Homología de Secuencia de Aminoácido
19.
J Bacteriol ; 183(3): 1032-7, 2001 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11208802

RESUMEN

The ptsN gene of Pseudomonas putida encodes IIA(Ntr), a protein of the phosphoenol pyruvate:sugar phosphotransferase (PTS) system which is required for the C source inhibition of the sigma(54)-dependent promoter Pu of the TOL (toluate degradation) plasmid pWW0. Using two-dimensional gel electrophoresis, we have examined the effect of ptsN disruption on the general expression pattern of P. putida. To this end, cells were grown in the presence or absence of glucose, and a 1,117-spot subset of the P. putida proteome was used as a reference for comparisons. Among all gene products whose expression was lowered by this carbon source (247 spots [about 22%]), only 6 behaved as Pu (i.e., were depressed in the ptsN background). This evidenced only a minor role for IIA(Ntr) in the extensive inhibition of gene expression in P. putida caused by glucose. However, the same experiments revealed a large incidence of glucose-independent effects brought about by the ptsN mutation. As many as 108 spots (ca. 9% of the cell products analyzed) were influenced, positively or negatively, by the loss of IIA(Ntr). By matching this pattern with that of an rpoN::OmegaKm strain of P. putida, which lacks the sigma(54) protein, we judge that most proteins whose expression was affected by ptsN were unrelated to the alternative sigma factor. These data suggest a role of IIA(Ntr) as a general regulator, independent of the presence of repressive carbon sources and not limited to sigma(54)-dependent genes.


Asunto(s)
Proteínas de Unión al ADN , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Pseudomonas putida/genética , Proteínas Bacterianas/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/metabolismo , Electroforesis en Gel Bidimensional , Modelos Genéticos , Proteoma , ARN Polimerasa Sigma 54 , Factor sigma/metabolismo
20.
J Immunol ; 165(10): 5680-5, 2000 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-11067925

RESUMEN

Antagonism of allospecific CTL by altered MHC ligands is a potential approach to specific immunomodulation of allogeneic T cell responses in acute graft rejection and graft-vs-host disease. In this study we have analyzed the capacity of peptide analogs of a natural HLA-B27-allospecific CTL epitope to antagonize direct alloreactivity. Alanine scanning demonstrated that positions 4, 5, and 7 of the peptide epitope were critical for allorecognition. A number of relatively conservative substitutions at each of these positions were then tested for their effect on allorecognition and antagonism. All substitutions at position 5 abrogated cytotoxicity. In contrast, a few changes at positions 4 and 7 were tolerated, indicating a limited flexibility of the allospecific CTL in recognition of peptide epitope variants. Most of the substitutions impairing cytotoxicity actually induced antagonism. However, whereas epitope variants with changes at positions 4 and 7 behaved as weak or intermediate antagonists, some of the variants with changes at position 5 antagonized CTL alloreactivity almost completely. The results in this study demonstrate for the first time that antagonism of direct class I-mediated alloreactivity can be achieved by variants of a natural allospecific peptide epitope.


Asunto(s)
Citotoxicidad Inmunológica , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Antígeno HLA-B27/inmunología , Oligopéptidos/inmunología , Oligopéptidos/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Sustitución de Aminoácidos/inmunología , Presentación de Antígeno , Unión Competitiva/inmunología , Línea Celular , Células Clonales , Pruebas Inmunológicas de Citotoxicidad , Epítopos de Linfocito T/análisis , Antígeno HLA-B27/metabolismo , Humanos , Ligandos , Oligopéptidos/agonistas , Oligopéptidos/síntesis química , Unión Proteica/inmunología
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