RESUMO
The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Divisão Celular , Linhagem Celular , Primers do DNA/genética , Estabilidade de Medicamentos , Fosfatases de Especificidade Dupla , Genes p53 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases JNK Ativadas por Mitógeno , MAP Quinase Quinase 4 , Espectrometria de Massas , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/metabolismo , Treonina/química , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Raios UltravioletaRESUMO
In the standard model of cytokine-induced signal transducer and activator of transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STAT3 is recruited to the cytoplasmic side of the cell surface receptor complex from within a cytosolic monomer pool. By using Superose-6 gel-filtration chromatography, we have discovered that there is little monomeric STAT3 (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B cell line and rat liver). The bulk of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distributions in the size range 200-400 kDa ("statosome I") and 1-2 MDa ("statosome II"). Upon treatment of Hep3B cells with interleukin-6 (IL-6) for 30 min (i) cytosolic tyrosine-phosphorylated STAT3 was found to be in complexes of size ranging from 200-400 kDa to 1-2 MDa; (ii) a small pool of monomeric STAT3 and tyrosine-phosphorylated STAT3 eluting at 80-100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A "homodimer") co-eluted with catalase at 230 kDa. In order to identify the protein components of the 200-400-kDa statosome I cytosolic complexes, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range from 20 to 114 kDa co-shifted with STAT3; three of these (p60, p20a, and p20b) were co-shifted in an IL-6-dependent manner. In-gel tryptic fragmentation and mass spectroscopy identified the major IL-6-dependent STAT3-co-shifted p60 protein as the chaperone GRP58/ER-60/ERp57. Taken together, these data (i) emphasize the absence of a detectable STAT3 monomer pool in the cytosol of cytokine-free liver cells as posited by the standard model, and (ii) suggest an alternative model for STAT signaling in which STAT3 proteins function in the cytoplasm as heteromeric complexes with accessory scaffolding proteins, including the chaperone GRP58.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Transdução de Sinais/fisiologia , Transativadores/fisiologia , Animais , Transporte Biológico , Compartimento Celular/fisiologia , Citocinas/farmacologia , Humanos , Ratos , Fator de Transcrição STAT3 , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Protein seryl/threonyl phosphatase inhibitors such as calyculin A block inside-out and outside-in platelet signaling. Our studies demonstrate that the addition of calyculin A blocks platelet adhesion and spreading on fibrinogen, responses that depend on integrin alphaIIb beta3 signaling. We hypothesized that this reflects a change in alphaIIb beta3 structure caused by a specific state of phosphorylation. We show that addition of calyculin A leads to increased phosphorylation of the beta3 subunit, and phosphoamino acid analysis reveals that only threonine residues become phosphorylated; sequence analysis by Edman degradation established that threonine 753 became stoichiometrically phosphorylated during inhibition of platelet phosphatases by calyculin A. This region of beta3 is linked to outside-in signaling such as platelet spreading responses. The effect of calyculin A on platelet adhesion and spreading and on the phosphorylation of T-753 in beta3 is reversed by the calcium ionophore A23187, demonstrating that these effects of calyculin A are not generally toxic ones. We propose that phosphorylation of beta3 on threonine 753, a region of beta3 linked to outside-in signaling, may be a mechanism by which integrin alphaIIb beta3 function is regulated.
Assuntos
Plaquetas/metabolismo , Citoplasma/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Plaquetas/citologia , Adesão Celular/efeitos dos fármacos , Humanos , Toxinas Marinhas , Oxazóis/farmacologia , Fosforilação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Transdução de Sinais , Treonina/metabolismoRESUMO
Synaptosomes were incubated in the presence of FeSO4 to test the hypothesis that iron-catalyzed oxidative damage causes an increase in the ubiquitination of synaptosomal proteins. Incubation with 10 or 50 microM FeSO4 caused concentration-dependent increases in carbonyl groups (an indication of protein oxidation) and ubiquitinated proteins (determined by probing Western blots with a monoclonal antibody to ubiquitin). Differences in protein ubiquitination occurred within 5 min of incubation, indicating a rapid response to oxidative stress. Results of experiments with MG-132, an inhibitor of the degradation of ubiquitinated proteins, suggested that oxidative damage stimulated ubiquitination rather than inhibited degradation of ubiquitinated proteins. The data are consistent with the hypothesis that synaptic terminals utilize the ubiquitin/proteasome proteolytic pathway to degrade oxidatively damaged proteins.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo/fisiologia , Sinaptossomos/metabolismo , Ubiquitinas/metabolismo , Animais , Compostos Ferrosos/farmacologia , Leupeptinas/farmacologia , Masculino , Proteínas do Tecido Nervoso/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sinaptossomos/efeitos dos fármacos , Fatores de TempoRESUMO
Proteasome inhibitors have been used to demonstrate that many proteins of the signal transduction pathways are regulated by degradation via the ubiquitin-proteasome pathway. The key question is what events target specific proteins for ubiquitination at one time and prevent ubiquitination at other times? In this review, we develop the notion that there is a direct relationship between the phosphorylation/dephosphorylation cascade of the signal transduction pathways and the targeting of the regulatory proteins for ubiquitination. We present examples where phosphorylation appears to alter the interaction between the targeting systems and the substrate by modifying the targeting system, the substrate, or both. These interacting systems are seen in the response of p53, c-jun and ATF-2 in cells subjected to stress or DNA damage and to the normal regulated response in a variety of pathways including the IkappaB-NFkappaB and JAK-STAT pathways. The interweaving of the two post-translational networks, phosphorylation and ubiquitination, provides a powerful insight into global regulatory control pathways.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Estresse Fisiológico , Animais , Proteínas de Ligação a DNA/metabolismo , Quinase 3 da Glicogênio Sintase , Humanos , Quinase I-kappa B , Proteínas Quinases JNK Ativadas por Mitógeno , Janus Quinase 3 , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT1 , Transativadores/metabolismo , beta CateninaRESUMO
The proteasome consists of a 20S proteolytic core particle (CP) and a 19S regulatory particle (RP), which selects ubiquitinated substrates for translocation into the CP. An eight-subunit subcomplex of the RP, the lid, can be dissociated from proteasomes prepared from a deletion mutant for Rpn10, an RP subunit. A second subcomplex, the base, contains all six proteasomal ATPases and links the RP to the CP. The base is sufficient to activate the CP for degradation of peptides or a nonubiquitinated protein, whereas the lid is required for ubiquitin-dependent degradation. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The lid subunits share sequence motifs with components of the COP9/signalosome complex and eIF3, suggesting that these functionally diverse particles have a common evolutionary ancestry.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/fisiologia , Proteínas de Ligação a DNA , Complexos Multienzimáticos/química , Complexos Multienzimáticos/fisiologia , Proteínas de Plantas/fisiologia , Proteínas , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Ubiquitinas/metabolismo , Complexo do Signalossomo COP9 , Proteínas de Transporte/fisiologia , Cisteína Endopeptidases/ultraestrutura , Proteínas Fúngicas/química , Proteínas Fúngicas/fisiologia , Proteínas Fúngicas/ultraestrutura , Microscopia Eletrônica , Complexos Multienzimáticos/ultraestrutura , Complexos Multiproteicos , Peptídeo Hidrolases , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas c-ets , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Ubiquitinas/fisiologiaRESUMO
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3alpha. Mouse UBR1p (E3alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans approximately 120 kilobases of genomic DNA and contains approximately 50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.
Assuntos
DNA Complementar/genética , Proteínas Fúngicas/genética , Ligases , Proteínas/genética , Proteínas de Saccharomyces cerevisiae , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 15 , Clonagem Molecular , DNA Complementar/isolamento & purificação , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Saccharomyces cerevisiae , Alinhamento de Sequência , Análise de SequênciaRESUMO
The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.
Assuntos
Cisteína Endopeptidases/química , Endopeptidases , Complexos Multienzimáticos/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatografia de Afinidade , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , DNA Fúngico/química , Biblioteca Gênica , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mapeamento de Peptídeos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Regulatory proteins are often ubiquitinated, depending on their phosphorylation status as well as on their association with ancillary proteins that serve as adapters of the ubiquitination machinery. We previously demonstrated that c-Jun is targeted for ubiquitination by its association with inactive c-Jun NH2-terminal kinase (JNK). Phosphorylation by activated JNK protects c-Jun from ubiquitination, thus by prolonging its half-life. In the study reported here, we determined the ability of JNK to target ubiquitination of its other substrates (Elk1 and activating transcription factor 2 (ATF2)) and associated proteins (ATF2 and JunB). We demonstrate that phosphorylation by JNK protects ATF2, but not Elk1, from JNK-targeted ubiquitination. We also show that association of inactive JNK with JunB or ATF2 is necessary to target them for ubiquitination. Unlike its targeting of c-Jun, JNK requires additional cellular components, yet to be identified, to target the ubiquitination of ATF2. Elk1 is phosphorylated by JNK, but JNK neither associates with nor targets Elk1 for ubiquitination. The implications for the dual role of JNK in the regulation of ubiquitination and stability of c-Jun, ATF2, and JunB in normally growing versus stressed cells are discussed.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA , Proteínas Quinases Ativadas por Mitógeno , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismo , Células 3T3 , Fator 2 Ativador da Transcrição , Animais , Cisteína Endopeptidases/metabolismo , Hidrólise , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Complexos Multienzimáticos/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas/metabolismo , Especificidade por Substrato , Proteínas Elk-1 do Domínio etsRESUMO
Vacuolar-type proton pumps are complex heterooligomers. When dissociated into subcomplexes and subunits, the partial reactions of ATP hydrolysis and transmembranous proton flow can be assigned to isolated domains. Data suggest that the molecular site of ATP hydrolysis resides within the 70-kDa subunit but that ATPase activity likely requires at least three additional subunits of 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have now cloned and sequenced the 70-kDa subunit from bovine brain and have expressed the protein in insect Sf9 (Spodoptera frugiperda) cells with a recombinant baculovirus. When purified, the protein has no significant ATPase activity but can be photoaffinity labeled with [alpha 32P]ATP and UV irradiation with an apparent Kd of 35 microM. When reconstituted with biochemically prepared 58-, 40-, and 33-kDa polypeptides, the recombinant 70-kDa subunit restores Ca(2+)-activated ATP hydrolysis to a specific activity of 0.6 mumol P(i).mg protein-1.min-1, thus demonstrating that ATP hydrolysis in vacuolar-type proton pumps is dependent upon both the 70-kDa subunit as well as multi-subunit interactions.
Assuntos
Vesículas Revestidas/enzimologia , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Bovinos , Clonagem Molecular , Primers do DNA/química , DNA Complementar/genética , Ativação Enzimática , Técnicas In Vitro , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas RecombinantesRESUMO
The chronic survival of many endoparasites is dependent on the ability of these organisms to escape the host immune response. Identification of the molecular mechanisms by which these organisms evade this response may yield novel approaches in the development of anti-inflammatory agents. We describe here the discovery and characterization of a novel 41-kilodalton glycoprotein from the canine hookwork (Ancylostoma caninum) that potently inhibits CD11/CD18-dependent neutrophil function in vitro. Neutrophil inhibitory factor (NIF) blocks the adhesion of activated human neutrophils to vascular endothelial cells as well as the release of H2O2 from activated neutrophils, over a similar concentration range (IC50 10-20 nM). Studies aimed at determining the nature of the NIF binding site on neutrophils revealed selective, high affinity binding of this protein to the integrin CD11b/CD18. A cDNA encoding NIF was isolated from a canine hookworm cDNA library. NIF comprises a mature polypeptide of 257 amino acids, preceded by a 17-amino acid leader. The mature protein has 10 cysteines and has seven potential N-linked glycosylation sites. NIF has no significant sequence homologies to any previously reported protein. As such, NIF represents a prototype of a novel class of leukocyte function inhibitors.
Assuntos
Ancylostomatoidea/fisiologia , Antígenos CD/metabolismo , Glicoproteínas/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Antígeno de Macrófago 1/metabolismo , Neutrófilos/fisiologia , Sequência de Aminoácidos , Ancylostomatoidea/metabolismo , Animais , Sequência de Bases , Antígenos CD18 , Clonagem Molecular , Primers do DNA , DNA Complementar , Cães , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/farmacologia , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Proteínas de Membrana/sangue , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
3 alpha-Hydroxysteroid dehydrogenase is a cytosolic, monomeric, NADPH-dependent oxidoreductase which reduces 3-keto-5-dihydrosteroids to their tetrahydro products. We present here the first partial amino acid sequence data for the human liver enzyme and show these sequences to be identical to the deduced amino acid sequence for human hepatic chlordecone reductase. In addition, these two enzymes exhibit similar substrate and cofactor specificities and immunological reactivity. The results suggest that the natural substrates for chlordecone reductase are 3-keto-5-dihydrosteroids and that these two proteins may be identical.
Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Oxirredutases do Álcool/metabolismo , Fígado/enzimologia , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/imunologia , 3-alfa-Hidroxiesteroide Desidrogenase (B-Específica) , Oxirredutases do Álcool/química , Oxirredutases do Álcool/imunologia , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Humanos , Immunoblotting , Dados de Sequência Molecular , Peso Molecular , Álcool Oxidorredutases Dependentes de NAD(+) e NADP(+) , Fragmentos de Peptídeos/química , Especificidade por Substrato , TripsinaRESUMO
A histone, macroH2A, nearly three times the size of conventional H2A histone, was found in rat liver nucleosomes. Its N-terminal third is 64 percent identical to a full-length mouse H2A. However, it also contains a large nonhistone region. This region has a segment that resembles a leucine zipper, a structure known to be involved in dimerization of some transcription factors. Nucleosomes containing macroH2A may have novel functions, possibly involving interactions with other nuclear proteins.
Assuntos
Histonas/química , Fígado/ultraestrutura , Nucleossomos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/química , Histonas/genética , Zíper de Leucina , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
The zeta subunit of the T cell antigen receptor (TCR) exists primarily as a disulfide-linked homodimer. This receptor subunit is important in TCR-mediated signal transduction and is a substrate for a TCR-activated protein tyrosine kinase. The zeta chain was found to undergo ubiquitination in response to receptor engagement. This posttranslational modification occurred in normal T cells and tumor lines. Both nonphosphorylated and phosphorylated zeta molecules were modified, and at least one other TCR subunit, CD3 delta, was also ubiquitinated after activation of the receptor. These findings suggest an expanded role for ubiquitination in transmembrane receptor function.
Assuntos
Ativação Linfocitária/fisiologia , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Ubiquitinas/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Complexo CD3 , Células Cultivadas , Hibridomas/imunologia , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/isolamento & purificação , Baço/imunologia , Ubiquitinas/isolamento & purificaçãoRESUMO
A mouse ubiquitin clone that recognizes multiple transcripts overexpressed in murine tumors compared to normal epidermis was isolated by differential screening of complementary DNA libraries from mouse squamous cell carcinomas. Coding region probes detected five ubiquitin transcripts. Oligonucleotides were designed for unique parts of three mouse ubiquitin gene complementary DNA clones. The overexpressed transcripts at 2.4, 2.8, 6.4, and 7.8 kilobases (kb) were detected by an oligonucleotide specific for a mouse UbC polyubiquitin clone. A 1.2-kb UbB overexpressed transcript was detected by an oligonucleotide for a mouse four-unit polyubiquitin, and a 0.7-kb UbA overexpressed transcript was recognized by an oligonucleotide for the mouse ubiquitin carboxyl-extension protein of 52 amino acids. All three classes of transcripts were induced in mouse skin by the hyperproliferative agent ethylphenyl propionate and by the tumor promoting agent 12-O-tetradecanoylphorbol-13- acetate. Heat shock of cultured keratinocytes induced both the 6.4- and 7.8-kb transcripts recognized by the UbC-specific oligonucleotide. Consistent with the overexpression of the ubiquitin transcripts, the level of free ubiquitin protein, as determined by Western analysis, was elevated in the tumors and proliferating epidermis as compared to normal epidermis. Our results indicate that the overexpression of ubiquitin genes could be related to a sustained state of proliferation and stress in the tumors compared to the normal resting epidermis.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Neoplasias/biossíntese , Neoplasias Cutâneas/metabolismo , Ubiquitinas/biossíntese , Alcinos/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular/fisiologia , Sondas de DNA , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Temperatura Alta , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/genética , RNA Neoplásico/análise , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Ubiquitinas/efeitos dos fármacos , Ubiquitinas/genéticaRESUMO
This study was designed to clone, sequence, and express the full-length cDNA for the human platelet p24/CD9 antigen. A 1.3-kilobase cDNA clone was identified that has an open reading frame encoding a mature protein of 228 amino acids (approximately 25,400 Da) containing 10 cysteine residues and four putative transmembrane domains. The identity of the clone was confirmed by: (i) its predicted size, (ii) identity to four peptide sequences from the isolated protein including the NH2 terminus, and (iii) expression of the isolated clone in Xenopus oocytes and Chinese hamster ovary cells. p24/CD9 has sequence identity (24-34%) to four other cell-surface proteins: ME491, a melanoma antigen; CO-029, a carcinoma antigen; CD37, a leukocyte antigen; and SM23, an antigen of the parasitic helminth Schistosoma mansoni. The five proteins have a similar number of amino acids and are characterized by the presence of four putative transmembrane domains. These data indicate the presence of a new family of surface antigens that may function in cellular activation and differentiation.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação/genética , Plaquetas/metabolismo , DNA/genética , Glicoproteínas de Membrana , Sequência de Aminoácidos , Animais , Sequência de Bases , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Testes de Precipitina , Mapeamento por Restrição , Alinhamento de Sequência , Tetraspanina 29 , Transcrição Gênica , XenopusRESUMO
Sixty-two snake venoms were screened to identify those which specifically inhibit the adhesive protein binding function of the glycoprotein (GP) IIb-IIIa complex, the receptor-mediating platelet aggregation. Although 52 of these venoms inhibited GPIIb-IIIa, only one of these, from the southeastern pigmy rattlesnake, Sistrurus m. barbouri, was specific for GPIIb-IIIa versus other integrins. The peptide responsible for this activity, termed barbourin, was sequenced and found to be highly homologous to other peptides of the viper venom GPIIb-IIIa antagonist family but was the first member which did not contain the Arg-Gly-Asp (RGD) amino acid sequence, believed to be required for inhibition of receptor function. Instead, barbourin contains the sequence, Lys-Gly-Asp (KGD). The conservative Lys for Arg substitution appears to be the sole structural feature which imparts integrin specificity to barbourin, since venom peptide analogs with Lys substitutions were also specific for GPIIb-IIIa. Thus, barbourin represents a new structural model useful for designing potent and GPIIb-IIIa-specific compounds that may have therapeutic value as platelet aggregation inhibitors.
Assuntos
Venenos de Crotalídeos , Venenos de Crotalídeos/farmacologia , Integrinas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/farmacologia , Sequência de Aminoácidos , Venenos de Crotalídeos/isolamento & purificação , Fibrinogênio/antagonistas & inibidores , Fibrinogênio/metabolismo , Dados de Sequência Molecular , Inibidores da Agregação Plaquetária/farmacologia , Glicoproteínas da Membrana de Plaquetas/isolamento & purificaçãoRESUMO
Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in mammalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor. Experiments with receptor mutants and with short peptides derived from the kinase insert region of the PDGF receptor showed that the recombinant p85 binds to a well-defined phosphotyrosine-containing sequence of the receptor. p85 appears to be the subunit of PI3-kinase that links the enzyme to the ligand-activated receptor.
Assuntos
Fosfotransferases/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Compostos de Sulfidrila/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Linhagem Celular , Clonagem Molecular , DNA , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases , Fosfoproteínas/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full-length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump.
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Proteínas/genética , ATPases Vacuolares Próton-Translocadoras , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Northern Blotting , Química Encefálica , Bovinos , Dados de Sequência Molecular , RNA/análiseRESUMO
On tissue sections of Alzheimer brain, 4 antibodies to tau immunolabel not only neurofibrillary tangles, neuritic plaques and neuropil threads but also the tangle-free cytoplasm of a subset of hippocampal and cortical neurons we believe to be at a stage of alteration preceding the formation of paired helical filaments (PHF). Pretreatment of tissue sections with alkaline phosphatase leads to an increase in staining intensity and in number of immunoreactive lesions with antibodies directed to an amino terminal and to a mid-region of the tau molecule. The diffuse neuronal staining could not be observed with any of 7 monoclonal antibodies recognizing ubiquitin. We conclude (1) that abnormal phosphorylation of tau occurs prior to its incorporation into PHF and leads to its accumulation in the nerve cell body and (2) that ubiquitin is seen associated only when a neurofibrillary tangle is already formed.