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1.
J Proteome Res ; 11(2): 1163-74, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22103298

RESUMO

The synaptic ribbon is an electron-dense structure found in hair cells and photoreceptors. The ribbon is surrounded by neurotransmitter-filled vesicles and considered to play a role in vesicle release. We generated an objective, quantitative analysis of the protein composition of the ribbon complex using a mass spectrometry-based proteomics analysis. Our use of affinity-purified ribbons and control IgG immunoprecipitations ensure that the identified proteins are indeed associated with the ribbon complex. The use of mouse tissue, where the proteome is complete, generated a comprehensive analysis of the candidates. We identified 30 proteins (comprising 56 isoforms and subunits) associated with the ribbon complex. The ribbon complex primarily comprises proteins found in conventional synapses, which we categorized into 6 functional groups: vesicle handling (38.5%), scaffold (7.3%), cytoskeletal molecules (20.6%), phosphorylation enzymes (10.6%), molecular chaperones (8.2%), and transmembrane proteins from the presynaptic membrane firmly attached to the ribbon (11.3%). The 3 CtBP isoforms represent the major protein in the ribbon whether calculated by molar amount (30%) or by mass (20%). The relatively high quantity of phosphorylation enzymes suggests a very active and regulated structure. The ribbon appears to comprise a concentrated cluster of proteins dealing with vesicle creation, retention and distribution, and consequent exocytosis.


Assuntos
Imunoprecipitação/métodos , Proteínas do Tecido Nervoso/química , Células Fotorreceptoras/química , Proteoma/análise , Sinapses/química , Animais , Bovinos , Núcleo Celular/química , Exocitose , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Modelos Moleculares , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Proteômica , Retina/citologia
2.
J Exp Med ; 194(7): 883-92, 2001 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-11581311

RESUMO

Peptide fragments of self-proteins bound to major histocompatibility complex molecules within the thymus are important for positively selecting T cell receptor (TCR)-bearing CD4(+)CD8(+) double positive (DP) thymocytes for further maturation. The relationship between naturally processed thymic self-peptides and TCR-specific cognate peptides is unknown. Here we employ HPLC purification of peptides released from H-2K(b) molecules of the C57BL/6 thymus in conjunction with mass spectrometry (MS) and functional profiling to identify a naturally processed K(b)-bound peptide positively selecting the N15 TCR specific for the vesicular stomatitis virus octapeptide (VSV8) bound to K(b). The selecting peptide was identified in 1 of 80 HPLC fractions and shown by tandem MS (MS/MS) sequencing to correspond to residues 68-75 of the MLRQ subunit of the widely expressed mitochondrial NADH ubiquinone oxidoreductase (NUbO(68-75)). Of note, the peptide differs at six of its eight residues from the cognate peptide VSV8 and functions as a weak agonist for mature CD8 single positive (SP) N15 T cells, with activity 10,000-fold less than VSV8. In N15 transgenic (tg) recombinase activating gene 2(-/)- transporter associated with antigen processing 1(-/)- fetal thymic organ culture, NUbO(68-75) induces phenotypic and functional differentiation of N15 TCR bearing CD8 SP thymocytes. Failure of NUbO(68-75) to support differentiation of a second K(b)-restricted TCR indicates that its inductive effects are not general.


Assuntos
Apresentação de Antígeno , Antígenos H-2/imunologia , Mitocôndrias/imunologia , Oligopeptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Timo/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Complexo I de Transporte de Elétrons , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/enzimologia , NADH NADPH Oxirredutases/imunologia , Oligopeptídeos/isolamento & purificação , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Seleção Genética , Timo/citologia
3.
J Exp Med ; 178(1): 27-47, 1993 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-8315383

RESUMO

Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DR8. Using the complementary techniques of mass spectrometry and Edman microsequencing, > 200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10-34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino- and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (> 85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.


Assuntos
Alelos , Antígenos HLA-DR/genética , Fragmentos de Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Cromatografia de Afinidade , Antígenos HLA-DR/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo
4.
Science ; 255(5048): 1127-9, 1992 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-1546313

RESUMO

The yeast transcription factor IIA (TFIIA), a component of the basal transcription machinery of RNA polymerase II and implicated in vitro in regulation of basal transcription, is composed of two subunits of 32 and 13.5 kilodaltons. The genes that encode these subunits, termed TOA1 and TOA2, respectively, were cloned. Neither gene shares obvious sequence similarity with the other or with any other previously identified genes. The recombinant factor bound to a TATA binding protein-DNA complex and complemented yeast and mammalian in vitro transcription systems depleted of TFIIA. Both the TOA1 and TOA2 genes are essential for growth of yeast.


Assuntos
Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Clonagem Molecular , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Fator de Transcrição TFIIA , Transcrição Gênica
5.
Science ; 282(5395): 1900-4, 1998 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-9836642

RESUMO

Transcription of naked DNA in vitro requires the general transcription factors and RNA polymerase II. However, this minimal set of factors is not sufficient for transcription when the DNA template is packaged into chromatin. Here, a factor that facilitates activator-dependent transcription initiation on chromatin templates was purified. This factor, remodeling and spacing factor (RSF), has adenosine triphosphate-dependent nucleosome-remodeling and spacing activities. Polymerases that initiate transcription with RSF can only extend their transcripts in the presence of FACT (facilitates chromatin transcription). Thus, the minimal factor requirements for activator-dependent transcription on chromatin templates in vitro have been defined.


Assuntos
Cromatina/genética , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cromatina/metabolismo , Dimerização , Células HeLa , Humanos , Peso Molecular , RNA Polimerase II/metabolismo , Moldes Genéticos , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação
6.
Science ; 260(5105): 219-22, 1993 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-8385799

RESUMO

Alternative splicing of precursor messenger RNAs (pre-mRNAs) is a common mechanism of regulating gene expression. SR proteins are a family of pre-mRNA splicing factors that are structurally related and evolutionarily conserved. Any member of the SR family can complement a splicing-deficient extract that lacks the entire family of SR proteins. Here it is demonstrated that particular SR proteins have distinct functions in alternative pre-mRNA splicing in vitro. In addition, SR proteins are differentially expressed in a variety of tissues. These results suggest a fundamental role for SR proteins in the regulation of alternative splicing.


Assuntos
Processamento Alternativo , Precursores de RNA/genética , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Extratos Celulares , Células HeLa , Humanos , Dados de Sequência Molecular , Splicing de RNA , RNA Viral/genética , Vírus 40 dos Símios/genética
7.
Science ; 271(5257): 1873-6, 1996 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-8596958

RESUMO

The human ELL gene on chromosome 19 undergoes frequent translocations with the trithorax-like MLL gene on chromosome 11 in acute myeloid leukemias. Here, ELL was shown to encode a previously uncharacterized elongation factor that can increase the catalytic rate of RNA polymerase II transcription by suppressing transient pausing by polymerase at multiple sites along the DNA. Functionally, ELL resembles Elongin (SIII), a transcription elongation factor regulated by the product of the von Hippel-Lindau (VHL) tumor suppressor gene. The discovery of a second elongation factor implicated in oncogenesis provides further support for a close connection between the regulation of transcription elongation and cell growth.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Neoplasias , Fatores de Alongamento de Peptídeos , Proto-Oncogenes , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Elonguina , Genes Supressores de Tumor , Histona-Lisina N-Metiltransferase , Humanos , Leucemia/genética , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição , Translocação Genética , Doença de von Hippel-Lindau/genética
8.
Science ; 269(5229): 1439-43, 1995 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-7660129

RESUMO

The Elongin (SIII) complex activates elongation by mammalian RNA polymerase II by suppressing transient pausing of the polymerase at many sites within transcription units. Elongin is a heterotrimer composed of A, B, and C subunits of 110, 18, and 15 kilodaltons, respectively. Here, the mammalian Elongin A gene was isolated and expressed, and the Elongin (SIII) complex reconstituted with recombinant subunits. Elongin A is shown to function as the transcriptionally active component of Elongin (SIII) and Elongin B and C as regulatory subunits. Whereas Elongin C assembles with Elongin A to form an AC complex with increased specific activity, Elongin B, a member of the ubiquitin-homology gene family, appears to serve a chaperone-like function, facilitating assembly and enhancing stability of the Elongin (SIII) complex.


Assuntos
Ligases , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Sequência de Bases , Elonguina , Genes Supressores de Tumor , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , RNA Mensageiro/biossíntese , Proteínas Recombinantes/metabolismo , Temperatura , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Proteína Supressora de Tumor Von Hippel-Lindau
9.
Science ; 285(5435): 1926-8, 1999 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-10489375

RESUMO

Antithrombin, a member of the serpin family, functions as an inhibitor of thrombin and other enzymes. Cleavage of the carboxyl-terminal loop of antithrombin induces a conformational change in the molecule. Here it is shown that the cleaved conformation of antithrombin has potent antiangiogenic and antitumor activity in mouse models. The latent form of intact antithrombin, which is similar in conformation to the cleaved molecule, also inhibited angiogenesis and tumor growth. These data provide further evidence that the clotting and fibrinolytic pathways are directly involved in the regulation of angiogenesis.


Assuntos
Antineoplásicos/farmacologia , Antitrombinas/farmacologia , Neovascularização Patológica/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antitrombinas/química , Antitrombinas/isolamento & purificação , Antitrombinas/metabolismo , Carcinoma de Células Pequenas/irrigação sanguínea , Carcinoma de Células Pequenas/tratamento farmacológico , Linhagem Celular , Meios de Cultivo Condicionados , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos SCID , Transplante de Neoplasias , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Conformação Proteica , Células Tumorais Cultivadas
10.
Science ; 293(5532): 1142-6, 2001 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-11498592

RESUMO

Modification of histones is an important element in the regulation of gene expression. Previous work suggested a link between acetylation and phosphorylation, but questioned its mechanistic basis. We have purified a histone H3 serine-10 kinase complex from Saccharomyces cerevisiae and have identified its catalytic subunit as Snf1. The Snf1/AMPK family of kinases function in conserved signal transduction pathways. Our results show that Snf1 and the acetyltransferase Gcn5 function in an obligate sequence to enhance INO1 transcription by modifying histone H3 serine-10 and lysine-14. Thus, phosphorylation and acetylation are targeted to the same histone by promoter-specific regulation by a kinase/acetyltransferase pair, supporting models of gene regulation wherein transcription is controlled by coordinated patterns of histone modification.


Assuntos
Proteínas de Ligação a DNA , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Ativação Transcricional , Acetilação , Domínio Catalítico , Histona Acetiltransferases , Lisina/metabolismo , Mio-Inositol-1-Fosfato Sintase/genética , Nucleossomos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia
11.
Science ; 268(5211): 726-31, 1995 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-7732382

RESUMO

Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces neurite outgrowth in a murine neuroblastoma cell line. Tritium-labeled lactacystin was used to identify the 20S proteasome as its specific cellular target. Three distinct peptidase activities of this enzyme complex (trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activities) were inhibited by lactacystin, the first two irreversibly and all at different rates. None of five other proteases were inhibited, and the ability of lactacystin analogs to inhibit cell cycle progression and induce neurite outgrowth correlated with their ability to inhibit the proteasome. Lactacystin appears to modify covalently the highly conserved amino-terminal threonine of the mammalian proteasome subunit X (also called MB1), a close homolog of the LMP7 proteasome subunit encoded by the major histocompatibility complex. This threonine residue may therefore have a catalytic role, and subunit X/MB1 may be a core component of an amino-terminal-threonine protease activity of the proteasome.


Assuntos
Acetilcisteína/análogos & derivados , Cisteína Endopeptidases/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Complexos Multienzimáticos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Treonina/efeitos dos fármacos , Acetilcisteína/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Cisteína Endopeptidases/metabolismo , Eletroforese em Gel de Poliacrilamida , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Complexo de Endopeptidases do Proteassoma , Células Tumorais Cultivadas
12.
Science ; 262(5134): 750-4, 1993 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-8235597

RESUMO

Nuclear factor of activated T cells (NFAT) is a transcription factor that regulates expression of the cytokine interleukin-2 (IL-2) in activated T cells. The DNA-binding specificity of NFAT is conferred by NFATp, a phosphoprotein that is a target for the immunosuppressive compounds cyclosporin A and FK506. Here, the purification of NFATp from murine T cells and the isolation of a complementary DNA clone encoding NFATp are reported. A truncated form of NFATp, expressed as a recombinant protein in bacteria, binds specifically to the NFAT site of the murine IL-2 promoter and forms a transcriptionally active complex with recombinant protein fragment react with T cell NFATp. The molecular cloning of NFATp should allow detailed analysis of a T cell transcription factor that is central to initiation of the immune response.


Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Nucleares , Linfócitos T/química , Fatores de Transcrição/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Imunossupressores/farmacologia , Interleucina-2/genética , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , RNA Mensageiro/análise , Proteínas Recombinantes , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
13.
Science ; 292(5516): 464-8, 2001 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-11292862

RESUMO

HIF (hypoxia-inducible factor) is a transcription factor that plays a pivotal role in cellular adaptation to changes in oxygen availability. In the presence of oxygen, HIF is targeted for destruction by an E3 ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein (pVHL). We found that human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. Because proline hydroxylation requires molecular oxygen and Fe(2+), this protein modification may play a key role in mammalian oxygen sensing.


Assuntos
Hidroxiprolina/metabolismo , Ligases , Oxigênio/fisiologia , Proteínas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Hipóxia Celular , Linhagem Celular , Cobalto/farmacologia , Desferroxamina/farmacologia , Humanos , Hidroxilação , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/química , Transativadores/genética , Células Tumorais Cultivadas , Ubiquitinas/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau
14.
Science ; 284(5414): 657-61, 1999 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-10213691

RESUMO

The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in most human kidney cancers. The VHL protein is part of a complex that includes Elongin B, Elongin C, and Cullin-2, proteins associated with transcriptional elongation and ubiquitination. Here it is shown that the endogenous VHL complex in rat liver also includes Rbx1, an evolutionarily conserved protein that contains a RING-H2 fingerlike motif and that interacts with Cullins. The yeast homolog of Rbx1 is a subunit and potent activator of the Cdc53-containing SCFCdc4 ubiquitin ligase required for ubiquitination of the cyclin-dependent kinase inhibitor Sic1 and for the G1 to S cell cycle transition. These findings provide a further link between VHL and the cellular ubiquitination machinery.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Culina , Proteínas F-Box , Ligases , Peptídeo Sintases/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Inibidoras de Quinase Dependente de Ciclina , Elonguina , Proteína 7 com Repetições F-Box-WD , Proteínas Fúngicas/metabolismo , Fígado , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases Associadas a Fase S , Proteínas Ligases SKP Culina F-Box , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau
15.
Neuron ; 23(1): 45-54, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10402192

RESUMO

The Drosophila latheo (lat) gene was identified in a behavioral screen for olfactory memory mutants. The original hypomorphic latP1 mutant (Boynton and Tully, 1992) shows a structural defect in adult brain. Homozygous lethal lat mutants lack imaginal discs, show little cell proliferation in the CNS of third instar larvae, and die as early pupae. latP1 was cloned, and all of the above mentioned defects of hypomorphic or homozygous lethal lat mutants were rescued with a lat+ transgene. lat encodes a novel protein with homology to a subunit of the origin recognition complex (ORC). Human and Drosophila LAT both associate with ORC2 and are related to yeast ORC3, suggesting that LAT functions in DNA replication during cell proliferation.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila/genética , Memória/fisiologia , Mutação/genética , Neurônios/patologia , Condutos Olfatórios/fisiopatologia , Sequência de Aminoácidos/genética , Animais , Animais Geneticamente Modificados , Encéfalo/anormalidades , Encéfalo/patologia , Encéfalo/fisiopatologia , Divisão Celular/fisiologia , Sistema Nervoso Central/patologia , Anormalidades Congênitas/genética , Drosophila/crescimento & desenvolvimento , Transtornos da Memória/genética , Dados de Sequência Molecular , Mutação/fisiologia , Complexo de Reconhecimento de Origem , Pupa/fisiologia , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genética , Transgenes/fisiologia
16.
Curr Biol ; 9(9): 485-8, 1999 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-10322114

RESUMO

Ewing family tumors result from the effects of chromosomal translocations that fuse the Ewing sarcoma (EWS) gene to various genes encoding transcription factors. The resulting chimeric EWS fusion proteins are transcriptional activators with transforming potential that have received much study. By contrast, the cellular function of somatic EWS remains obscure. EWS belongs to a family of RNA-binding proteins thought to play role in RNA synthesis or processing. Here, we show that EWS interacts with Pyk2, a protein tyrosine kinase implicated in a variety of signal transduction processes. G-protein-coupled receptor signaling and other stimuli of Pyk2 kinase activity significantly block the interaction between EWS and Pyk2. Furthermore, as assessed by sucrose gradient centrifugation, EWS partitions with dense ribosome-containing fractions in a manner that is enhanced by signaling from the G-protein-coupled m1 muscarinic acetylcholine receptor (mAChR). We conclude that extranuclear EWS is a previously unrecognized target of G-protein-coupled receptor regulation.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Muscarínicos/metabolismo , Ribonucleoproteínas/metabolismo , Sarcoma de Ewing , Animais , Quinase 2 de Adesão Focal , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Células PC12 , Proteína EWS de Ligação a RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismo
17.
Curr Biol ; 8(7): 393-403, 1998 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-9545197

RESUMO

BACKGROUND: Transversion mutations are caused by 8-oxoguanine (OG), a DNA lesion produced by the spontaneous oxidation of guanine nucleotides, which mis-pairs with adenine during replication. Resistance to this mutagenic threat is mediated by the GO system, the components of which are functionally conserved in bacteria and mammals. To date, only one of three GO system components has been identified in the budding yeast Saccharomyces cerevisiae, namely the OG:C-specific glycosylase/lyase yOgg1. Furthermore, S. cerevisiae has been reported to contain a unique glycosylase/lyase activity, yOgg2, which excises OG residues opposite adenines. Paradoxically, according to the currently accepted model, yOgg2 activity should increase the mutagenicity of OG lesions. Here we report the isolation of yOgg2 and the elucidation of its role in oxidative mutagenesis. RESULTS: Borohydride-dependent cross-linking using an OG-containing oligonucleotide substrate led to the isolation of yOgg1 and a second protein, Ntg1, which had previously been shown to process oxidized pyrimidines in DNA. We demonstrate that Ntg1 has OG-specific glycosylase/lyase activity indistinguishable from that of yOgg2. Targeted disruption of the NTG1 gene resulted in complete loss of yOgg2 activity and yeast lacking NTG1 had an elevated rate of A:T to C:G transversions. CONCLUSIONS: The Ntg1 and yOgg2 activities are encoded by a single gene. We propose that yOgg2 has evolved to process OG:A mis-pairs that have arisen through mis-incorporation of 8-oxo-dGTP during replication. Thus, the GO system in S. cerevisiae is fundamentally distinct from that in bacteria and mammals.


Assuntos
Reparo do DNA , Guanina/análogos & derivados , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Marcadores de Afinidade , Sequência de Aminoácidos , Sequência de Bases , Boroidretos , Primers do DNA/genética , Reparo do DNA/genética , DNA Fúngico/química , DNA Fúngico/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA-Formamidopirimidina Glicosilase , Marcação de Genes , Genes Fúngicos , Guanina/química , Guanina/metabolismo , Dados de Sequência Molecular , Mutagênese , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/isolamento & purificação , N-Glicosil Hidrolases/metabolismo , Oxirredução , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
18.
Curr Biol ; 6(8): 968-80, 1996 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-8805338

RESUMO

BACKGROUND: Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins. RESULTS: Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G. CONCLUSIONS: S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.


Assuntos
Reparo do DNA/genética , Proteínas de Escherichia coli , N-Glicosil Hidrolases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA-Formamidopirimidina Glicosilase , Dados de Sequência Molecular , Família Multigênica , N-Glicosil Hidrolases/isolamento & purificação , N-Glicosil Hidrolases/metabolismo , Oligodesoxirribonucleotídeos , Especificidade por Substrato
19.
Mol Cell Biol ; 21(22): 7629-40, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11604499

RESUMO

The yeast NuA4 complex is a histone H4 and H2A acetyltransferase involved in transcription regulation and essential for cell cycle progression. We identify here a novel subunit of the complex, Yng2p, a plant homeodomain (PHD)-finger protein homologous to human p33/ING1, which has tumor suppressor activity and is essential for p53 function. Mass spectrometry, immunoblotting, and immunoprecipitation experiments confirm the stable stoichiometric association of this protein with purified NuA4. Yeast cells harboring a deletion of the YNG2 gene show severe growth phenotype and have gene-specific transcription defects. NuA4 complex purified from the mutant strain is low in abundance and shows weak histone acetyltransferase activity. We demonstrate conservation of function by the requirement of Yng2p for p53 to function as a transcriptional activator in yeast. Accordingly, p53 interacts with NuA4 in vitro and in vivo, an interaction reminiscent of the p53-ING1 physical link in human cells. The growth defect of Delta yng2 cells can be rescued by the N-terminal part of the protein, lacking the PHD-finger. While Yng2 PHD-finger is not required for p53 interaction, it is necessary for full expression of the p53-responsive gene and other NuA4 target genes. Transcriptional activation by p53 in vivo is associated with targeted NuA4-dependent histone H4 hyperacetylation, while histone H3 acetylation levels remain unchanged. These results emphasize the essential role of the NuA4 complex in the control of cell proliferation through gene-specific transcription regulation. They also suggest that regulation of mammalian cell proliferation by p53-dependent transcriptional activation functions through recruitment of an ING1-containing histone acetyltransferase complex.


Assuntos
Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Proteínas de Plantas/fisiologia , Proteínas de Saccharomyces cerevisiae , Ativação Transcricional , Proteínas Supressoras de Tumor , Acetilação , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Histona Acetiltransferases , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteínas Nucleares , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Proteínas , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transativadores/genética , Transativadores/metabolismo , Transativadores/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologia
20.
Mol Cell Biol ; 17(10): 6029-39, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9315662

RESUMO

Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.


Assuntos
HIV-1/genética , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Transativadores/metabolismo , Ativação Transcricional/fisiologia , Alanina , Sequência de Aminoácidos , Extratos Celulares , Núcleo Celular/química , Clonagem Molecular , Coenzimas/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Produtos do Gene tat/fisiologia , Glutamina , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Transativadores/análise , Transativadores/genética , Transativadores/isolamento & purificação , Fatores de Elongação da Transcrição , Produtos do Gene tat do Vírus da Imunodeficiência Humana
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