RESUMO
To investigate the role of retinal-based pigments (opsins) in circadian photoreception in mice, animals mutated in plasma retinol binding protein were placed on a vitamin A-free diet and tested for photic induction of gene expression in the suprachiasmatic nucleus. After 10 months on the vitamin A-free diet, the majority of mice contained no detectable retinal in their eyes. These mice demonstrated fully intact photic signaling to the suprachiasmatic nucleus as measured by acute mPer mRNA induction in the suprachiasmatic nucleus in response to bright or dim light. The data suggest that a non-opsin pigment is the primary circadian photoreceptor in the mouse.
Assuntos
Células Fotorreceptoras de Vertebrados/fisiologia , Proteínas de Ligação ao Retinol/metabolismo , Transdução de Sinais/fisiologia , Núcleo Supraquiasmático/fisiopatologia , Deficiência de Vitamina A/fisiopatologia , Animais , Ritmo Circadiano/fisiologia , Cruzamentos Genéticos , Feminino , Homozigoto , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Valores de Referência , Retinaldeído/fisiologia , Proteínas de Ligação ao Retinol/deficiência , Proteínas de Ligação ao Retinol/genética , Proteínas Plasmáticas de Ligação ao Retinol , Núcleo Supraquiasmático/fisiologia , Fatores de TempoRESUMO
Mitosis requires cyclin-dependent kinase (cdk) 1-cyclin B activity [1]. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B [2]. Cdk2 is also active during mitosis [3, 4]. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable [5]. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2 [6]. Here, we show that cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. Our findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Quinase 2 Dependente de Ciclina , Xenopus , Proteínas de XenopusRESUMO
Over the past several years, discoveries from mouse genetics have had direct impact on our understanding of vitamin A metabolism. Although the metabolism of vitamin A in the mouse does have some special features (for example very large stores of liver and pulmonary retinyl esters), the ability to construct knockout and transgenic mouse models has yielded an impressive amount of information directly relevant to understanding the general principles of vitamin A transport, storage and degradation. We discuss below the metabolism of vitamin A through a number of genetically engineered mouse strains with alterations in genes that affect this metabolism. The novelty of this experimental approach is evidenced by the fact that the oldest of these strains was first reported only eight years ago.1)
Assuntos
Vitamina A/metabolismo , Animais , Intestino Delgado/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Epitélio Pigmentado Ocular/metabolismo , Pré-Albumina/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Tretinoína/metabolismoRESUMO
cAMP-dependent protein kinase is targeted to discrete subcellular locations by a family of specific anchor proteins (A-kinase anchor proteins, AKAPs). Localization recruits protein kinase A (PKA) holoenzyme close to its substrate/effector proteins, directing and amplifying the biological effects of cAMP signaling.AKAPs include two conserved structural modules: (i) a targeting domain that serves as a scaffold and membrane anchor; and (ii) a tethering domain that interacts with PKA regulatory subunits. Alternative splicing can shuffle targeting and tethering domains to generate a variety of AKAPs with different targeting specificity. Although AKAPs have been identified on the basis of their interaction with PKA, they also bind other signaling molecules, mainly phosphatases and kinases, that regulate AKAP targeting and activate other signal transduction pathways. We suggest that AKAP forms a "transduceosome" by acting as an autonomous multivalent scaffold that assembles and integrates signals derived from multiple pathways. The transduceosome amplifies cAMP and other signals locally and, by stabilizing and reducing the basal activity of PKA, it also exerts long-distance effects. The AKAP transduceosome thus optimizes the amplitude and the signal/noise ratio of cAMP-PKA stimuli travelling from the membrane to the nucleus and other subcellular compartments.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ancoragem à Quinase A , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Citoesqueleto/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Peroxissomos/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de SinaisRESUMO
Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III.
Assuntos
Proteínas de Ligação ao Retinol/genética , Vitamina A/metabolismo , Tecido Adiposo/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , DNA Complementar , Escherichia coli , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Coração/crescimento & desenvolvimento , Hibridização In Situ , Cinética , Camundongos , Dados de Sequência Molecular , Desenvolvimento Muscular , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/metabolismo , Proteínas Celulares de Ligação ao Retinol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Spodoptera , Especificidade por Substrato , TransfecçãoRESUMO
Vitamin A (retinol) is required to maintain immunity and epithelial turnover and is a key micronutrient needed for combating infection. Vitamin A actions on the immune system are diverse and cannot be accounted for by a single effect or mechanism. The actions of retinol in maintaining gut integrity in humans and immunoglobulin levels in mice was investigated. For 30 children, performance on the lactulose/mannitol test, a test commonly used to assess intestinal barrier function, was inversely correlated (P=.012) with serum retinol concentrations. Thus, children with lower serum retinol, and presumably poorer vitamin A nutritional status, are more likely to have impaired intestinal integrity. Knockout mice that have impairments in plasma retinol transport have circulating immunoglobulin levels that are half those observed in matched wild type mice. No differences were observed in B and T cell populations present in spleen, thymus, and bone marrow.
Assuntos
Absorção Intestinal , Mucosa Intestinal/fisiologia , Proteínas de Ligação ao Retinol/metabolismo , Vitamina A/fisiologia , Animais , Brasil , Estudos de Coortes , Humanos , Recém-Nascido , Lactulose/farmacocinética , Estudos Longitudinais , Manitol/farmacocinética , Camundongos , Camundongos Knockout , Estado Nutricional , Análise de Regressão , Proteínas Plasmáticas de Ligação ao Retinol , Vitamina A/sangue , Vitamina A/farmacologiaRESUMO
The folding of proteins from their initial unstructured state to their mature form has long been known to be promoted by other proteins known as chaperones and chaperonins. Recent biochemical and structural discoveries have provided dramatic insight into how these folding proteins work. This review will discuss these findings and suggest future experimental directions.
Assuntos
Proteínas de Bactérias/metabolismo , Chaperoninas/metabolismo , Escherichia coli , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteínas de Bactérias/química , Chaperoninas/química , Modelos Moleculares , Desnaturação Proteica , Relação Estrutura-AtividadeRESUMO
The amino-terminal arginine-rich motif of the phage HK022 Nun protein binds phage lambda nascent mRNA transcripts while the carboxy-terminal domain binds RNA polymerase and arrests transcription. The role of specific residues in the carboxy-terminal domain in transcription termination were investigated by mutagenesis, in vitro and in vivo functional assays, and NMR spectroscopy. Coordination of zinc to three histidine residues in the carboxy-terminus inhibited RNA binding by the amino-terminal domain; however, only two of these histidines were required for transcription arrest. These results suggest that additional zinc-coordinating residues are supplied by RNA polymerase in the context of the Nun-RNA polymerase complex. Substitution of the penultimate carboxy-terminal tryptophan residue with alanine or leucine blocks transcription arrest, whereas a tyrosine substitution is innocuous. Wild-type Nun fails to arrest transcription on single-stranded templates. These results suggest that Nun inhibition of transcription elongation is due in part to interactions between the carboxy-terminal tryptophan of Nun and double-stranded DNA, possibly by intercalation. A model for the termination activity of Nun is developed on the basis of these data.
Assuntos
Bacteriófagos/química , Regiões Terminadoras Genéticas , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Triptofano/metabolismo , Proteínas Virais/fisiologia , Zinco/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Histidina/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Ligação Proteica , Moldes Genéticos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases (PKAs), typically determined by their specific regulatory subunits. We describe changes in the cAMP signal transduction pathway during cell cycle progression in synchronized rat thyroid cells. Both PKA type II (PKAII) localization and nuclear cAMP signaling are significantly modified during G(0) and G(1)-S transitions. G(1) is characterized by PKA activation and amplified cAMP signal transduction. This is associated with a decrease in the concentration of RI and RII regulatory subunits and enhanced anchoring of PKAII to the Golgi-centrosome region. Just prior to S, the cAMP pathway is depressed. Up-regulation of the pathway by exogenous cAMP in G(1) inhibited the subsequent decay of the Cdk inhibitor p27 and delayed the onset of S phase. Forced translocation of endogenous PKAII to the cytosol down-regulated cAMP signaling, advancing the timing of p27 decay and inducing premature exit from G(1). These data indicate that membrane-bound PKA amplifies the transduction of cAMP signals in G(1) and that the length of G(1) is influenced by cAMP-PKA.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Glândula Tireoide/citologia , Proteínas Supressoras de Tumor , Animais , Transporte Biológico , Compartimento Celular , Núcleo Celular/enzimologia , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Citosol/enzimologia , Regulação para Baixo , Fase G1/fisiologia , Membranas/enzimologia , Proteínas Associadas aos Microtúbulos/metabolismo , Ratos , Transdução de SinaisRESUMO
Thyroid transcription factor 1 (TTF1) is a nuclear homeodomain protein that binds to and activates the promoters of several thyroid-specific genes, including that of the thyroglobulin gene (pTg). These genes are also positively regulated by thyroid-stimulating hormone/cyclic AMP (cAMP)/protein kinase A (PKA) signaling. We asked whether PKA directly activates TTF1. We show that cAMP/PKA activates pTg and a synthetic target promoter carrying TTF1 binding site repeats in several cell types. Activation depends on TTF1. Phosphopeptide mapping indicates that TTF1 is constitutively phosphorylated at multiple sites, and that cAMP stimulated phosphorylation of one site, serine 337, in vivo. However, alanine substitution at this residue or at all sites of phosphorylation did not reduce PKA activation of pTg. Thus, PKA stimulates TTF1 transcriptional activity in an indirect manner, perhaps by recruiting to or removing from the target promoter another regulatory factor(s).
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Tireoglobulina/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Alanina/química , Animais , Células COS , Linhagem Celular , Meios de Cultura Livres de Soro , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Mutação , Células PC12 , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ratos , Fator Nuclear 1 de Tireoide , Ativação Transcricional , TransfecçãoRESUMO
The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation. RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA. To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts. When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template. Nun may arrest transcription by anchoring RNAP to DNA.
Assuntos
DNA Viral/metabolismo , Fatores de Alongamento de Peptídeos , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Azidas , Proteínas de Bactérias/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , Reagentes de Ligações Cruzadas , RNA Polimerases Dirigidas por DNA/metabolismo , Ditiotreitol/farmacologia , Escherichia coli/enzimologia , Escherichia coli/virologia , Proteínas de Escherichia coli , Dados de Sequência Molecular , Fenantrolinas/metabolismo , Ligação Proteica , Piridinas , RNA Viral/metabolismo , Moldes Genéticos , Fatores de Transcrição/química , Fatores de Elongação da Transcrição , Ensaio de Placa Viral , Proteínas Virais/química , Zinco/farmacologiaRESUMO
Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Replicação do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Cromatina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Densitometria , Ativação Enzimática , Glutationa Transferase/farmacologia , Immunoblotting , Masculino , Mitose/efeitos dos fármacos , Complexo de Reconhecimento de Origem , Proteínas Recombinantes de Fusão/farmacologia , Espermatozoides/fisiologia , Xenopus/genética , Xenopus/crescimento & desenvolvimentoRESUMO
Retinol-binding protein (RBP) is the sole specific transport protein for retinol (vitamin A) in the circulation, and its single known function is to deliver retinol to tissues. Within tissues, retinol is activated to retinoic acid, which binds to nuclear receptors to regulate transcription of >300 diverse target genes. In the eye, retinol is also activated to 11-cis-retinal, the visual chromophore. We generated RBP knockout mice (RBP(-/-)) by gene targeting. These mice have several phenotypes. Although viable and fertile, they have reduced blood retinol levels and markedly impaired retinal function during the first months of life. The impairment is not due to developmental retinal defect. Given a vitamin A-sufficient diet, the RBP(-/-) mice acquire normal vision by 5 months of age even though blood retinol levels remain low. Deprived of dietary vitamin A, vision remains abnormal and blood retinol declines to undetectable levels. Another striking phenotype of the mutant mice is their abnormal retinol metabolism. The RBP(-/-) mice can acquire hepatic retinol stores, but these cannot be mobilized. Thus, their vitamin A status is extremely tenuous and dependent on a regular vitamin A intake. Unlike wild-type mice, serum retinol levels in adult RBP(-/-) animals become undetectable after only a week on a vitamin A-deficient diet and their retinal function rapidly deteriorates. Thus RBP is needed for normal vision in young animals and for retinol mobilization in times of insufficient dietary intake, but is otherwise dispensable for the delivery of retinol to tissues.
Assuntos
Proteínas de Ligação ao Retinol/fisiologia , Vitamina A/metabolismo , Fatores Etários , Animais , Disponibilidade Biológica , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dieta , Eletrorretinografia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Retina/fisiologia , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Transgenes , Visão Ocular/fisiologia , Vitamina A/sangueRESUMO
The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the lambda nutR site in vivo. We show that it is also essential for Nun termination at lambda nutL. Three recessive mis-sense nusG mutations have been isolated that inhibit termination by Nun at lambda nutR. The mutations are ineffective in a lambda pL nutL fusion, even when lambda nutR replaces lambda nutL. The mutant strains support lambda growth, indicating that lambda N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both lambda nutR and lambda nutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.
Assuntos
Proteínas de Bactérias/genética , Colífagos/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Virais/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , Modelos Genéticos , Mutagênese Sítio-Dirigida , Plasmídeos , Fator Rho/fisiologia , Proteínas Ribossômicas/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição , Proteínas Virais/metabolismoAssuntos
Bacteriófagos/genética , Produtos do Gene vif/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/genética , Transcrição Gênica , Sequência de Aminoácidos , Bacteriófagos/metabolismo , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Produtos do Gene vif/química , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/química , Regiões Promotoras Genéticas , RNA Viral/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
A yeast two-hybrid screen revealed that regulatory subunits (RII) of PKAII bind the Yotiao protein. Yotiao interacts with the NR1 subunit of the NMDA receptor. A purified C-terminal fragment of Yotiao binds PKAII, via an RII binding site constituted by amino acid residues 1452-1469, with a dissociation constant (K(d)) between 50 and 90 nM in vitro. A stable complex composed of Yotiao, RII and NR1 was immunoprecipitated from whole rat brain extracts. Immunostaining analysis disclosed that Yotiao, RIIbeta and NR1 colocalize in striatal and cerebellar neurons. Co-assembly of Yotiao/PKAII complexes with NR1 subunits may promote cAMP-dependent modulation of NMDA receptor activity at synapses, thereby influencing brain development and synaptic plasticity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas de Ancoragem à Quinase A , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas do Citoesqueleto/química , Ligantes , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.
Assuntos
Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Estrutura Secundária de Proteína , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40 , Cinética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Fenótipo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Distinct A Kinase Anchor Proteins (AKAPs) immobilize and concentrate protein kinase A II (PKAII) isoforms at specific intracellular locations. AKAP121 binds and targets PKAIIalpha to the cytoplasmic surface of mitochondria. Mechanisms that control expression of this mitochondrial AKAP are unknown. We have cloned cDNA for rat AKAP121 and show that AKAP121 protein expression is regulated by thyroid stimulating hormone (TSH) and cAMP. Differentiated thyroid cells (TL5) accumulate AKAP121 upon incubation with TSH or a cAMP analog. Levels of total and newly synthesized AKAP121 mRNA also increased after treatment. AKAP121 mRNA accumulated in the presence of cycloheximide, suggesting that transcription of the anchor protein gene is directly controlled by cAMP and PKA. AKAP121 is induced with similar kinetics when an unrelated, spermatocyte-derived cell line (GC-2) is incubated with 8-chlorophenylthio-cAMP. Thus, AKAP121 concentration may be controlled by hormones that activate adenylate cyclase. This mode of regulation could provide a general mechanism for (a) enhancing the sensitivity of distal organelles to cAMP and (b) shifting the focus of cAMP-mediated signaling from cytoplasm to organelles.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Regulação da Expressão Gênica , Proteínas/genética , Espermatócitos/metabolismo , Glândula Tireoide/metabolismo , Tireotropina/farmacologia , Proteínas de Ancoragem à Quinase A , Sequência de Aminoácidos , Animais , Compartimento Celular , Linhagem Celular , Clonagem Molecular , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Complementar/genética , Masculino , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Ratos , Transdução de Sinais , Transcrição GênicaRESUMO
Vision in all vertebrates is dependent on an exchange of retinoids between the retinal pigment epithelium and the visual photoreceptors. It has been proposed that the interphotoreceptor retinoid-binding protein (IRBP) is essential for this intercellular exchange, and that it serves to prevent the potentially cytotoxic effects of retinoids. Although its precise function in vivo has yet to be defined, the early expression of IRBP suggests that it may also be required for normal photoreceptor development. To further assess the biological role of IRBP, we generated transgenic mice with targeted disruption of the IRBP gene (IRBP-/- mice). Specifically, homologous recombination was used to replace the first exon and promoter region of the IRBP gene with a phosphoglycerate kinase-promoted neomycin-resistant gene. Immunocytochemical and Western blot analyses demonstrated the absence of IRBP expression in the IRBP-/- mice. As early as postnatal day 11, histological examination of the retinas of IRBP-/- mice revealed a loss of photoreceptor nuclei and changes in the structural integrity of the receptor outer segments. At 30 d of age, the photoreceptor abnormalities in IRBP-/- mice were more severe, and electroretinographic recordings revealed a marked loss in photic sensitivity. In contrast, no morphological or electrophysiological changes were detected in age-matched heterozygotes. These observations indicate that normal photoreceptor development and function are highly dependent on the early expression of IRBP, and that in the absence of IRBP there is a slowly progressive degeneration of retinal photoreceptors.
Assuntos
Proteínas do Olho , Células Fotorreceptoras/fisiologia , Proteínas de Ligação ao Retinol/fisiologia , Envelhecimento/fisiologia , Animais , Northern Blotting , Western Blotting , Eletrorretinografia , Imuno-Histoquímica , Camundongos , Camundongos Knockout/genética , Hibridização de Ácido Nucleico , Células Fotorreceptoras/fisiopatologia , Retina/metabolismo , Retina/patologia , Retina/fisiopatologia , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , RibonucleasesRESUMO
The Nun protein of phage HK022 is an RNA binding protein of the arginine-rich motif family. Nun binds the phage lambda boxB RNA sequence (BOXB) on nascent lambda transcripts and arrests transcription elongation. Binding to BOXB is inhibited by Zn2+ and stimulated by the Escherichia coli NusA protein. Deletion of the Nun C-terminal region enhances BOXB binding and makes it independent of Zn2+ and NusA. The C terminus of Nun thus appears to interfere with the N-terminal RNA binding motif. NusA relieves this interference by binding to the Nun C terminus and forming a complex with Nun and BOXB. However, NusA also inhibits transcription arrest in vitro, in the absence of the other Nus factors. Nun deleted for its C terminus fails to bind RNA polymerase (RNAP) (RNAP) or NusA in vitro or to arrest transcription in vivo or in vitro. Our findings are consistent with the idea that NusA inhibits transcription arrest by binding to the Nun C terminus, thus blocking the interaction between Nun and RNAP. NusG, NusB, and NusE factors restore transcription arrest, presumably by promoting transfer of Nun from NusA to RNAP.