RESUMO
Asialoglycoprotein receptor (ASGP-R) is an endocytic C-type lectin receptor in hepatocytes that clears plasma glycoconjugates containing a terminal galactose or N-acetylgalactosamine. The carbohydrate recognition domain (CRD) of ASGP-R has three Ca(2+) binding sites (sites 1, 2 and 3), with Ca(2+) at site 2 being directly involved in ligand binding. Following endocytosis, the ligands are released from ASGP-R in endosomes to allow receptor recycling to the cell membrane. Although dissociation of the receptor-ligand complex is mediated by the acidic environment within the mature endosomes, many of these complexes also dissociate in the early time of endocytosis, where pH is approximately neutral. To investigate the mechanism of ligand release from ASGP-R in early endosomes, we examined the binding mode of Ca(2+) and ligands to ASGP-R CRD by NMR. We demonstrate that sites 1 and 2 of ASGP-R are high affinity Ca(2+) binding sites, site 3 is low affinity, and that Ca(2+) ions bind to sites 1 and 2 cooperatively. The pH and Ca(2+) concentration dependences of Ca(2+) binding states indicated that early endosome conditions favor apo-ASGP-R CRD, allowing ligand release. Our results elucidated that the cooperative binding mode of Ca(2+) makes it possible for ASGP-R to be more sensitive to Ca(2+) concentrations in early endosomes, and plays an important role in the efficient release of ligand from ASGP-R. In our proposed mechanism, ASGP-R can rapidly release Ca(2+) and its ligand even at nearly neutral pH. Sequence comparisons of endocytic C-type lectin receptors suggest that this mechanism is common in their family.
Assuntos
Receptor de Asialoglicoproteína/química , Receptor de Asialoglicoproteína/metabolismo , Sequência de Aminoácidos , Receptor de Asialoglicoproteína/genética , Cálcio/metabolismo , Dicroísmo Circular , Endossomos/metabolismo , Galactose/metabolismo , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Soluções , Ressonância de Plasmônio de SuperfícieRESUMO
A practical antibiotics-free plasmid expression system in cyanobacteria was developed by using the complementation of cyanobacterial recA null mutation with the EscherichiacolirecA gene on the plasmid. This system was applied to the production of polyhydroxyalkanoate (PHA), a biodegradable plastic, and the transgenic cyanobacteria stably maintained the pha genes for PHA production in the antibiotics-free medium, and accumulated up to 52% cell dry weight of PHA.
Assuntos
Antibacterianos/química , Biotecnologia/métodos , Cianobactérias/metabolismo , Poli-Hidroxialcanoatos/química , Materiais Biocompatíveis/química , Biodegradação Ambiental , Dióxido de Carbono/química , Clonagem Molecular , Escherichia coli/metabolismo , Teste de Complementação Genética , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Plásticos , Recombinases Rec A/genética , Synechococcus/metabolismoRESUMO
The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.
Assuntos
Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Ribulose-Bifosfato Carboxilase/biossíntese , Ribulose-Bifosfato Carboxilase/genética , Synechococcus/genética , Escherichia coli/genética , Mutação da Fase de Leitura/genética , Plasmídeos/genética , Biossíntese de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Synechococcus/enzimologiaRESUMO
The CO(2)-regulatory function of the AT-rich element in the promoter for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbc) genes in the cyanobacterium Synechococcus sp. PCC7002 was analyzed using the transcription factor decoy approach. Double-stranded phosphorothioate AT-rich oligonucleotides with high affinity for a sequence-specific DNA-binding protein were successfully introduced into cyanobacterial cells in culture without any transfection reagent. The AT-rich decoy oligonucleotides interfered with CO(2) regulation of rbc expression by blocking the binding of the sequence-specific DNA-binding protein, indicating that the AT-rich element plays a critical role in CO(2) regulation for rbc genes. The decoy oligonucleotide approach to cyanobacteria provides a simple and excellent tool for investigating transcriptional regulation in vivo.
Assuntos
Dióxido de Carbono/antagonistas & inibidores , Cianobactérias/enzimologia , Regulação Bacteriana da Expressão Gênica , Oligonucleotídeos/farmacologia , Ribulose-Bifosfato Carboxilase/genética , Sequência Rica em At , Transporte Biológico , Cianobactérias/efeitos dos fármacos , Cianobactérias/genética , Proteínas de Ligação a DNA/metabolismo , Repressão Enzimática , Fluoresceína-5-Isotiocianato , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Ativação Transcricional/efeitos dos fármacosRESUMO
We analyzed the promoter of the genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (rbc) in the cyanobacterium Synechococcus sp. PCC7002 and localized the CO(2)-regulatory element. Cyanobacterial transformants were constructed with several DNA segments of the rbc promoter fused to the chloramphenicol acetyltransferase (CAT) gene, and their acetyltransferase activities were analyzed under 0.03% and 1% CO(2) conditions. We found that the AT-rich element localized from -262 to -291 relative to the rbc translation-starting site was required for CO(2)-dependent repression. Fluorescent-labeled oligonucleotide probes of identical sequence to the AT-rich element were reacted with protein extracts from cells cultured under conditions of low and high CO(2) atmospheric content. We detected a gel retardation complex of a strong signal intensity in extracts from cells cultured under 15% CO(2), but only a weak signal from cells cultured under 1% CO(2). Moreover, a DNA affinity precipitation assay identified a 16-kDa protein that bound to nucleotide sequences within the AT-rich element. The partial amino acid sequence of the protein was similar to the deduced protein sequences of ORF129 and ORF155 from Synechocystis 6803. Our findings suggest that the AT-rich element plays a role as a negative CO(2)-regulatory element and its trans-acting factor possibly regulates the rbc transcription in response to CO(2) levels.
Assuntos
Dióxido de Carbono/farmacologia , Cianobactérias/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Ribulose-Bifosfato Carboxilase/genética , Transativadores/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cianobactérias/genética , Cianobactérias/metabolismo , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Ribulose-Bifosfato Carboxilase/metabolismo , Transativadores/genéticaRESUMO
The preference for the optimal nucleotide of the mammalian translation initiation AUG context [GCCGCC(A/G)CCaugG] is generally more pronounced in the highly expressed genes than in the transcription factor genes at the -9 through -1 positions in humans. The influence of amino acid preference on the nucleotide choice at the +4 position was also examined.
Assuntos
Regulação da Expressão Gênica/genética , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas/genética , Aminoácidos/química , Humanos , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Fatores de TranscriçãoRESUMO
We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay proved reliable measurement and multiple detection of DNA binding proteins.
