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1.
Structure ; 2(10): 981-90, 1994 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-7866749

RESUMO

BACKGROUND: In Gram-negative proteobacteria, the nitrogen level in the cell is reflected by the uridylylation status of a key signal transducing protein, PII. PII modulates the activity of glutamine synthetase (GS) through its interaction with adenylyl transferase and it represses the expression of GS by acting in concert with nitrogen regulatory protein II. RESULTS: The three-dimensional structure of the Escherichia coli PII trimer has been determined at 2.7 A resolution. PII shows a low level of structural similarity to a broad family of alpha/beta proteins and contains a double beta alpha beta motif. The PII trimer contains three beta-sheets, each of which is composed of strands from each of the three monomers. These are surrounded by six alpha-helices. CONCLUSIONS: The structure of PII suggests potential regions of interaction with other proteins and serves as an initial step in understanding its signal transducing role in nitrogen regulation.


Assuntos
Proteínas de Bactérias/química , Escherichia coli/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência Conservada , Eletroquímica , Escherichia coli/genética , Escherichia coli/metabolismo , Glutamato-Amônia Ligase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Nucleotidiltransferases/metabolismo , Proteínas PII Reguladoras de Nitrogênio , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais
2.
Antiviral Res ; 130: 1-6, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26965420

RESUMO

Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N-(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection (Fraser et al., 2014b). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2α in DENV infection using guanabenz, a specific inhibitor of eIF2α dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/fisiologia , Fenretinida/farmacologia , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Linhagem Celular , Células Cultivadas , Camundongos , Modelos Biológicos , Fosforilação , Biossíntese de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , Estresse Fisiológico , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Replicação Viral/efeitos dos fármacos
3.
J Mol Biol ; 282(1): 149-65, 1998 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-9733647

RESUMO

GlnK is a recently discovered homologue of the PII signal protein, an indicator of the nitrogen status of bacteria. PII occupies a central position in the dual cascade that regulates the activity of glutamine synthetase and the transcription of its gene. The complete role of Escherichia coli GlnK is yet to be determined, but already it is known that GlnK behaves like PII and can substitute for PII under some circumstances thereby adding to the subtleties of nitrogen regulation. There are also indications that the roles of the two proteins differ; the expression of PII is constitutive while that of GlnK is linked to the level of nitrogen in the cell. The discovery of GlnK begs the question of why E. coli has both GlnK and PII. Clearly, the structural similarities and differences of GlnK and PII will lead to a better understanding of how PII-like proteins function in E. coli and other organisms. We have crystallised and solved the X-ray structure of GlnK at 2.0 A resolution. The asymmetric unit has two independent copies of the GlnK subunit and both pack around 3-fold axes to form trimers. The trimers have a barrel-like core with recognition loops (the T-loops) that protrude from the top of the molecule. The two GlnK molecules have similar core structures to PII but differ significantly at the C terminus and the loops. The T-loops of the two GlnK molecules also differ from each other; one is disordered while the conformation of the other is stabilised by lattice contacts. The conformation of the ordered T-loop of GlnK differs from that observed in the PII structure despite the fact that their sequences are very similar. The structures suggest that the T-loops do not have a rigid structure and that they may be flexible in solution. The presence of a turn of 310 helix in the middle of the T-loop suggests that secondary structure could form when it interacts with soluble receptor enzymes.Co-crystals of GlnK and ATP were used to determine the structure of the complex. In these crystals, GlnK occupies a position of 3-fold symmetry. ATP binds in a cleft on the side of the molecule. The cleft is suitably positioned for ATP to influence the flexible T-loops. It is found at the junction of two beta sheets and is formed by two peptides one of which contains a variant of the "Gly-loop" found in other mononucleotide binding proteins. This sequence, Thr-Gly-X-X-Gly-Asp-Gly-Lys-Ile-Phe, forms part of the B-loop and is conserved in a wide variety of organisms that include bacteria, algae and archeabacteria. This sequence is more highly conserved than the functional T-loop, suggesting that ATP has an important role in PII-like proteins.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Transporte/química , Sequência de Aminoácidos , Ânions/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Proteínas PII Reguladoras de Nitrogênio , Conformação Proteica , Homologia de Sequência de Aminoácidos , Transdução de Sinais
4.
Gene ; 129(1): 9-16, 1993 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-8335265

RESUMO

A cosmid (pND320) bearing 42.5 kb of Escherichia coli chromosomal DNA, including the genes between xylE and ssb near minute 92 on the linkage map, was isolated by selection for complementation of a dnaB mutation. Known nucleotide (nt) sequences were used to align restriction maps in this region to the physical map of the chromosome (coordinates 4319.5 to 4362 kb), and to locate precisely and define the orientations of 19 genes. Predicted physical linkage of sequenced genes across unsequenced gaps of defined length was confirmed by the nt sequence analysis of fragments subcloned from pND320. Mutant complementation by plasmids showed that ubiA is located between malM and plsB. A previously sequenced long open reading frame that encodes the C-terminal portion of the E. coli ubiA product (4-hydroxybenzoate polyprenyltransferase, HPTase) shows a high degree of sequence identity with the corresponding segment of yeast HPTase (the COQ2 gene product). Comparison of homologous regions from E. coli and Salmonella typhimurium was used to locate precisely the gene alr that encodes alanine racemase (ARase) between dnaB and tyrB. Subcloning of alr downstream from tandem bacteriophage lambda promoters produced a plasmid that directed high-level overproduction of a soluble approx. 40-kDa protein with ARase activity.


Assuntos
Alanina Racemase/genética , Alquil e Aril Transferases , Cromossomos Bacterianos/ultraestrutura , Escherichia coli/genética , Genes Bacterianos , Transferases/genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Clonagem Molecular , DNA Bacteriano/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mapeamento por Restrição
5.
FEBS Lett ; 337(3): 255-8, 1994 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-8293810

RESUMO

The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix-assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 x 0.2 x 0.3 mm were grown and analysed by X-ray diffraction. The crystals belong to space group P6(3) with a = b = 61.6 A, c = 56.3 A and Vm of 2.5 for one subunit in the asymmetric unit. A low-resolution electron density map showed electron density concentrated around a three-fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 +/- 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/química , Fenômenos Químicos , Físico-Química , Cromatografia por Troca Iônica , Cristalização , Cristalografia por Raios X , Genes Reguladores , Substâncias Macromoleculares , Espectrometria de Massas , Peso Molecular , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio , Transdução de Sinais
6.
FEBS Lett ; 391(1-2): 223-8, 1996 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-8706922

RESUMO

The 3D structure of PII, the central protein that controls the level of transcription and the enzymatic activity of glutamine synthetase in enteric bacteria revealed that residues 37-55 form the "T' loop, part of which protrudes from the core of the protein. Within this loop are the only two tyrosine residues that occur in the polypeptide, and one of them, Tyr-51, has been shown by chemical modification studies to be the site of uridylylation. Since tyrosine at position 46 is conserved in all known PII proteins, oligonucleotide directed mutagenesis was used to investigate the role of the two residues. Changing Tyr-51 to phenylalanine or serine abolished uridylylation. Altering tyrosine at position 46 to phenylalanine affected the rate of uridylylation of the protein. This latter mutation does not alter the structure of PII but the reduction in the uridylylation efficiency suggests a role for this residue in recognition and binding of the sensor enzyme uridylyl transferase.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Bactérias/metabolismo , Sequência de Bases , Cristalografia por Raios X , Primers do DNA , Escherichia coli/genética , Cinética , Substâncias Macromoleculares , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas PII Reguladoras de Nitrogênio , Fenilalanina , Plasmídeos , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Serina , Software , Tirosina , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , Uridina
7.
FEMS Microbiol Lett ; 125(2-3): 219-24, 1995 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-7875569

RESUMO

The subcellular distribution of the soluble flavohaemoglobin (HMP) of Escherichia coli has been determined. Cells over-expressing HMP from the cloned hmp gene on a multicopy plasmid were fractionated by osmotic shock and lysozyme treatment. Spectral analysis of subcellular fractions showed the CO-binding haemoprotein to be cytoplasmic. However, Western blotting using antibody raised to purified HMP revealed approximately 30% of the protein to be periplasmic in the over-expressing strain. Western analysis also revealed substantial levels of periplasmic HMP in a strain expressing only chromosomally encoded protein but none in an hmp mutant. The results are discussed in relation to protein function and the similar distribution reported for Vitreoscilla globin.


Assuntos
Proteínas de Bactérias/análise , Di-Hidropteridina Redutase , Proteínas de Escherichia coli , Escherichia coli/química , Flavoproteínas/análise , Hemeproteínas/análise , NADH NADPH Oxirredutases , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Monóxido de Carbono/metabolismo , Citoplasma/química , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Globinas/química , Hemeproteínas/biossíntese , Hemeproteínas/química , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Frações Subcelulares/química
8.
Farmaco ; 56(1-2): 33-6, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11347963

RESUMO

Our research is directed towards enhancing the understanding of the molecular biology of dengue virus replication with the ultimate goal being to develop novel antiviral strategies based on preventing critical inter- or intra-molecular interactions required for the normal virus life cycle. The viral RNA-dependent RNA polymerase (NS5) and the viral helicase (NS3) interaction offers a possible target for inhibitors to bind and prevent replication. In this study the yeast-two hybrid system was used to show that a small region of NS5 interacts with NS3, and also with the cellular nuclear transport receptor importin-beta. Furthermore, intramolecular interaction between the two putative domains of NS5 can also be detected by the yeast two-hybrid assay. We have also modified the colony lift assay for the beta-galactosidase reporter activity in intact yeast cells which reflects the strength of interaction between two proteins to a microtiter plate format. This assay offers a unique opportunity to screen for small molecule compounds that block physiologically important interactions.


Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Dados de Sequência Molecular , RNA Helicases , Serina Endopeptidases , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacos
9.
Antiviral Res ; 99(3): 301-6, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23769930

RESUMO

Infection by one of the 4 distinct serotypes of dengue virus (DENV) threatens >40% of the world's population, with no efficacious vaccine or antiviral agent currently available. DENV replication through the virus-encoded nonstructural protein (NS) 5 protein occurs in the infected cell cytoplasm, but NS5 from DENV2 has thus far been shown to localize strongly in the nucleus throughout infection. Here we use specific antibodies cross-reactive with NS5 from DENV1-4 to demonstrate nuclear localization of NS5 from all DENV serotypes for the first time in both infected as well as transfected cells, although to differing extents. The small-molecule inhibitor Ivermectin was inhibitory towards both DENV 1 and 2 NS5 interaction with its nuclear transporter importin α/ß in vitro, and protected against infection from DENV1-4. Ivermectin thus has potential in the clinical setting as a dengue antiviral.


Assuntos
Antivirais/farmacologia , Núcleo Celular/virologia , Vírus da Dengue/efeitos dos fármacos , Dengue/virologia , Ivermectina/farmacologia , Proteínas não Estruturais Virais/metabolismo , Citoplasma/virologia , Dengue/tratamento farmacológico , Vírus da Dengue/classificação , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Humanos , Transporte Proteico/efeitos dos fármacos , Proteínas não Estruturais Virais/genética
10.
Biochem J ; 255(2): 581-8, 1988 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-3060113

RESUMO

A dihydropteridine reductase from Escherichia coli was purified to apparent homogeneity. It is a dimeric enzyme with identical subunits (Mr 27000) and a free N-terminal group. It can use NADH (Vmax./Km 3.36 s-1) and NADPH (Vmax./Km 1.07 s-1) when 6-methyldihydro-(6H)-pterin is the second substrate, as well as quinonoid dihydro-(6H)-biopterin (Vmax./Km 0.69 s-1), dihydro-(6H)-neopterin (Vmax./Km 0.58 s-1), dihydro-(6H)-monapterin 0.66 s-1), 6-methyldihydro-(6H)-pterin and cis-6,7-dimethyldihydro-(6H)-pterin (Vmax./Km 0.66 s-1) when NADH is the second substrate. The pure reductase has a yellow colour and contains bound FAD. The enzyme also has pterin-independent NADH and NADPH oxidoreductase activities when potassium ferricyanide is the electron acceptor.


Assuntos
Di-Hidropteridina Redutase/metabolismo , Escherichia coli/enzimologia , NADH NADPH Oxirredutases/metabolismo , Sequência de Aminoácidos , Cromatografia por Troca Iônica , Di-Hidropteridina Redutase/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , Metotrexato/farmacologia , Dados de Sequência Molecular , Peso Molecular , NAD/metabolismo , Espectrofotometria Ultravioleta , Especificidade por Substrato
11.
Biol Chem Hoppe Seyler ; 373(10): 1067-73, 1992 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1418677

RESUMO

E. coli Dihydropteridine reductase, known to have a pterin-independent oxidoreductase activity with potassium ferricyanide as electron donor, has now been shown to possess also dihydrofolate reductase activity. The kinetic parameters for dihydrofolate reductase activity have been determined. The ratio of the three activities, dihydropteridine reductase, dihydrofolate reductase and pterin-independent oxidoreductase activity is 1.0, 0.05 and 4.3, respectively. The enzyme, a flavoprotein which is unstable in the presence of dithiothreitol, was shown to be a monomer with a molecular mass of 25.7 kDa. The apparent lack of discrimination between hydride transfer from the pyridine nucleotide to N5 of the pterin in the dihydropteridine reductase reaction and C6 of folate in the dihydrofolate reaction suggested that the FAD prosthetic group may be involved in the hydride transfers. The flavoprotein inhibitor N,N- dimethylpropargylamine inhibited the dihydropteridine reductase and oxidoreductase reactions differently and did not affect the dihydrofolate reductase activity however.


Assuntos
Di-Hidropteridina Redutase/metabolismo , Escherichia coli/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Di-Hidropteridina Redutase/isolamento & purificação , Estabilidade Enzimática , Peso Molecular , NADH Desidrogenase/metabolismo
12.
Eur J Biochem ; 268(7): 2028-37, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11277925

RESUMO

PII is a signal transduction protein that is part of the cellular machinery used by many bacteria to regulate the activity of glutamine synthetase and the transcription of its gene. The structure of PII was solved using a hexagonal crystal form (form I). The more physiologically relevant form of PII is a complex with small molecule effectors. We describe the structure of PII with ATP obtained by analysis of two different crystal forms (forms II and III) that were obtained by co-crystallization of PII with ATP. Both structures have a disordered recognition (T) loop and show differences at their C termini. Comparison of these structures with the form I protein reveals changes that occur on binding ATP. Surprisingly, the structure of the PII/ATP complex differs with that of GlnK, a functional homologue. The two proteins bind the base and sugar of ATP in a similar manner but show differences in the way that they interact with the phosphates. The differences in structure could account for the differences in their activities, and these have been attributed to a difference in sequence at position 82. It has been demonstrated recently that PII and GlnK form functional heterotrimers in vivo. We construct models of the heterotrimers and examine the junction between the subunits.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Transdução de Sinais , Proteínas de Bactérias/química , Proteínas de Transporte/química , Cristalografia por Raios X , Glutamato-Amônia Ligase/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Proteínas PII Reguladoras de Nitrogênio , Conformação Proteica
13.
Acta Crystallogr D Biol Crystallogr ; 52(Pt 1): 93-104, 1996 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-15299730

RESUMO

The structure of the bacterial signal transduction protein P(II) has been refined to an R factor of 13.2% using 3sigma data between 10 and 1.9 A. The crystals exhibited twinning by merohedry and X-ray intensities were corrected using the method of Fisher & Sweet [Fisher & Sweet (1980). Acta Cryst. A36, 755-760] prior to refinement. Our earlier 2.7 A structure [Cheah, Carr, Suffolk, Vasudevan, Dixon & Ollis (1994). Structure, 2, 981-990] served as a starting model. P(II) is a trimeric molecule, each subunit has a mass of 12.4 kDa and contains 112 amino-acid residues. The refined model includes all 1065 protein atoms per subunit plus 312 water molecules. The high-resolution refinement confirms the correctness of our 2.7 A model, although it leads to a redefinition of the extent of various secondary-structural elements. The monomeric structure of P(II) exhibits an interlocking double betaalphabeta fold. This is a stable fold found in a number of proteins with diverse functions. The association of the protein into a trimer leads to a new structure which we describe in detail. The effects of crystal packing forces are discussed and potential interaction sites with other proteins and effector molecules are identified.

14.
Mol Gen Genet ; 226(1-2): 49-58, 1991 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2034230

RESUMO

In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of Mr 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.


Assuntos
Proteínas de Bactérias/genética , Di-Hidropteridina Redutase/genética , Escherichia coli/genética , Genes Bacterianos , Hemeproteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Di-Hidropteridina Redutase/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Dados de Sequência Molecular , Proteínas PII Reguladoras de Nitrogênio , Mapeamento por Restrição , Alinhamento de Sequência , Solubilidade
15.
EMBO J ; 16(18): 5562-71, 1997 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-9312015

RESUMO

Adenylyl transferase (ATase) is the bifunctional effector enzyme in the nitrogen assimilation cascade that controls the activity of glutamine synthetase (GS) in Escherichia coli. This study addresses the question of whether the two antagonistic activities of ATase (adenylylation and deadenylylation) occur at the same or at different active sites. The 945 amino acid residue ATase has been truncated in two ways, so as to produce two homologous polypeptides corresponding to amino acids 1-423 (AT-N) and 425-945 (AT-C). We demonstrate that ATase has two active sites; AT-N carries a deadenylylation activity and AT-C carries an adenylylation activity. Glutamine activates the adenylylation reaction of the AT-C domain, whereas alpha-ketoglutarate activates the deadenylylation reaction catalysed by the AT-N domain. With respect to the regulation by the nitrogen status monitor PII, however, the adenylylation domain appears to be dependent on the deadenylylation domain: the deadenylylation activity of AT-N depends on PII-UMP and is inhibited by PII. The adenylylation activity of AT-C is independent of PII (or PII-UMP), whereas in the intact enzyme PII is required for this activity. The implications of this intramolecular signal transduction for the prevention of futile cycling are discussed.


Assuntos
Escherichia coli/enzimologia , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Catálise , Escherichia coli/genética , Genótipo , Glutamato-Amônia Ligase/metabolismo , Cinética , Modelos Químicos , Dados de Sequência Molecular , Nucleotidiltransferases/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
16.
Biotechnol Appl Biochem ; 18(3): 259-73, 1993 12.
Artigo em Inglês | MEDLINE | ID: mdl-8297505

RESUMO

Human tumour necrosis factor-beta (TNF-beta) was produced in eukaryotic cells using the insect baculovirus cloning and expression system. A novel insect signal sequence, the honey-bee (Apis mellifera) prepromelittin secretory sequence, was used to aid in the post-translational modifications, glycosylation and secretion of recombinant human TNF-beta. Human TNF-beta cDNA was cloned using the insect baculovirus vector pAcC4s. Expression of the human TNF-beta was regulated by the insect Autographa californica nuclear-polyhedrosis-virus polyhedrin promoter. The 5' end of the TNF-beta cDNA was fused to the honey-bee prepromelittin signal sequence on the baculovirus vector. Insect [Spodoptera frugiperda (Sf9)] cells infected with the recombinant baculovirus secreted high levels of recombinant human TNF-beta into the culture medium. The amount of TNF-beta secreted by the Sf9 cells was estimated to be 28 micrograms of TNF-beta/ml of culture medium at 60-72 h post infection. The secreted human TNF-beta was a 22.5 kDa polypeptide which was glycosylated. Amino acid sequencing of the N-terminus of the recombinant human TNF-beta purified from the infected Sf9-cell culture confirmed that the secreted product was indeed human TNF-beta. This demonstrates that the honey-bee prepromelittin signal sequence was efficiently recognized and accurately cleaved in the Sf9 insect cells. The insect-derived TNF-beta exhibited a high cytotoxic activity similar to that of the native human TNF-beta when assessed by cytotoxic assays using murine L929 cells. Thus the insect baculovirus expression vector can be used for the production of abundant quantities of biologically active, glycosylated human TNF-beta protein.


Assuntos
Linfotoxina-alfa/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Metabolismo dos Carboidratos , Carboidratos/análise , Células Cultivadas , Clonagem Molecular , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Glicosilação , Humanos , Linfotoxina-alfa/biossíntese , Linfotoxina-alfa/química , Linfotoxina-alfa/imunologia , Dados de Sequência Molecular , Mariposas , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Análise de Sequência
17.
Biochem Biophys Res Commun ; 257(3): 731-7, 1999 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-10208852

RESUMO

The dengue virus NS5 RNA-dependent RNA polymerase has been detected in the nucleus of virus-infected mammalian cells. We demonstrate here for the first time using in vitro and in vivo assay systems that the 37-amino-acid linker interdomain of NS5 (residues 369 to 405) contains a nuclear localization sequence (NLS) which is capable of targeting b-galactosidase to the nucleus. Further, we show that the linker is recognized by subunits of the NLS-binding importin complex with an affinity similar to that of the bipartite NLS of the retinoblastoma protein and, in analogous fashion to proteins such as the SV40 large tumor antigen, contains a functional protein kinase CK2 phosphorylation site (threonine 395). Interestingly, this site appears to inhibit NS5 nuclear targeting, probably through a cytoplasmic retention mechanism. The linker may have an important role in targeting NS5 to the nucleus in a regulated manner during the dengue virus infectious cycle.


Assuntos
Vírus da Dengue/enzimologia , Sinais de Localização Nuclear/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Fatores Biológicos/metabolismo , Transporte Biológico/efeitos dos fármacos , Caseína Quinase II , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Consenso , Citosol/metabolismo , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/farmacologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas , Proteínas não Estruturais Virais/metabolismo
18.
Acta Crystallogr D Biol Crystallogr ; 54(Pt 5): 996-8, 1998 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-9757118

RESUMO

The trimeric signal-transduction protein GlnK, from Escherichia coli, has been over-expressed, purified to homogeneity and crystallized. The crystals belong to space group P213 with a = 85.53 A and have two subunits in the asymmetric unit. The complex of GlnK with ATP crystallized in space group P63 with a = 57.45 and c = 54.79 A. These crystals have a single subunit in the asymmetric unit. High-quality diffraction data from crystals of GlnK and the GlnK complex have been collected to 2.0 A.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Escherichia coli/enzimologia , Conformação Proteica , Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte/isolamento & purificação , Cristalização , Cristalografia por Raios X , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Transdução de Sinais
19.
Proc Natl Acad Sci U S A ; 97(8): 3942-7, 2000 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-10760266

RESUMO

The PII protein is Escherichia coli's cognate transducer of the nitrogen signal to the NRII (NtrB)/NRI (NtrC) two-component system and to adenylyltransferase. Through these two routes, PII regulates both amount and activity of glutamine synthetase. GlnK is the recently discovered paralogue of PII, with a similar trimeric x-ray structure. Here we show that PII and GlnK form heterotrimers, in E. coli grown in nitrogen-poor medium. In vitro, fully uridylylated heterotrimers of the two proteins stimulated the deadenylylation activity of adenylyltransferase, albeit to a lower extent than homotrimeric PII-UMP. Fully uridylylated GlnK did not stimulate, or hardly stimulated, the deadenylylation activity. We propose that uridylylated PII/GlnK heterotrimers fine-regulate the activation of glutamine synthetase. The PII/GlnK couple is a first example of prokaryotic signal transducer that can form heterotrimers. Advantages of hetero-oligomer formation as molecular mechanism for fine-regulation of signal transduction are discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Biopolímeros , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Nitrogênio/metabolismo , Transdução de Sinais , Proteínas de Bactérias/química , Proteínas de Transporte/química , Óperon , Proteínas PII Reguladoras de Nitrogênio
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