RESUMO
Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.
Assuntos
Amidoidrolases , Aminopeptidases/antagonistas & inibidores , Antibacterianos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Aminopeptidases/química , Antibacterianos/síntese química , Antibacterianos/química , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Metaloendopeptidases/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Proteica , Staphylococcus aureus/enzimologia , Relação Estrutura-AtividadeRESUMO
Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminally truncated forms of cdc25B did not induce GVBD, even though many had phosphatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggesting a direct interaction in vivo. cdc25B(356-556), but not cdc25B(364-529), inhibited GVBD induction by GST-cdc25B(31-566) suggesting that a region of cdc25B near to the C-terminus was responsible for the inhibition. To determine the region of peptide sequence that was inhibitory, cdc25B(356-556) was subjected to proteolysis with endoproteinase lys-C. Following a demonstration that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD, a series of peptides spanning amino acids at the C-terminus were synthesized. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanine scan of the peptide revealed residues critical for GVBD inhibition, and site-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566) eliminated its ability to induce GVBD. These results demonstrate that a cdc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-competent cdc25B, is required for induction of GVBD following microinjection into oocytes.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/farmacologia , Oócitos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Fosfatases cdc25/química , Fosfatases cdc25/farmacologia , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sequência Conservada/genética , Ativação Enzimática/efeitos dos fármacos , Metaloendopeptidases/metabolismo , Microinjeções , Mutagênese Sítio-Dirigida/genética , Oócitos/citologia , Oócitos/efeitos dos fármacos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Alinhamento de Sequência , Deleção de Sequência/genética , Xenopus laevis , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
Cdc25B is a dual specificity phosphatase involved in the control of cyclin-dependent kinases and the progression of cells through the cell cycle. A series of minimal domain Cdc25B constructs maintaining catalytic activity have been expressed. The structure of a minimum domain construct binding sulfate was determined at 1.9 A resolution and a temperature of 100 K. Other forms of the same co?nstruct were determined at lower resolution and room temperature. The overall folding and structure of the domain is similar to that found for Cdc25A. An important difference between the two is that the Cdc25B domain binds oxyanions in the catalytic site while that of Cdc25A appears unable to bind oxyanions. There are also important conformational differences in the C-terminal region. In Cdc25B, both sulfate and tungstate anions are shown to bind in the catalytic site containing the signature motif (HCxxxxxR) in a conformation similar to that of other protein tyrosine phosphatases and dual specificity phosphatases, with the exception of the Cdc25A. The Cdc25B constructs, with various truncations of the C-terminal residues, are shown to have potent catalytic activity. When cut back to the site at which the Cdc25A structure begins to deviate from the Cdc25B structure, the activity is considerably less. There is a pocket extending from the catalytic site to an anion-binding site containing a chloride about 14 A away. The catalytic cysteine residue, Cys473, can be oxidized to form a disulfide linkage to Cys426. A readily modifiable cysteine residue, Cys484, resides in another pocket that binds a sulfate but not in the signature motif conformation. This region of the structure is highly conserved between the Cdc25 molecules and could serve some unknown function.
Assuntos
Domínio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Fase G2 , Mitose , Fosfatases cdc25/química , Fosfatases cdc25/metabolismo , Sequência de Aminoácidos , Ânions/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Cristalização , Cristalografia por Raios X , Dissulfetos , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Sulfatos/química , Sulfatos/metabolismo , Temperatura , Compostos de Tungstênio/metabolismo , Fosfatases cdc25/genéticaRESUMO
The authors mutated two key residues in the sequence of the cytokine interleukin 1 beta, namely the double mutant Phe46 to Trp46 and Trp120 to Phe120 and the single point mutation Lys103 to Leu103 and measured the resulting receptor binding and biological activities. The biological and receptor binding activities of the Trp46 mutein was reduced by a factor of 12 and 25, respectively, and surprisingly, those of the Leu103 mutein, 2600 and 600-fold relative to the wild-type protein. The authors had previously showed that Lys103 was unusually reactive to a variety of derivatizing agents. Furthermore, the Trp to Phe mutation allowed us to monitor the local environment of that residue by studying its intrinsic fluorescence properties, as well as any change in the fluorescence properties of Trp120 of the Leu103 mutein. The results of these studies show that mutation of Lys103 to Leu103 produces subtle long-range changes in the micro-environment of Trp120, indicative of a key role for this residue in the folding of the entire protein.
Assuntos
Interleucina-1/genética , Animais , Células Cultivadas , Clonagem Molecular , Interleucina-1/química , Cinética , Leucina , Lisina , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores de Interleucina-1/metabolismo , Espectrometria de Fluorescência , TriptofanoRESUMO
U-31,355, or 4-amino-2-(benzylthio)-6-chloropyrimidine is an inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound acts as a specific inhibitor of the RNA-directed DNA polymerase function of HIV-1RT and does not impair the functions of the DNA-catalyzed DNA polymerase or the Rnase H of the enzyme. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-31,355. The data were analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP, and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived that allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates, and the inhibitor. The results obtained indicate that U-31,355 acts as a mixed inhibitor with respect to the template:primer and dNTP binding sites associated with the RNA-directed DNA polymerase domain of the enzyme. The inhibitor possessed a significantly higher binding affinity for the enzyme-substrate complexes, than for the free enzyme and consequently did not directly affect the functions of the substrate binding sites. Therefore, U-31,355 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond. Moreover, the potency of U-31,355 depends on the base composition of the template:primer in that the inhibitor showed a much higher binding affinity for the enzyme-poly (rC):(dG)10 complexes than for the poly (rA):(dT)10 complexes.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores da Transcriptase Reversa/metabolismo , Animais , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , Transcriptase Reversa do HIV , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Cinética , Linfócitos/virologia , Computação Matemática , Camundongos , Pirimidinas/farmacologia , DNA Polimerase Dirigida por RNA/metabolismo , Retroviridae/enzimologia , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
We have recently shown that hsp56, the FK506-binding immunophilin component of both the heat shock protein (hsp90.hsp70.hsp56) heterocomplex and the untransformed glucocorticoid receptor heterocomplex, is bound directly to hsp90 (Czar, M. J., Owens-Grillo, J. K., Dittmar, K. D., Hutchison, K. A., Zacharek, A. M., Leach, K. L., Deibel, M. R., and Pratt, W. B. (1994) J. Biol. Chem. 269, 11155-11161). In this work, we show that both untransformed glucocorticoid receptor and hsp90 heterocomplexes contain CyP-40, a 40-kDa immunophilin of the cyclosporin A-binding class. CyP-40 is present in both native glucocorticoid receptor heterocomplexes and receptor heterocomplexes reconstituted with rabbit reticulocyte lysate, and the presence of CyP-40 in the receptor heterocomplex is stabilized by molybdate. Immunoadsorption of hsp90 from cell lysate yields coimmunoadsorption of both hsp56 and CyP-40, showing that both immunophilins are in native heterocomplex with hsp90. However, immunoadsorption of hsp56 does not yield coimmunoadsorption of CyP-40; thus, the two immunophilins do not exist in the same heterocomplex with hsp90. Both purified CyP-40 and hsp56 bind directly to purified hsp90, and excess CyP-40 blocks the binding of hsp56, consistent with the presence of a common immunophilin binding site on hsp90. Our data also suggest that there are at least two types of untransformed glucocorticoid receptor-hsp90 heterocomplexes, one that contains hsp56 and another that contains CyP-40. The role played by the immunophilins in steroid receptor action is unknown, but it is clear that the peptidylprolyl isomerase activity of immunophilins is not required for glucocorticoid receptor-hsp90 heterocomplex assembly and proper folding of the hormone binding domain by the hsp90-associated protein folding system of reticulocyte lysate.
Assuntos
Proteínas de Transporte/metabolismo , Ciclosporina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Sítios de Ligação , Western Blotting , Células CHO , Proteínas de Transporte/isolamento & purificação , Bovinos , Sistema Livre de Células , Cricetinae , Citosol/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP90/isolamento & purificação , Proteínas de Choque Térmico/isolamento & purificação , Camundongos , Peso Molecular , Coelhos , Receptores de Glucocorticoides/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reticulócitos/metabolismo , Proteínas de Ligação a Tacrolimo , TransfecçãoRESUMO
In their unliganded state, mouse glucocorticoid receptors (GR) that are overexpressed in the WCL2 line of Chinese hamster ovary cells are distributed in a nonrandom manner throughout all planes of the nucleus. These untransformed nuclear receptors exist in a heterocomplex containing three heat shock proteins, hsp90, hsp70, and hsp56, the latter being an immunophilin of the FK506 binding type whose cellular function is unknown. Because a knowledge of the cellular distribution of hsp56 could provide important clues to its function in steroid-receptor heterocomplexes, we have examined hsp56 localization in intact cells by indirect immunofluorescence using the UPJ56 antibody. The majority of hsp56 is located in the nucleus, with substantial amounts also visualized in the cytoplasm of intact cells. The cytoplasmic hsp56 was examined in rat pulmonary endothelial cells where the protein was found to colocalize with microtubules. The nuclear hsp56 was examined in the WCL2 cells, where the protein was found by confocal imaging to colocalize throughout all planes of the nucleus in the same mottled pattern as the overexpressed GR. Like the GR, the nuclear hsp56 is recovered largely in the cytosolic fraction after hypotonic rupture of WCL2 cells. An observation potentially related to the microtubule-associated fraction of hsp56 is that immunoadsorption of hsp56 from WCL2 cytosol is accompanied by coadsorption of the microtubule-associated protein-1C complex. These observations are discussed with respect to the possible biological functions of hsp56 in the folding and/or cytoplasmic-nuclear trafficking of the receptor.
Assuntos
Proteínas de Transporte/análise , Núcleo Celular/química , Citoplasma/química , Proteínas de Ligação a DNA/análise , Proteínas de Choque Térmico/análise , Microtúbulos/química , Receptores de Glucocorticoides/análise , Animais , Células CHO , Células Cultivadas , Cricetinae , Endotélio/ultraestrutura , Imunofluorescência , Técnicas de Imunoadsorção , Pulmão/ultraestrutura , Proteínas Associadas aos Microtúbulos/análise , Ratos , Proteínas de Ligação a TacrolimoRESUMO
Heat shock protein 56 (hsp56) was previously identified as an immunophilin based on its ability to specifically bind to FK506-Affi-Gel 10. In this report, we have quantitated human Jurkat T cell hsp56 binding to 3H-FK506, as well as to the immunosuppressant rapamycin. Binding was measured utilizing immunoadsorbed hsp56, and, in addition, we demonstrate that 3H-FK506 binds to hsp56 in solution. Hsp56 bound to an antibody-Sepharose column binds 3H-FK506 with an affinity of 19.4 +/- 4.6 nM, as compared to 23.2 +/- 6.8 nM for soluble hsp56. In competition experiments, the apparent affinity constant for rapamycin was 11.6 +/- 2.8 nM, using immobilized hsp56, and 17.3 +/- 7.7 nM, using the soluble hsp56 preparation. These results demonstrate that hsp56 binds FK506 and rapamycin with similar affinities, and suggest that hsp56 may play a role in mediating the cellular function of both of these drugs.
Assuntos
Proteínas de Transporte/metabolismo , Cromatografia de Afinidade/métodos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Polienos/metabolismo , Tacrolimo/metabolismo , Western Blotting , Humanos , Ligação Proteica , Ensaio Radioligante/métodos , Sirolimo , Proteínas de Ligação a Tacrolimo , Células Tumorais CultivadasRESUMO
We have recently reported that v-Raf forms a native heterocomplex with rat heat shock protein (hsp) 90 and a 50-kDa phosphoprotein (p50) in stably transfected 3Y1 fibroblasts (Stancato, L. F., Chow, Y-H., Hutchison, K. A., Perdew, G. H., Jove, R., and Pratt, W. B. (1993) J. Biol. Chem. 268, 21711-21716). Several members of the nuclear receptor family exist in heterocomplexes containing hsp90 and various members of the immunophilin protein family, including hsp56, an immunophilin of the FK506 binding class (Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458). In this work, we have asked if Raf is also associated with an immunophilin. We have immunoadsorbed v-Raf from stably transfected rat 3Y1 fibroblasts and show that the immunoadsorbed v-Raf.hsp90.p50 heterocomplex binds the immunosuppressant drug [3H]FK506. The binding is of high affinity (KD 82 nM) and specific in the sense that it is competed by nonradioactive FK506 and rapamycin but not by cyclosporin A. The [3H]FK506 binding activity is eliminated when the heterocomplex proteins are dissociated from v-Raf. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for the immunophilin association. We have shown that hsp90 and p50 do not bind FK506, and the v-Raf heterocomplex does not contain any of the established FK506-binding proteins. Thus, we propose that Raf is associated with a novel immunophilin.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Tacrolimo/metabolismo , Células 3T3 , Animais , Antígenos CD40 , Células Cultivadas , Camundongos , Proteínas Oncogênicas v-raf , Ratos , Proteínas de Ligação a TacrolimoRESUMO
Interleukin-1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin-1 receptor. In contrast to IL-1 beta, IRAP binds to the IL-1 receptor but does not elicit a physiological response. We have determined the solution structure of IRAP using NMR spectroscopy. While the overall topology of the two 153-residue proteins is quite similar, functionally critical differences exist concerning the residues of the linear amino acid sequence that constitute structurally homologous regions in the two proteins. Structurally homologous residues important for IL-1 receptor binding are conserved between IRAP and IL-1 beta. By contrast, structurally homologous residues critical for receptor activation are not conserved between the two proteins.
Assuntos
Receptores de Interleucina-1/antagonistas & inibidores , Sialoglicoproteínas/química , Sequência de Aminoácidos , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/química , Interleucina-1/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/química , SoluçõesRESUMO
The bisheteroarylpiperazine U-90152E is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound inhibits both the RNA- and DNA-directed DNA polymerase functions of HIV-1 RT. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-90152E. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived, which allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The results obtained indicate that U-90152E acts exclusively as a mixed inhibitor with respect to the template: primer and dNTP binding sites for both the RNA- and DNA-directed DNA polymerase domains of the enzyme. The inhibitor shows a significantly higher binding affinity for the enzyme-substrate complexes than for the free enzyme and consequently does not directly impair the functions of the substrate binding sites. Therefore, U-90152E appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond.
Assuntos
Antivirais/farmacologia , Indóis/farmacologia , Piperazinas/farmacologia , Inibidores da Transcriptase Reversa , Delavirdina , Transcriptase Reversa do HIV , Cinética , Matemática , DNA Polimerase Dirigida por RNA/química , Proteínas Recombinantes/antagonistas & inibidores , Moldes GenéticosRESUMO
We have reported previously that the three heat shock proteins hsp56, hsp70, and hsp90 exist together in a heterocomplex in human lymphocyte cytosol (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). All three of these proteins also exist in the native glucocorticoid receptor heterocomplex isolated from WCL2 cell cytosol and we have recently shown that the three heat shock proteins are present when immunopurified mouse glucocorticoid receptor is reconstituted into a heterocomplex by rabbit reticulocyte lysate (Hutchison, K. A., Scherrer, L. C., Czar, M. J., Ning, Y., Sanchez, E. R., Leach, K. L., Deibel, M. R., Jr., and Pratt, W. B. (1993) Biochemistry 32, 3953-3957). In this work, we show that highly purified mouse hsp90 binds in a reversible equilibrium to immunopurified rabbit hsp56, but hsp56 does not bind to purified mouse hsp70. In contrast to the equilibrium binding of hsp90 to hsp56, purified hsp90 binds poorly or not at all to purified hsp70 unless a third factor from reticulocyte lysate is present to permit complex formation. This hsp70.hsp90 complex-forming factor is heat-labile, and in the presence of this factor and ATP, a heat shock protein heterocomplex can be reconstituted from purified mouse hsp90 and hsp70 and rabbit hsp56 that is present in the factor preparation. Our data are consistent with a model in which hsp56 and hsp70 bind to different sites on hsp90 but do not interact with each other. The presence of hsp56 in the heat shock protein heterocomplex is not stabilized by molybdate but hsp56 is stabilized if the glucocorticoid receptor is present in addition to hsp90 and hsp70.
Assuntos
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico/isolamento & purificação , Immunoblotting , Cinética , Células L , Camundongos , Molibdênio/farmacologia , Ligação Proteica , Coelhos , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/isolamento & purificação , Reticulócitos/metabolismo , Termodinâmica , Triancinolona Acetonida/metabolismoRESUMO
We report the crystallization of samples of a recombinant preparation of human interleukin-1 receptor antagonist protein (IRAP) and solution of the crystal structure by isomorphous replacement methods. Crystals were obtained by the hanging-drop vapor-diffusion method at 277 K from solutions of PEG 4000 containing sodium chloride, dithiothreitol and PIPES [sodium piperazione-N,N'-bis(2-ethanesulfonate)] buffer at pH 7.0. Crystals appear within about a week and grow as truncated tetragonal bipyramids to 0.3-0.6 mm on an edge. X-ray diffraction data from these crystals specify space group P4(3)2(1)2 and unit-cell dimensions of a = b = 72.35(26), c = 114.7(8) A and Z = 16 (two molecules per asymmetric unit). Fresh crystals diffract to about 2.3 A resolution. The search for heavy-atom derivatives has produced two, potassium gold cyanide and trimethyl lead chloride, as same-site, single-site derivatives. Inspection of an electron-density map at 4 A resolution calculated with these derivatives confirms that the IRAP molecule is a member of the interleukin-1 structural family.
RESUMO
The tetramer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT* and possesses excellent antiviral activity at nontoxic doses in HIV-1 infected lymphocytes grown in tissue culture. Kinetic studies of the HIV-1 RT-catalyzed RNA-directed DNA polymerase activity were carried out in order to determine if the inhibitor interacts with the template primer or the deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using steady-state Briggs-Haldane kinetics assuming that the template: primer binds to the enzyme first, followed by the binding of the dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived which allows the calculation of all the specific forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The calculated rate constants are in agreement with this model and the results indicated that U-9843 acts as a noncompetitive inhibitor with respect to both the template:primer and dNTP binding sites. Hence, U-9843 exhibits the same binding affinity for the free enzyme as for the enzyme-substrate complexes and must inhibit the RT polymerase by interacting with a site distinct from the substrate binding sites. Thus, U-9843 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either an event leading up to the formation of the phosphoester bond, the formation of the ester bond itself or translocation of the enzyme relative to its template:primer following the formation of the ester bond.
Assuntos
HIV-1/enzimologia , Polidesoxirribonucleotídeos/biossíntese , Polivinil/farmacologia , DNA Polimerase Dirigida por RNA/farmacologia , Ácidos Sulfônicos/farmacologia , Sítios de Ligação , Primers do DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Transcriptase Reversa do HIV , HIV-1/efeitos dos fármacos , HIV-1/genética , Cinética , DNA Polimerase Dirigida por RNA/metabolismo , Moldes GenéticosRESUMO
Avian progesterone receptor complexes contain five major co-purifying proteins, including hsp90, hsp70, and a 23-kDa protein. The other receptor-associated proteins, p50 and p54, share amino acid sequence similarities with a 52-59-kDa component (p59, hsp56, or FKBP52) of mammalian steroid receptor complexes that is also a member of the FK506-binding proteins (FKBP) family of immunophilins. We show here that p50, but not p54, cross-reacts with a rabbit antiserum prepared against human FKBP52. Both p50 and p54 bind an FK506 affinity resin at 0.5 M KCl, but only p50 binds efficiently in low salt conditions. Glycerol density gradient analyses show that both p50 and p54 exist predominantly in oligomeric complexes at low ionic strength. The poor retention of p54 on FK506 resin at low ionic strength compared with the high retention of p50 suggests that these proteins may largely exist in separate complexes and may interact with other proteins, such as progesterone receptor, in distinctive manners. In HeLa cell extracts, a 55-kDa FK506-binding protein, distinct from FKBP52, cross-reacts with anti-p54 antibody FF1. We conclude that p50 is avian FKBP52, whereas p54 and the 55-kDa human FF1 antigen are FKBP54, a novel immunophilin.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Receptores de Progesterona/metabolismo , Tacrolimo/metabolismo , Animais , Proteínas de Transporte/imunologia , Galinhas , Cromatografia de Afinidade , Reações Cruzadas , Eletroforese em Gel Bidimensional , Células HeLa , Proteínas de Choque Térmico/imunologia , Humanos , Receptores de Progesterona/química , Proteínas de Ligação a TacrolimoRESUMO
We have previously identified hsp56, a protein component of steroid receptor complexes, as an FK506 binding protein [Yem et al. (1992) J. Biol. Chem. 267, 2868-2871]. We now report that hsp56 is also found to be a major immunophilin in chicken thymus, by virtue of binding to FK506-Affi-Gel-10 as well as positive cross-reactivity with a polyclonal antiserum directed against human hsp56. Limited digests of purified chicken hsp56 with endoproteinase Lys C result in the production of a unique polypeptide having a mass of about 17 kDa (p17), as judged by Western blotting. Peptide mapping provided additional proof that p17 is a fragment which comprises the entire FK506 binding domain I of chicken hsp56, terminating with an Arg-Lys which might represent a processing site. Binding of radiolabeled dihydro FK506 to p17 is saturable with a calculated KD of 42 nM. Since size exclusion chromatography of drug-p17 complexes indicates that the active species is a homodimer with a mass of 30-40 kDa, the stoichiometry calculated for the drug-protein complex is approximately 1:1. Furthermore, unlike FKBP-12, chicken p17 bound to FK506 does not bind to calcineurin-calmodulin complexes. This work demonstrates the excision of a domain from an hsp56 protein that is active in binding FK506 and functionally distinct from FKBP-12, a protein of similar size and structure.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Tacrolimo/metabolismo , Timo/química , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Galinhas , Endopeptidases/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Análise de Sequência , Homologia de Sequência de Aminoácidos , Tacrolimo/análogos & derivados , Proteínas de Ligação a TacrolimoRESUMO
The quinoline U-78036 represents a new class of non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitors. The agent possesses excellent antiviral activity at nontoxic doses in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the HIV-1 reverse transcriptase (RT)-catalyzed RNA-directed DNA polymerase function were carried out in order to determine whether the inhibitor interacts with the template-primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The data were analyzed using steady-state or Briggs-Haldane kinetics assuming that the template-primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The calculated rate constants are in agreement with this model. The results show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template-primer and the dNTP binding sites of the enzyme. Hence, U-78036 inhibits the RNA-directed DNA polymerase activity of RT by interacting with a site distinct from the template-primer and dNTP binding sites. Moreover, the potency of U-78036 is dependent on the base composition of the template-primer. The equilibrium constants for various enzyme-substrate-inhibitor complexes were at least seven times lower for the poly(rC).(dG)10-catalyzed system than the one catalyzed by poly(rA).(dT)10. In addition, the inhibitor does not impair the DNA-dependent DNA polymerase activity and the RNase H function of HIV-1 RT nor does it inhibit the RNA-directed DNA polymerase activity of the HIV-2, avian myoblastoma virus, and murine leukemia virus RT enzymes.
Assuntos
Antivirais/farmacologia , HIV-1/efeitos dos fármacos , Nitrilas/farmacologia , Quinolinas/farmacologia , Inibidores da Transcriptase Reversa , Células Cultivadas , Transcriptase Reversa do HIV , HIV-1/enzimologia , Cinética , Linfócitos/microbiologia , Estrutura Molecular , Nitrilas/química , Quinolinas/química , DNA Polimerase Dirigida por RNA/metabolismo , Retroviridae/enzimologia , Ribonuclease H/antagonistas & inibidores , Especificidade por Substrato , Moldes GenéticosRESUMO
The bis(heteroaryl)piperazine U-88204E is a potent inhibitor of HIV-1 reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the RNA- and DNA-dependent DNA polymerases of RT were carried out in order to determine whether the inhibitor interacts directly with the template:primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The experimental results were analyzed using steady-state or Briggs-Haldane kinetics, by assuming that the template:primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The results of the analysis show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template:primer and the dNTP binding sites. The potency of U-88204E on the RNA-directed DNA polymerase activity depends on the base composition of the template:primer. The Ki values for the poly(rC):(dG)10-directed reactions were at least 7 times lower than the ones for reactions directed by poly(rA):(dT)10. The inhibitor did not inhibit the RNase H function of HIV-1 RT nor did it impair the RNA-directed DNA polymerase activity of HIV-2 RT. These data thus demonstrate the unique specificity of U-88204E for HIV-1 RT.
Assuntos
HIV-1/enzimologia , Indóis/farmacologia , Piperazinas/farmacologia , Inibidores da Transcriptase Reversa , Ribonuclease H/antagonistas & inibidores , Transcriptase Reversa do HIV , Cinética , Matemática , Modelos Teóricos , Poli T/biossíntese , Proteínas Recombinantes/antagonistas & inibidores , Moldes GenéticosRESUMO
It has recently been reported that the hsp56 component of glucocorticoid receptor heterocomplexes is an immunophilin of the FK506 binding class [Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., Zurcher-Neely, H., Ruff, V. A., Johnson, R. A., & Deibel, M. R. (1992) J. Biol. Chem. 267, 2868-2871; Tai, P. K., Albers, M. W., Chang, H., Faber, L. E., & Schreiber, S. L. (1992) Science 256, 1315-1318]. The existence of binding proteins for these two potent groups of immunosuppressants in the same molecular complex compels us to ask whether FK506 affects glucocorticoid receptor function. We show here that hsp56 is a component of the native L-cell glucocorticoid receptor heterocomplex and that [3H]FK506 binds to the immunopurified, untransformed receptor complex. However, at concentrations in excess of those required to occupy all of its binding sites on hsp56, FK506 does not affect the steroid binding activity of the receptor nor does it stabilize or dissociate the receptor-hsp90 complex. FK506 does not affect steroid-mediated hsp90 dissociation from the receptor in vitro, and it does not affect steroid-mediated nuclear transfer of the receptor or steroid-mediated transcriptional enhancement from a reporter in intact cells. When immunopurified mouse glucocorticoid receptor is reconstituted into a heat shock protein complex by rabbit reticulocyte lysate, hsp56 is present in the reconstituted complex in addition to hsp90 and hsp70. FK506, however, does not affect reconstitution of the complex or return of the receptor to the steroid binding state, a change of conformation that occurs upon receptor association with hsp90.(ABSTRACT TRUNCATED AT 250 WORDS)