RESUMO
Despite antiretroviral therapy, proviral latency of human immunodeficiency virus type 1 (HIV-1) remains a principal obstacle to curing the infection. Inducing the expression of latent genomes within resting CD4(+) T cells is the primary strategy to clear this reservoir. Although histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (also known as vorinostat, VOR) can disrupt HIV-1 latency in vitro, the utility of this approach has never been directly proven in a translational clinical study of HIV-infected patients. Here we isolated the circulating resting CD4(+) T cells of patients in whom viraemia was fully suppressed by antiretroviral therapy, and directly studied the effect of VOR on this latent reservoir. In each of eight patients, a single dose of VOR increased both biomarkers of cellular acetylation, and simultaneously induced an increase in HIV RNA expression in resting CD4(+) cells (mean increase, 4.8-fold). This demonstrates that a molecular mechanism known to enforce HIV latency can be therapeutically targeted in humans, provides proof-of-concept for histone deacetylase inhibitors as a therapeutic class, and defines a precise approach to test novel strategies to attack and eradicate latent HIV infection directly.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Ácidos Hidroxâmicos/farmacologia , Latência Viral/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/sangue , HIV-1/genética , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/efeitos adversos , Inibidores de Histona Desacetilases/farmacologia , Histonas/efeitos dos fármacos , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/efeitos adversos , Provírus/efeitos dos fármacos , Provírus/genética , Provírus/crescimento & desenvolvimento , RNA Viral/biossíntese , RNA Viral/sangue , Medição de Risco , Regulação para Cima/efeitos dos fármacos , Viremia/tratamento farmacológico , Viremia/virologia , VorinostatRESUMO
To understand the secretion and processing of interleukin-1 (IL-1), a Chinese hamster fibroblast cell line (R1610) was transfected with a human IL-1 beta cDNA under the control of the SV40 early promoter and linked to the gene for neomycin resistance. After selecting for transfected cells resistant to G418, two clones were found to constitutively express the IL-1 beta 31-kD precursor which was almost exclusively located in the cytosol. Pulse-chase experiments failed to show any secretion of IL-1 and very little IL-1 activity was detectable in cell supernatants. Furthermore, surface membrane IL-1 activity could not be detected, although low levels of activity could be released upon brief trypsin treatment. Therefore, unlike monocytes, these fibroblast cells lack the mechanism for secreting and processing of IL-1 beta.
Assuntos
DNA/genética , Regulação da Expressão Gênica , Interleucina-1/biossíntese , Fragmentos de Peptídeos/biossíntese , Precursores de Proteínas/biossíntese , Transfecção , Animais , Linhagem Celular , Clonagem Molecular , Cricetinae , Citoplasma/análise , Eletroforese em Gel de Poliacrilamida , Fibroblastos , Imunofluorescência , Vetores Genéticos , Imunoensaio , Interleucina-1/análise , Interleucina-1/genética , Interleucina-1beta , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/genética , Plasmídeos , Testes de Precipitina , Regiões Promotoras Genéticas , Precursores de Proteínas/análise , Precursores de Proteínas/genética , RNA Mensageiro/análise , Transcrição GênicaRESUMO
Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.
Assuntos
Acetoacetatos/farmacologia , Fármacos Anti-HIV/farmacologia , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/metabolismo , HIV-1/efeitos dos fármacos , Pirróis/farmacologia , Integração Viral/efeitos dos fármacos , Acetoacetatos/química , Acetoacetatos/metabolismo , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Catálise/efeitos dos fármacos , Técnicas de Cocultura , DNA Circular/biossíntese , DNA Circular/metabolismo , DNA Viral/biossíntese , DNA Viral/metabolismo , Resistência Microbiana a Medicamentos , Integrase de HIV/genética , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/metabolismo , Repetição Terminal Longa de HIV/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Mutação , Pirróis/química , Pirróis/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Linfócitos T/virologia , Transcrição Gênica , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacosRESUMO
Long-acting/extended-release drug formulations have proved very successful in diverse areas of medicine, including contraception, psychiatry and, most recently, human immunodeficiency virus (HIV) disease. Though challenging, application of this technology to anti-tuberculosis treatment could have substantial impact. The duration of treatment required for all forms of tuberculosis (TB) put existing regimens at risk of failure because of early discontinuations and treatment loss to follow-up. Long-acting injections, for example, administered every month, could improve patient adherence and treatment outcomes. We review the state of the science for potential long-acting formulations of existing tuberculosis drugs, and propose a target product profile for new formulations to treat latent tuberculous infection (LTBI). The physicochemical properties of some anti-tuberculosis drugs make them unsuitable for long-acting formulation, but there are promising candidates that have been identified through modeling and simulation, as well as other novel agents and formulations in preclinical testing. An efficacious long-acting treatment for LTBI, particularly for those co-infected with HIV, and if coupled with a biomarker to target those at highest risk for disease progression, would be an important tool to accelerate progress towards TB elimination.
Assuntos
Antituberculosos/uso terapêutico , Preparações de Ação Retardada , Tuberculose Latente/tratamento farmacológico , Antituberculosos/química , HumanosAssuntos
Fármacos Anti-HIV/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Zidovudina/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Sítios de Ligação , Resistência Microbiana a Medicamentos , Conformação Molecular , Núcleosídeo-Fosfato Quinase/metabolismo , Fosforilação , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/metabolismo , Zidovudina/química , Zidovudina/metabolismoRESUMO
The clinical utility of intervention in HIV-1 disease has been proven by inhibitors targeting reverse transcriptase and protease. However, novel approaches including inhibition of viral entry, integration and assembly would provide additional options to maintain long-term suppression. The identification of specific inhibitors for each of these processes has recently validated these approaches as viable alternatives for the development of new agents to treat HIV-1 infection. The most recent preclinical advances in novel antiretroviral agents are reviewed and promising new approaches that target viral processes are highlighted.
Assuntos
Fármacos Anti-HIV/farmacologia , Desenho de Fármacos , HIV-1/efeitos dos fármacos , Montagem de Vírus/efeitos dos fármacos , Integração Viral/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores da Protease de HIV/farmacologia , HIV-1/patogenicidade , HIV-1/fisiologia , Humanos , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.
Assuntos
Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Integrases/química , Integrases/metabolismo , Vírus da Imunodeficiência Símia/enzimologia , Sequência de Aminoácidos , Vírus do Sarcoma Aviário/enzimologia , Sítios de Ligação , Cristalização , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Dimerização , Glutamina/química , Glutamina/metabolismo , Integrase de HIV/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , SoluçõesRESUMO
We have identified a series of novel inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase by randomly screening natural product extracts using an in vitro biochemical assay designed to identify inhibitors of integrase-catalysed strand transfer. Equisetin recovered from the fungus Fusarium heterosporum and a novel enantiomeric homologue of equisetin from Phoma sp. were isolated as inhibitors of HIV-1 integrase in vitro. Two additional analogues, a novel decalin derivative, integric acid, and oteromycin were also discovered to be inhibitors of integrase. Equisetin and related compounds inhibit 3' end-processing and strand transfer as well as disintegration catalysed by either the full-length enzyme or the truncated integrase core domain (amino acids 50-212). These compounds also inhibit strand transfer reactions catalysed by stable complexes assembled in vitro and integration reactions catalysed by pre-integration complexes isolated from HIV-1-infected cells. The compounds described in this report are structurally novel and mechanistically distinct from many previously described inhibitors of HIV-1 integrase. These results demonstrate the utility of using an appropriately configured assay to identify compounds that are effective post-assembly and the potential of isolating novel integrase inhibitors from complex natural product extracts.
Assuntos
Ácidos Carboxílicos/isolamento & purificação , Fusarium/metabolismo , Inibidores de Integrase de HIV/isolamento & purificação , Naftalenos/isolamento & purificação , Pirróis/isolamento & purificação , Pirrolidinas/isolamento & purificação , Tetra-Hidronaftalenos , Sequência de Bases , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Catálise , Primers do DNA , Fusarium/química , Integrase de HIV/química , Integrase de HIV/isolamento & purificação , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , Naftalenos/química , Naftalenos/farmacologia , Pirróis/química , Pirróis/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Pirrolidinonas/química , Pirrolidinonas/isolamento & purificação , Pirrolidinonas/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoAssuntos
Vírus Defeituosos/fisiologia , Regulação Viral da Expressão Gênica , HIV-1/fisiologia , Integração Viral , Animais , Sequência de Bases , Células COS , Clonagem Molecular/métodos , Vírus Defeituosos/genética , Produtos do Gene tat/biossíntese , Genes Reporter , Genes tat , Genoma Viral , HIV-1/genética , Humanos , Células Jurkat , Luciferases/biossíntese , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Provírus/genética , Provírus/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Ativação Transcricional , Transfecção/métodos , Produtos do Gene tat do Vírus da Imunodeficiência HumanaAssuntos
Butiratos/síntese química , Inibidores de Integrase de HIV/síntese química , HIV , Replicação Viral/efeitos dos fármacos , Butiratos/química , Butiratos/farmacologia , Linhagem Celular , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , Humanos , Relação Estrutura-AtividadeRESUMO
Highly purified preparations of Xenopus transcription factor A exhibit DNA-activated ATPase (ATP phosphorylase, EC 3.6.1.3) activity, which is inhibited by affinity-purified anti-factor A antibodies but not by nonspecific gamma-globulins. This enzymatic activity copurifies with both factor A and the 7 S particle, a ribonucleoprotein complex composed of factor A and 5 S RNA in a one-to-one stoichiometric ratio. At equal concentrations of protein, factor A and the 7 S particle catalyze the hydrolysis of ATP to ADP and Pi at similar rates. Kinetic analysis demonstrates that factor A is a fairly typical ATPase with a Vmax of 1.7 nmol/min/mg of protein and a KM of 5.0 X 10(-5) M, whereas the corresponding values for the 7 S particle are 2.7 nmol/min/mg of protein and 1.4 X 10(-4) M, respectively. Besides ATP, dATP is also an effective substrate for the enzyme with a Vmax of 0.7 nmol/min/mg of protein and a KM of 3.3 X 10(-5) M in reactions catalyzed by the 7 S particle. The ATPase activity of free factor A, but not the 7 S particle, can be stimulated approximately 3-fold by the addition of pBR322 plasmid DNA. Proteolytic fragments of factor A generated by treatment of the 7 S particle with papain and trypsin retain a portion of their catalytic activity, 50 and 10%, respectively, in concert with their relative size. Radioactive ATP and dATP can be photocross-linked to factor A by UV irradiation. These radioactive substrates are also cross-linked to the papain- and trypsin-generated fragments with markedly decreased efficiencies. UV photocross-linking of non-substrate nucleotides to factor A was not detectable. These results provide evidence that the ATPase activity is intrinsic to the factor A protein which is essential for the specific initiation of 5 S RNA gene transcription.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Proteínas de Xenopus , Trifosfato de Adenosina/efeitos da radiação , Animais , Ativação Enzimática , Cinética , Peso Molecular , Fotoquímica , Conformação Proteica , Raios Ultravioleta , Xenopus laevisRESUMO
We have performed pulse-chase experiments to investigate the secretion and processing of interleukin 1 (IL-1) by human peripheral blood monocytes. Polyclonal antisera generated against either recombinant IL-1 alpha (p15) or IL-1 beta (p17) could distinguish the two isoelectric forms in lysates and supernatants of lipopolysaccharide-activated monocytes. In agreement with previous results, no processed IL-1 (alpha or beta) is detected in cell lysates. Both the 31-kDa precursor and 17-kDa mature forms of IL-1 were present, however, in the culture media indicating that processing is not required for secretion. The relative amounts of the secreted 31- and 17-kDa forms of IL-1 remain constant with time throughout each experiment; in addition, 31-kDa IL-1 added to monocyte cultures is not processed to the mature 17-kDa form. Precursor IL-1 beta is however, processed to 17 kDa by monocyte extracts. Therefore, the maturation and secretion of IL-1 are intimately coordinated processes. The kinetics of IL-1 secretion are unique in comparison with other secreted proteins; release of both IL-1 alpha and IL-1 beta is delayed following synthesis, and large pools of precursor IL-1 accumulate intracellularly. The intracellular half-lives of IL-1 alpha and IL-1 beta are 15 and 2.5 h, respectively. This discrepancy in half-lives is a reflection of the different kinetics with which IL-1 alpha and IL-1 beta are secreted. IL-1 beta is released continuously beginning 2 h after synthesis, whereas the secretion of IL-1 alpha is delayed for an additional 10 h. The distinct kinetics of secretion demonstrated for IL-1 alpha and IL-1 beta suggest that the release of each pI species of IL-1 is controlled by a selective mechanism(s).
Assuntos
Interleucina-1/metabolismo , Monócitos/metabolismo , Animais , Soros Imunes , Cinética , Peso Molecular , Coelhos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The origin binding protein (OBP) of herpes simplex virus (HSV) type 1 specifically interacts with two high-affinity sites in each HSV DNA replication origin. The sequence-specific DNA binding activity of OBP maps to the carboxy-terminal one-third of the protein. For a single binding site, recombinantly expressed forms of this DNA binding domain have the same sequence specificity and binding affinity as the full-length OBP. However, unlike the full-length protein, truncated OBP does not bind HSV replication origins in a cooperative manner. To determine if cooperative interactions between DNA-bound OBP molecules are essential for viral DNA replication, the 317-amino-acid carboxy-terminal DNA binding domain of OBP was expressed in chick embryo fibroblasts. Cells were infected with HSV type 1, and viral DNA synthesis and virus production were monitored. We found that cells expressing truncated OBP were severely restricted for virus replication and that HSV DNA synthesis was undetectable. The results demonstrate that the amino-terminal two-thirds of OBP is essential for HSV DNA replication and that the OBP DNA binding domain acts as a transdominant inhibitor of viral DNA replication. The results also suggest that this experimental approach could be used to generate a refined map of essential OBP functions and that the approach may be generally applicable to the analysis of the multifunction HSV DNA replication complex.
Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA/fisiologia , Simplexvirus/fisiologia , Proteínas Virais/fisiologia , Replicação Viral/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Embrião de Galinha , Replicação do DNA/genética , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Fibroblastos , Dados de Sequência Molecular , Simplexvirus/genética , Proteínas Virais/genética , Replicação Viral/genéticaRESUMO
We have purified the 31-kDa precursor of human interleukin 1 beta (proIL1 beta) from recombinant Escherichia coli expressing the protein. The recombinant precursor was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity. The protein migrates at the expected molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration columns. The specific activity of the recombinant precursor is less than 10(2) units/mg in the EL4 thymoma assay compared with 5 x 10(8) units/mg for the recombinant 17-kDa mature protein. The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor competition studies using 125I-labeled 17-kDa IL1 beta. Inactivity of the IL1 beta precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays. The inactive IL1 beta precursor is converted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting IL1 beta precursor. Removal of the first 114 amino acids from proIL1 beta generates a fully active molecule. In contrast, removal of the first 77 amino acids by treatment with trypsin only partially restores activity. The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct correlation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proIL1 beta is similar in secondary structure to mature IL1 beta; both proteins are nonhelical beta sheet proteins.
Assuntos
Interleucina-1/isolamento & purificação , Precursores de Proteínas/isolamento & purificação , Dicroísmo Circular , Humanos , Peso Molecular , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The virally encoded origin binding protein (OBP) of herpes simplex virus (HSV) is required for viral DNA synthesis. OBP binds at the replication origin to initimultienzyme replication complex (Challberg, M. D., and Kelly, T. J. (1989) Annu Rev. Biochem. 58, 671-717), OBP binds to two sites at the replication origin. The sequence-specific interaction of OBP with each binding site is localized to the major groove, and in both HSV origins the two interaction surfaces are in phase, aligned on the same face of the helix (Hazuda, D. J., Perry, H. C., Naylor, A. M., and McClements, W. L. (1991) J. Biol. Chem. 261, 24621-24625). Using native gel electrophoresis, we now demonstrate that OBP binding to the origin is highly cooperative and that cooperativity requires the putative NH2-terminal leucine zipper. Neither the phase nor orientation of the binding sites affect cooperativity, suggesting that the interaction promotes wrapping of origin DNA around the OBP multimer. A comparison of OBP DNase I footprints with the DNase I footprints of a truncated protein defective in cooperativity demonstrates that the interaction between OBPs bound at sites I and II affects the conformation of the intervening DNA, particularly when the phase or orientation of the two sites is different from wild type. OBP may elicit a unique nucleoprotein structure which facilitates unwinding of the origin and/or assembly of the replication complex. We also demonstrate that OBP can exchange binding sites, forming interduplex complexes. This property may be important for reinitiation of DNA replication.
Assuntos
Replicação do DNA , DNA Viral/genética , Proteínas de Ligação a DNA/metabolismo , Simplexvirus/metabolismo , Proteínas Virais/metabolismo , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Cromatografia em Gel , DNA Viral/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Desoxirribonuclease I , Eletroforese em Gel de Poliacrilamida , Exonucleases/metabolismo , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Conformação Proteica , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Simplexvirus/genética , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
Integric acid (1), an acyl eremophilane sesquiterpenoid, was identified as an inhibitor of HIV-1 integrase, the enzyme responsible for provirus entry into the host cell nucleus and integration in to the host genome. Chemical and enzymatic modification of integric acid led to the preparation of several selective chemical derivatives of integric acid. Preparation, HIV-1 inhibitory activity, and the structure-activity relationship against coupled and strand transfer assays are described. It appears that most of the groups present in the natural product are required for inhibition of HIV-1 integrase strand transfer activity. In contrast, inhibition of 3' processing activity is less stringent suggesting distinct SAR for the two integrase reactions.
Assuntos
Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , HIV-1/enzimologia , Naftalenos/química , Naftalenos/farmacologia , Amidas/química , Hidrólise , Relação Estrutura-AtividadeRESUMO
We demonstrate by agarose gel electrophoresis and DNase I footprinting that Xenopus transcription factor A promotes DNA reassociation. This ability of factor A is dependent upon the domain-like structure of the protein. Digestion of factor A by papain results in a protein fragment which promotes DNA reassociation whereas a smaller fragment obtained by trypsin digestion does not. Although factor A requires zinc for specific binding to the 5 S RNA gene, the metal is not required for single-stranded DNA binding or promotion of DNA reassociation by this protein. The factor A-dependent renaturation of the 5 S RNA gene from its individual 32P end-labeled strands results in the proper gene conformation as evidenced by the restoration of the DNase I footprint characteristic of the intragenic control region. Alterations in DNase I cleavage patterns induced by factor A on the individual 5 S DNA strands are distinct from those induced by the protein on the duplex 5 S RNA gene. The ability of factor A to promote DNA reassociation further defines the possible roles of this protein in the formation of active transcription complexes and their maintenance during repeated rounds of transcription.
Assuntos
DNA/metabolismo , Transativadores , Fatores de Transcrição/fisiologia , Proteínas de Xenopus , Animais , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/metabolismo , Desnaturação de Ácido Nucleico , Renaturação de Ácido Nucleico/efeitos dos fármacos , Papaína/metabolismo , Fragmentos de Peptídeos/fisiologia , RNA/genética , Relação Estrutura-Atividade , Fatores de Transcrição/farmacologia , Transcrição Gênica , Tripsina/metabolismo , Xenopus laevis , Zinco/farmacologiaRESUMO
The D-ribose operon (rbs) of Escherichia coli K-12 maps at 83 min and is inducible. The rbs operon of E. coli B/r maps at 2 min and is constitutive. Evidence is presented showing that a second inducible copy of the rbs operons is present in E. coli B/r mapping at 83 min. The data indicated that the duplication of the rbs operon represented a transposition of the 83-min region to 2 min. The identification of a second copy of the rbs operon in B/r and the determination of its inducibility were based on the reactivation, through mutagenesis, of inducible rbs expression, mapping by P1 transduction of the mutation site to 83 min, and merodiploid complementation analysis of the D-ribokinase expression in E. coli B/r. We also show that the rbs transposition to 2-min continued to generate transposable elements coding for the 1- to 2-min region of the chromosome and transposing onto extrachromosomal DNA target molecules such as pBR322.
Assuntos
Elementos de DNA Transponíveis , Escherichia coli/genética , Óperon , Fosfotransferases (Aceptor do Grupo Álcool) , Ribose/metabolismo , Mapeamento Cromossômico , Cromossomos Bacterianos , Enzimas de Restrição do DNA , Indução Enzimática , Genes Reguladores , Teste de Complementação Genética , Fosfotransferases/biossíntese , Plasmídeos , Ribose/biossíntese , Transdução GenéticaRESUMO
The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Simplexvirus/metabolismo , Proteínas Virais/metabolismo , Sequência de Bases , Ligação Competitiva , Clonagem Molecular , Replicação do DNA , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Mutação , Ácidos Nucleicos Heteroduplexes , Oligonucleotídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais/genéticaRESUMO
The processing of precursor interleukin 1 beta (IL1 beta) by elastase, cathepsin G, and collagenase, the major proteases released at sites of inflammation, was investigated using recombinant pro-IL1 beta. Each of these proteases cleaved the 31-kDa inactive precursor to a form similar in size and specific activity (greater than 10(8) units/mg) to the 17-kDa mature protein isolated from activated monocytes. Elastase, collagenase, and cathepsin G cleaved the IL1 beta precursor at distinct sites which are amino-terminal to the monocyte-processing site, Ala-117 (Cameron, P., Lumjuco, G., Rodkey, J., Bennett, C., and Schmidt, J. A. (1985) J. Exp. Med. 162, 790-801). Amino-terminal sequencing of the products of digestion by elastase and cathepsin G determined that resultant active IL1 beta proteins contained an additional 13 or 3 amino acids relative to mature IL1 beta. Synovial fluid collected from patients with inflammatory polyarthritis and bronchoalveolar lavage fluid from patients with sarcoidosis supplied similar processing activity(s). Control fluids from patients who had no symptoms of inflammatory disease did not exhibit processing activity. Lavage fluids that processed precursor IL1 beta were demonstrated to contain cathepsin G and/or elastase activity, whereas controls were negative. Because a significant fraction of IL1 beta may be secreted from monocytes as the inactive 31-kDa precursor (Hazuda, D. J., Lee, J. C., and Young, P. R. (1988) J. Biol. Chem. 263, 8473-8479, Bomford, R., Absull, E., Hughes-Jenkins, C., Simpkin, D., and Schmidt, J. (1987) Immunology 62, 543-549, and Mizel, S. B. (1988) in Cellular and Molecular Aspects of Inflammation Poste, G., and Crooke, S., eds) pp. 75-93, Plenum Publishing Corp., New York), these results suggest that in vivo the IL1 beta precursor can be processed after secretion by any of several proteases released at inflammatory sites.