RESUMO
The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the 'SDN loop' by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5.
Assuntos
Actinomycetales/enzimologia , Proteínas de Bactérias , Carboxipeptidases/metabolismo , Hexosiltransferases , Peptidil Transferases , Alanina , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Carboxipeptidases/química , Carboxipeptidases/genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Sequência Conservada , Glicina , Cinética , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/química , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Mutagênese Sítio-Dirigida , Proteínas de Ligação às Penicilinas , Penicilinas/metabolismo , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Especificidade por SubstratoRESUMO
HIV-1 and related viruses require co-receptors, in addition to CD4, to infect target cells. The chemokine receptor CCR-5 (ref.1) was recently demonstrated to be a co-receptor for macrophage-tropic (M-tropic) HIV-1 strains, and the orphan receptor LESTR (also called fusin) allows infection by strains adapted for growth in transformed T-cell lines (T-tropic strains). Here we show that a mutant allele of CCR-5 is present at a high frequency in caucasian populations (allele frequency, 0.092), but is absent in black populations from Western and Central Africa and Japanese populations. A 32-base-pair deletion within the coding region results in a frame shift, and generates a non-functional receptor that does not support membrane fusion or infection by macrophage- and dual-tropic HIV-1 strains. In a cohort of HIV-1 infected caucasian subjects, no individual homozygous for the mutation was found, and the frequency of heterozygotes was 35% lower than in the general population. White blood cells from an individual homozygous for the null allele were found to be highly resistant to infection by M-tropic HIV-1 viruses, confirming that CCR-5 is the major co-receptor for primary HIV-1 strains. The lower frequency of heterozygotes in seropositive patients may indicate partial resistance.
Assuntos
Mutação da Fase de Leitura , Infecções por HIV/imunologia , HIV-1/imunologia , Receptores de Citocinas/imunologia , Receptores de HIV/imunologia , Alelos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Estudos de Coortes , Primers do DNA , Frequência do Gene , Genótipo , Infecções por HIV/genética , Soropositividade para HIV/genética , Soropositividade para HIV/imunologia , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Fusão de Membrana , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Conformação Proteica , Receptores CCR5 , Receptores de Citocinas/química , Receptores de Citocinas/genética , Receptores de HIV/química , Receptores de HIV/genética , População Branca/genéticaRESUMO
An important aspect of human immunodeficiency virus (HIV-1) infection is the regulation of its expression by nuclear factor kappa B (NF-kappa B) through redox-controlled signal transduction pathways. In this study, we demonstrate that iron chelation by deferoxamine (DFO) protects against the cytotoxic and reactivating effects of hydrogen peroxide (H2O2). These protective effects were observed both in lymphocytic (ACH-2) and promonocytic (U1) cells latently infected by HIV-1. Concomitantly, NF-kappa B activation by H2O2, when followed by gel retardation assay, was decreased in the DFO-treated U1 and ACH-2 cells. This latter DFO-mediated effect was specific, as DFO did not clearly affect AP-1 DNA-binding activity when studied after H2O2-induced stress. More importantly, DFO protected against the H2O2-induced activation of HIV-1 as evidenced by reverse transcriptase activity in the supernatant. DFO also protected against PMA-induced NF-kappa B activation as well as TNF-alpha-induced HIV-1 activation. Furthermore, DFO attenuated the p24 response in PBMC infected with HIV-1 and stimulated with IL-2. These different effects of DFO were obtained at DFO concentrations lower than 5 microM. Other chemically unrelated iron chelators also provided protection against cytotoxicity, NF-kappa B activation, and HIV-1 activation in U1 cells challenged with H2O2.
Assuntos
Desferroxamina/farmacologia , HIV-1/efeitos dos fármacos , NF-kappa B/biossíntese , Sideróforos/farmacologia , Sequência de Bases , Linhagem Celular , Sondas de DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , HIV-1/genética , HIV-1/fisiologia , Humanos , Peróxido de Hidrogênio/farmacologia , Quelantes de Ferro/farmacologia , Dados de Sequência Molecular , NF-kappa B/genética , Oxirredução , Estresse Oxidativo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-1/genética , Fator de Necrose Tumoral alfa/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
A method based on subtractive hybridization of brain complementary DNAs with peripheral messenger RNAs has enabled us to construct an enriched brain-specific cDNA library. Single-stranded cDNAs (ssc DNAs) were synthesized from brain polyadenylated mRNAs and subsequently hybridized with peripheral mRNAs immobilized on nitrocellulose membrane. Unhybridized sscDNAs were converted into double-stranded cDNAs and cloned into plasmid pUC13. The screening of the resulting library showed that a high percentage of the cloned cDNAs corresponded to mRNAs specifically transcribed in the brain.
