RESUMO
The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.
Assuntos
Citometria de Fluxo , Imunoensaio , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Adulto , Estudos de Casos e Controles , Criança , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 17/genética , Feminino , Humanos , Leucemia Promielocítica Aguda/imunologia , Masculino , Proteínas de Fusão Oncogênica/imunologia , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Expression of the human papillomavirus type 11 E1 and E2 genes is necessary and sufficient to support viral DNA replication. The full-length E2 protein is a transcriptional modulator that also interacts with the E1 helicase to form an E1/E2 complex at the viral origin of replication. Previous studies indicated that efficient binding of this complex to the replication origin is site-specific and that the E2 homodimer was required for efficient E1 binding. Human papillomavirus type 11 E2 and E1 proteins have been purified and their cooperative binding to the HPV type 11 viral replication origin has been characterized. Low-affinity E1 binding to the HPV type 11 replication origin was demonstrated and found to be largely nonspecific. DNA binding by E1 does not require complex formation with E2 and appears to be independent of ATP binding or hydrolysis. E1 binding quantitatively increased with the addition of increasing amounts of E2 and mutations in the E2 binding site demonstrated that the E2BS site is required for E1 and E2 to specifically bind as a high-affinity complex at the replication origin. Analysis of the A/T-rich E1 binding site via mutation showed that it was nonessential for high-affinity E1/E2 complex formation. Thus, although the replication functions between the animal and the human papillomaviruses are well conserved, there are subtle differences in the DNA binding requirements for E1, which may portend mechanistic differences among the DNA replication systems of various papillomavirus types.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica , Papillomaviridae/genética , Origem de Replicação/genética , Proteínas Virais/genética , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Papillomaviridae/metabolismo , Especificidade por Substrato , Proteínas Virais/metabolismoRESUMO
Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.
Assuntos
Hidrolases Anidrido Ácido/química , DNA Helicases/química , Replicação do DNA , Proteínas de Ligação a DNA/química , Papillomaviridae/química , Proteínas Virais/química , Hidrolases Anidrido Ácido/isolamento & purificação , Hidrolases Anidrido Ácido/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Antígenos Transformantes de Poliomavirus/metabolismo , Baculoviridae/genética , Células Cultivadas , Dicroísmo Circular , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Insetos/citologia , Insetos/virologia , Camundongos , Nucleosídeo-Trifosfatase , Mutação Puntual , Estrutura Secundária de Proteína , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismoRESUMO
The deposition of the beta amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid beta-protein of 39-43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP. We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease. When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid beta-protein peptide and shows a reduction of residues 17-42 of Abeta (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.
Assuntos
Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/enzimologia , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , Cromossomos Humanos Par 19 , Cricetinae , DNA Complementar , Feto , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Mamíferos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Especificidade da EspécieRESUMO
The oxidative stress responsive transcription factor nuclear factor-kappa B (NF-kappa B) consists of a p50 (50 kDa) and p65/RelA (65 kDa) component and can be activated in vitro by TNF alpha, IL1 beta, hydrogen peroxide and oxygen radicals. All of the above factors are also known to be elevated at certain times after transient global ischemia. The present study was performed to determine if NF-kappa B was activated in vivo by transient global forebrain ischemia. Adult male rats were subjected to 30 min of 4-vessel occlusion (4-VO) and sacrificed at selected post-ischemic time points. Levels of NF-kappa B p50 and p65 subunits were determined by immunocytochemistry, Western blot and electrophoretic mobility-shift analysis. The enhancer complex was also confirmed by immuno-gel-shift analysis. Specific labeling of DNA strand breaks and DNA fragmentation was examined in situ by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Western blot analysis of hippocampus showed induction of p50 and p65. A time course of NF-kappa B induction in hippocampus showed a p50-specific band at 6 h that increased in intensity over 12, 48 h and then decreased by 96 h post-ischemia. Immunocytochemistry revealed at 24 h post-ischemia that p65 and p50 immunoreactivity was present in neuronal nuclei of hippocampal CA1 neurons as well as all other hippocampal regions and several other forebrain regions which were not vulnerable to transient forebrain ischemia. At 72 h post-ischemia, nuclear NF-kappa B immunoreactivity had disappeared in all brain areas except in hippocampal CA1 neurons which were degenerating. No evidence for DNA fragmentation as revealed by TUNEL staining could be observed at 24 h. However, at 72 h, hippocampal CA1 neurons were heavily labeled. The results of this study demonstrate that global forebrain ischemia causes a transient activation of NF-kappa B in many forebrain regions. NF-kappa B remains persistently activated in the vulnerable hippocampal CA1 sector. Because of the persistent activation of NF-kappa B in these neurons, the possibility exists that NF-kappa B has a role in programmed cell death in hippocampal CA1 neurons.
Assuntos
Isquemia Encefálica/metabolismo , Fragmentação do DNA , NF-kappa B/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Eletroforese/métodos , Hipocampo/citologia , Imuno-Histoquímica , Masculino , Neurônios/fisiologia , Ratos , Ratos WistarAssuntos
Precursor de Proteína beta-Amiloide/genética , Endopeptidases/genética , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Ativação Enzimática/genética , Regulação da Expressão Gênica , Humanos , Plasmídeos/química , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/química , Especificidade por Substrato , TransfecçãoRESUMO
Thrombin, an important mitogen governing smooth muscle cell proliferation, binds to cultured bovine aortic smooth muscle cells (BASMCs) via both the proteolytically activated thrombin receptor (PATR) and thrombomodulin (TM). Although TM mRNA expression and functional activity is regulated by thrombin in human endothelial cells and mouse hemangioma cells, it remains unclear in those models whether the increased TM mRNA expression observed upon thrombin stimulation is mediated through the activation of PATR or via TM occupancy. We observed in cultured BASMCs that TM mRNA is increased threefold to sixfold by either thrombin, basic fibroblast growth factor (bFGF), or platelet-derived growth factor (PDGF). The increase in TM mRNA with thrombin is time dependent (maximal at 3 hours), a consequence of increased mRNA stability, and accompanied by increases in cell surface TM functional activity. Thrombin-induced TM mRNA was reproduced by the hexameric thrombin receptor-activating peptide (TRAP6) and augmented by a TM-specific antibody. Together, these data suggest that up-regulation of TM mRNA by thrombin is mediated via the PATR. We speculate that increases in BASMC TM mRNA and activity after thrombin may contribute to the impaired thrombus formation observed after atherosclerotic vascular injury.
Assuntos
Receptores de Trombina/fisiologia , Trombina/farmacologia , Trombomodulina/biossíntese , Transcrição Gênica/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Arteriosclerose/fisiopatologia , Bovinos , Células Cultivadas , Clonagem Molecular , Sequência Consenso , Cricetinae , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Mensageiro/biossíntese , Ratos , Receptores de Superfície Celular/fisiologia , Receptores de Trombina/biossíntese , Receptores de Trombina/química , Proteínas Recombinantes/biossíntese , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND AND PURPOSE: After global ischemia, brain levels of hydrogen peroxide, oxygen radicals, and the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are increased. Oxygen radicals, TNF-alpha, and IL-1 beta are known to activate nuclear factor-kappa B (NF-kappa B) in vitro. The present study was performed to determine whether NF-kappa B was activated in vivo by global ischemia in hippocampal CA1 neurons. METHODS: Adult male rats were subjected to 30 minutes of four-vessel occlusion and killed 72 hours later. Levels of NF-kappa B p50 and p65 subunits in hippocampus were determined by immunocytochemistry, Western blot, and gel-shift analysis. Specific labeling of DNA strand breaks was demonstrated by means of an Apoptag apoptosis detection kit. RESULTS: Labeling of DNA strand breaks was present at 72 hours. Chromatin compaction and segregation, a characteristic of apoptosis, was observed in sections stained with hematoxylin and eosin. NF-kappa B p50 and p65 immunoreactivity localized only to nuclei of CA1 neurons at 72 hours after reperfusion. Induction of the activated p50 and p65 subunits was confirmed by Western blot and electromobility shift analysis. The results demonstrate that NF-kappa B is activated selectively in hippocampal CA1 neurons at 72 hours after four-vessel occlusion, which is at the approximate time of CA1 neuronal cell death. CONCLUSIONS: Transient forebrain ischemia resulted in a marked activation of nuclear NF-kappa B in the highly vulnerable CA1 sector. Intense nuclear localization of NF-kappa B was associated only with dying neurons; regions of the hippocampus that were not vulnerable to four-vessel occlusion did not exhibit nuclear NF-kappa B localization. The elevation of NF-kappa B in degenerating CA1 neurons may be associated mechanistically with apoptotic or necrotic cell death.
Assuntos
Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , NF-kappa B/metabolismo , Neurônios/fisiologia , Prosencéfalo/fisiopatologia , Animais , Apoptose , Isquemia Encefálica/genética , Dano ao DNA , Hipocampo/patologia , Hipocampo/fisiopatologia , Masculino , Prosencéfalo/patologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Hippocampal CA1 neurons are highly susceptible to short periods of transient global ischemia. We have previously reported in a rat model of transient forebrain global ischemia that activation and nuclear localization of NF-kB occurs in the CA1 neurons at 24 and 72 h post reperfusion. Events following NF-kB activation would ultimately determine whether damaged cells will undergo programmed cell death. We have selected bcl-x gene expression for study because there is increasing evidence that proteins encoded by the bcl-2 gene family (bcl-2, bcl-x, bax etc) play a role in the regulation of programmed cell death. We have observed that the bcl-x gene promoter contains a putative consensus sequence for NF-kB/CS4 responsive activation. We also can show that other members of the bcl-2 multigene family contain the NF-kB/CS4 sequence in their five prime regulatory regions. In this study, we show that NF-kB p50 and NF-kB p65 act in synergy to transactivate the bcl-x promoter in co-transfected 293 cells. We also report that following ischemia and NF-kB activation, bcl-x messenger RNA levels increase in the CA1 hippocampal region. As a result of this transcriptional increase, surprisingly, it is bcl-xs, the apoptotic form of bcl-x, that is elevated. These results suggest that activation of NF-kB can lead to increased expression of bcl-x as manifested by the increase in the short form of bcl-x.
Assuntos
Hipocampo/metabolismo , Ataque Isquêmico Transitório/metabolismo , Prosencéfalo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/fisiologia , Sequência Consenso , Regulação da Expressão Gênica/genética , Genes Reporter , Hipocampo/irrigação sanguínea , Hipocampo/citologia , NF-kappa B/análise , NF-kappa B/genética , Oxirredução , Plasmídeos , Regiões Promotoras Genéticas/genética , Prosencéfalo/irrigação sanguínea , Prosencéfalo/citologia , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Proteína bcl-XRESUMO
Thrombin and other mitogens regulate the expression of the urokinase-type plasminogen activator receptor (uPAR) protein and mRNA levels in bovine vascular smooth muscle cells (SMC). We investigated interactions between mitogens capable of increasing uPAR mRNA levels in SMC. Up-regulation of uPAR mRNA upon thrombin and basic fibroblast growth factor (bFGF) stimulation was preceded by a 2-3-fold transient increase in bFGF mRNA within 1 h. TGF-beta1 did not result in a significant change in bFGF mRNA levels. Platelet-derived growth factor (PDGF) while substantially enhancing uPAR mRNA levels, diminished bFGF mRNA levels by 3-4-fold. Both thrombin and bFGF induced the message for bFGF-R 2-3-fold. Thrombin also provoked a 3-4-fold rise in TGF-beta1 mRNA levels within 30 min. In summary, on the mRNA level, we demonstrated both positive as well as negative feed-back mechanisms between different mitogens, among them bFGF revealing in addition to autoinduction also up-regulation of the transcript concentration of its own receptor. Thus, cooperation and possible amplification of mitogenic effects might be implicated in the fine-tuned regulation of uPAR mRNA in stimulated bovine aorta SMC.
Assuntos
Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Sequência de Bases , Bovinos , Células Cultivadas , DNA Complementar , Fator 2 de Crescimento de Fibroblastos/metabolismo , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Fator de Crescimento Transformador beta/metabolismoAssuntos
Hipocampo/química , Hibridização In Situ/métodos , Técnicas de Sonda Molecular , RNA/isolamento & purificação , Ribonucleases/química , Amiloide/genética , Animais , Sequência de Bases , Haplorrinos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Príons , Precursores de Proteínas/genética , RNA/metabolismo , Sondas RNA , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Fatores de TempoRESUMO
Bovine vascular smooth muscle cells (SMC) express the urokinase-type plasminogen activator receptor (u-PAR) claimed to be important in cell invasion. Receptor numbers and affinity are regulated by thrombin and several other mitogens involved in SMC proliferation. We investigated the effects of these mitogens on u-PAR mRNA levels. On continuous thrombin stimulation the u-PAR message in SMC was 10 +/- 2.3-fold elevated reaching a maximum between 6 and 9 hours and declining to control values within 48 hours. Thrombin present for 30 minutes on the cell surface produced similar effects. Stimulation with the thrombin receptor activation peptide S-F-L-L-R-N representing the NH2-terminus of the tethered ligand also increased u-PAR mRNA levels with an identical time course. D-Phe-Pro-Arg-chloromethyl ketone (PPACK) active site blocked thrombin and the catalytically inactive thrombin mutant S205A did not affect u-PAR mRNA levels. Thrombin stimulation also resulted in a 2 +/- 0.2-fold transient increase in thrombin receptor mRNA preceding the rise in u-PAR message. Transforming growth factor beta 1 (TGF beta 1) and platelet-derived growth factor (PDGF) showed similar time courses for the elevation of u-PAR mRNA levels with a maximal 5.5 +/- 0.9 and 12 +/- 2.5-fold increase, respectively. Basic fibroblast growth factor (bFGF) and phorbol myristate acetate (PMA) showed a more prolonged effect increasing u-PAR mRNA levels 8 +/- 2.0-fold and 12.3 +/- 2.5-fold, respectively, within 6 hours but remaining 5 to 10-fold elevated at 48 hours. In order to decide if the u-PAR mRNA increase was due to message stabilization or a consequence of transcriptional activation we used the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) during the stimulation experiments. u-PAR mRNA levels on TGF beta 1 stimulation of SMC decayed after the addition of DRB indicating that enhancement of transcriptional activity was involved in the induction. In contrast, the time course of u-PAR mRNA elevation on thrombin, bFGF, and PMA stimulation was not significantly altered in the presence of DRB suggesting that in these latter cases u-PAR mRNA message accumulation was at least in part due to mRNA stabilization. Increased transcriptional activity, mRNA stabilization and expression of u-PAR protein on the SMC surface in response to growth factors may facilitate enhanced cell surface protease activity, cell migration, and development of atheromatous lesions.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Mitógenos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores de Superfície Celular/biossíntese , Receptores de Trombina/efeitos dos fármacos , Trombina/farmacologia , Sequência de Aminoácidos , Animais , Bovinos , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Diclororribofuranosilbenzimidazol/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Dados de Sequência Molecular , Músculo Liso Vascular/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Superfície Celular/genética , Receptores de Trombina/biossíntese , Receptores de Trombina/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
We isolated a full length cDNA clone for bovine u-PAR from a bovine aorta endothelial cell cDNA library and compared the structural properties of this receptor protein to the published human and murine sequences. The bovine u-PAR cDNA clone spans a nucleotide sequence stretch of 1335 bp. The open reading frame contains 330 amino acids with a 20 amino acid long putative signal peptide. The mature protein contains 6 potential N-linked glycosylation sites and a high cysteine content (9%). Bovine u-PAR revealed three homologous internal structural repeats. The NH2-terminal repeat containing the u-PA binding site showed 54% identity to the human and murine NH2-terminal domain, compared to 64% identity between human and mouse u-PAR. Southern blot analysis of genomic DNAs from 9 eukaryotic species suggests that the u-PAR gene is conserved in man, monkey, and cow.
Assuntos
Bovinos/genética , DNA Complementar/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Genes , Humanos , Camundongos , Dados de Sequência Molecular , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Molecular cloning of the polypeptide component of the Rel-related human p75 nucleoprotein complex has revealed its identity with the 65-kDa (p65) subunit of NF-kappa B. Functional analyses of chimeric proteins composed of NF-kappa B p65 C-terminal sequences linked to the DNA-binding domain of the yeast GAL4 polypeptide have indicated that the final 101 amino acids of NF-kappa B p65 comprise a potent transcriptional activation domain. Transient transfection of human T cells with an expression vector encoding NF-kappa B p65, but not NF-kappa B p50, produced marked transcriptional activation of a basal promoter containing duplicated kappa B enhancer motifs from the long terminal repeat of type 1 human immunodeficiency virus. These stimulatory effects of NF-kappa B p65 were synergistically enhanced by coexpression of NF-kappa B p50 but were completely inhibited by coexpression of the v-rel oncogene product. Together, these functional studies demonstrate that NF-kappa B p65 is a transactivating subunit of the heterodimeric NF-kappa B complex and serves as one cellular target for v-Rel-mediated transcriptional repression.
Assuntos
NF-kappa B/fisiologia , Proteínas Repressoras/fisiologia , Proteínas Oncogênicas de Retroviridae/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , NF-kappa B/química , Oligodesoxirribonucleotídeos/química , Proteínas Oncogênicas v-rel , Proteínas Repressoras/química , Transativadores , Fatores de Transcrição/química , Transcrição GênicaRESUMO
Studies of NF-kappa B suggest that this enhancer binding activity corresponds to a family of at least four proteins (p50, p55, p75, and p85) differentially induced with biphasic kinetics during T cell activation. While p55 and p50 are closely related to the 50 kd DNA binding subunit of NF-kappa B, p75 and p85 exhibit DNA binding properties that distinguish them from this 50 kd polypeptide and its regulatory subunits I kappa B and p65. All four members of this kappa B-specific protein family are structurally related to the v-Rel oncoprotein and one, p85, appears identical to human c-Rel. v-Rel, but not nontransforming v-Rel mutants, binds to the kappa B enhancer and inhibits NF-kappa B-activated transcription from the IL-2 receptor alpha promoter and HIV-1 LTR. These findings suggest a Rel-related family of kappa B enhancer binding proteins and raise the possibility that the transforming activity of v-Rel is linked to its inhibitory action on cellular genes under NF-kappa B control.
Assuntos
Proteínas de Ligação a DNA/genética , NF-kappa B/fisiologia , Oncogenes , Proteínas Tirosina Quinases/genética , Proteínas Oncogênicas de Retroviridae/genética , Sequência de Bases , Sondas de DNA , Elementos Facilitadores Genéticos , Vetores Genéticos , Células HeLa/enzimologia , Humanos , Dados de Sequência Molecular , NF-kappa B/antagonistas & inibidores , Sondas de Oligonucleotídeos , Proteínas Oncogênicas v-rel , Mapeamento de Peptídeos , Plasmídeos , Biossíntese de Proteínas , Proteínas Oncogênicas de Retroviridae/isolamento & purificação , Proteínas Oncogênicas de Retroviridae/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
The type 1 human immunodeficiency virus (HIV-1) encodes a 27-kDa protein termed Nef (negative factor). Nef has been reported to down-regulate viral gene transcription directed by the HIV-1 long terminal repeat. To assess the possible role of Nef in the initiation or maintenance of viral latency, we prepared two different nef expression vectors (pNEF from the HXB-3 proviral clone; pNEF-2/3 from HXB-2 and HXB-3) and a control vector containing a frameshift mutation in the HXB-3 nef coding sequence (pNEF-fs). Consistent with prior studies, the Nef proteins produced by pNEF and pNEF-2/3 were approximately 27 kDa in size, posttranslationally modified by myristoylation, and primarily associated with cytoplasmic membrane structures. However, in contrast to previous reports, these Nef proteins failed to inhibit transcriptional activity of the HIV-1 long terminal repeat in any of a variety of cell types, including primary human T lymphocytes, Jurkat or YT-1 leukemic T cells, U-937 promonocytic cells, and nonlymphoid COS cells. Furthermore, HXB-3 proviral clones of HIV-1 containing either a wild-type or mutated version of the nef gene replicated in an indistinguishable manner when transfected into COS cells. Our findings suggest that Nef is neither a transcriptional inhibitor nor a negative viral factor under these assay conditions. Rather, we suggest that the primary biological function of this conserved HIV-1 protein has yet to be defined, perhaps reflecting an intrinsic shortcoming in the in vitro experimental systems presently available for the study of HIV-1.
Assuntos
Regulação Viral da Expressão Gênica , Produtos do Gene nef/metabolismo , HIV-1/genética , Transcrição Gênica , Proteínas Virais Reguladoras e Acessórias/metabolismo , Animais , Linhagem Celular , DNA Viral/efeitos dos fármacos , DNA Viral/genética , Produtos do Gene nef/farmacologia , Genes nef , HIV-1/efeitos dos fármacos , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes/farmacologia , Sequências Repetitivas de Ácido Nucleico/efeitos dos fármacos , Mapeamento por Restrição , Transfecção , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
The rex gene of the type I human T-cell leukaemia virus (HTLV-I) encodes a phosphorylated nuclear protein of relative molecular mass 27,000 which is required for viral replication. The Rex protein acts by promoting the cytoplasmic expression of the incompletely spliced viral messenger RNAs that encode the virion structural proteins. To identify the biologically important peptide domains within Rex, we introduced a series of mutations throughout its sequence. Two distinct classes of mutations lacking Rex biological activity were identified. One class corresponds to trans-dominant repressors as they inhibit the function of the wild-type Rex protein. The second class of mutants, in contrast, are recessive negative, rather than dominant negative, as they are not appropriately targeted to the cell nucleus. These results indicate the presence of at least two functionally distinct domains within the Rex protein, one involved in protein localization and a second involved in effector function. The trans-dominant Rex mutants may represent a promising new class of anti-viral agents.
Assuntos
Regulação da Expressão Gênica , Genes Dominantes , Genes Virais , HIV-1/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Mutação , Proteínas Oncogênicas de Retroviridae/genética , Transativadores/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Produtos do Gene rex , Dados de Sequência Molecular , Proteínas Oncogênicas de Retroviridae/fisiologia , Transativadores/fisiologia , TransfecçãoRESUMO
The human interleukin-2 receptor alpha (IL2R alpha) gene is transcriptionally activated by both phorbol esters and the HTLV-I trans-activator (Tax) protein through a mechanism that involves the interaction of inducible DNA binding proteins with a kappa B-like enhancer element (-267 to -256). Using mutated IL2R alpha promoter constructs in transient transfection and DNA binding assays, we now demonstrate that sequences located immediately upstream and downstream of the kappa B enhancer also contribute to the regulation of IL2R alpha gene expression. One upstream sequence termed UE-1 is preferentially required for phorbol ester relative to Tax-induced activation and specifically interacts with a constitutively expressed 56-kD cellular factor. In contrast, two overlapping downstream elements between nucleotides -252 and -239 appear to be required for both phorbol ester and Tax-induced activation. One of these elements, an Sp1-like sequence, binds a constitutively expressed 100-kD T-cell protein consistent in size with Sp1 isolated from HeLa cells. The second element, located between the kappa B and Sp1 sites, resembles the decanucleotide core of the serum response element (SRE) from the c-fos gene and interacts with a constitutively expressed factor. Together, these findings implicate a functional role for multiple constitutively expressed DNA binding proteins, in addition to the inducible kappa B-specific factors, in the overall regulation of IL2R alpha gene activation.