RESUMEN
The Wnt pathway has essential roles in cell proliferation, cell fate determination and tumorigenesis by regulating the expression of a wide range of target genes. As a core signaling cascade, the canonical Wnt pathway is regulated at different levels by numerous proteins. We have previously shown that carboxypeptidase E (CPE) is a novel regulator of the canonical Wnt signaling pathway. Here, we show that CPE and the Wnt3a ligand are co-secreted from cells. We show that although the C'-terminal Lys residue of Wnt3a is critical for its activity and is important for the effect of CPE on the Wnt pathway, CPE does not execute its effect by removing this Wnt3a residue. Interestingly, CPE through its N'-terminal sequence, forms aggregates with Wnt3a and possible endoplasmic reticulum (ER) stress leading to its loss of function. Together, our current results provide a mechanistic insight into the way CPE regulates the canonical Wnt signaling pathway.
Asunto(s)
Carboxipeptidasa H/fisiología , Proteína Wnt3A/metabolismo , Animales , Células COS , Chlorocebus aethiops , Estrés del Retículo Endoplásmico , Células HEK293 , Humanos , Agregado de Proteínas , Vía de Señalización WntRESUMEN
The NAP motif of activity-dependent neuroprotective protein (ADNP) enhanced memory scores in patients suffering from mild cognitive impairment and protected activities of daily living in schizophrenia patients, while fortifying microtubule (MT)-dependent axonal transport, in mice and flies. The question is how does NAP fortify MTs? Our sequence analysis identified the MT end-binding protein (EB1)-interacting motif SxIP (SIP, Ser-Ile-Pro) in ADNP/NAP and showed specific SxIP binding sites in all members of the EB protein family (EB1-3). Others found that EB1 enhancement of neurite outgrowth is attenuated by EB2, while EB3 interacts with postsynaptic density protein 95 (PSD-95) to modulate dendritic plasticity. Here, NAP increased PSD-95 expression in dendritic spines, which was inhibited by EB3 silencing. EB1 or EB3, but not EB2 silencing inhibited NAP-mediated cell protection, which reflected NAP binding specificity. NAPVSKIPQ (SxIP=SKIP), but not NAPVAAAAQ mimicked NAP activity. ADNP, essential for neuronal differentiation and brain formation in mouse, a member of the SWI/SNF chromatin remodeling complex and a major protein mutated in autism and deregulated in schizophrenia in men, showed similar EB interactions, which were enhanced by NAP treatment. The newly identified shared MT target of NAP/ADNP is directly implicated in synaptic plasticity, explaining the breadth and efficiency of neuroprotective/neurotrophic capacities.
Asunto(s)
Espinas Dendríticas/fisiología , Proteínas de Homeodominio/metabolismo , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Homólogo 4 de la Proteína Discs Large , Escherichia coli , Guanilato-Quinasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/fisiología , Células PC12 , Dominios y Motivos de Interacción de Proteínas , Ratas , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Aberrant activation of the canonical Wnt signal transduction pathway is involved in many diseases including cancer and is especially implicated in the development and progression of colorectal cancer. The key effector protein of the canonical Wnt pathway is ß-catenin, which functions with T-cell factor/lymphoid enhancer factor to activate expression of Wnt target genes. In this study, we used a new functional screen based on cell survival in the presence of cDNAs encoding proteins that activate the Wnt pathway thus identifying novel Wnt signaling components. Here we identify carboxypeptidase E (|CPE) and its splice variant, ΔN-CPE, as novel regulators of the Wnt pathway. We show that whereas ΔN-CPE activates the Wnt signal, the full-length CPE (F-CPE) protein is an inhibitor of Wnt/ß-catenin signaling. F-CPE forms a complex with the Wnt3a ligand and the Frizzled receptor. Moreover, F-CPE disrupts disheveled-induced signalosomes that are important for transducing the Wnt signal and reduces ß-catenin protein levels and activity. Taken together, our data indicate that F-CPE and ΔN-CPE regulate the canonical Wnt signaling pathway negatively and positively, respectively, and demonstrate that this screening approach can be a rapid means for isolation of novel Wnt signaling components.
Asunto(s)
Carboxipeptidasa H/metabolismo , Vía de Señalización Wnt , Proteína Wnt3A/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Carboxipeptidasa H/genética , Chlorocebus aethiops , Proteínas Dishevelled , Receptores Frizzled/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Proteolisis , ARN Interferente Pequeño/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Methyl jasmonate (MJ) is a plant stress hormone with selective cytotoxic anti-cancer activities. The TNF-related apoptosis-inducing ligand (TRAIL) death pathway is an attractive target for cancer therapy. Although TRAIL receptors are specifically expressed in primary cancer cells and cancer cell lines, many types of cancer cells remain resistant to TRAIL-induced cytotoxicity. Here we have assessed a possible synergy between MJ and TRAIL cytotoxicity in colorectal cancer (CRC) cell lines. EXPERIMENTAL APPROACH: CRC cell lines were pre-incubated with sub-cytotoxic concentrations of MJ followed by TRAIL administration. Cell death was determined by XTT assay and microscopy. Cytochrome c release, caspase cleavage, TRAIL-associated factors, X-linked inhibitor of apoptosis (XIAP) and survivin protein levels were detected by immunoblotting. Survivin transcription was examined by RT-PCR. KEY RESULTS: Pre-treatment with MJ resulted in increased TRAIL-induced apoptotic cell death, increased cytochrome c release and caspase cleavage. TNFRSF10A, TNFRSF10B, TNFRSF10D, Fas-associated death domain and cellular FLICE-like inhibitory protein remained unchanged during MJ-induced TRAIL sensitization, whereas MJ induced a significant decrease in survivin protein levels. Overexpression of survivin prevented MJ-induced TRAIL cytotoxicity, implying a role for survivin in MJ-induced TRAIL sensitization. MJ decreased survivin mRNA indicating that MJ may affect survivin transcription. In a ß-catenin/transcription factor (TCF)-dependent luciferase activity assay, MJ decreased TCF-dependent transcriptional activity. CONCLUSION AND IMPLICATIONS: MJ, at sub-cytotoxic levels, sensitized CRC cells to TRAIL-induced apoptosis. Thus, combinations of MJ and TRAIL, both selective anti-cancer agents, have potential as novel treatments for CRC.
Asunto(s)
Acetatos/farmacología , Antineoplásicos/farmacología , Neoplasias del Colon/patología , Ciclopentanos/farmacología , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Oxilipinas/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Citocromos c/metabolismo , Regulación hacia Abajo , Sinergismo Farmacológico , Citometría de Flujo , Genes Reporteros , Humanos , Luciferasas/genética , Receptores de Muerte Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Survivin , TransfecciónRESUMEN
Beta-catenin is the central signalling molecule of the canonical Wnt pathway, where it activates target genes in a complex with lymphoid enhancer factor/T-cell factor transcription factors in the nucleus. The regulation of beta-catenin activity is thought to occur via a cytoplasmatic multiprotein complex that includes the serine/threonine kinase glycogen synthase kinase-3beta (GSK-3beta) that phosphorylates beta-catenin, marking it for degradation by the proteasome. Here, we provide evidence showing that GSK-3beta has a nuclear function in downregulating the activity of beta-catenin. Using colorectal cell lines that express a mutant form of beta-catenin, which cannot be phosphorylated by GSK-3beta and ectopically expressed mutant beta-catenin protein, we show that nuclear GSK-3beta functions in a mechanism that does not involve beta-catenin phosphorylation to reduce the levels of Wnt signalling. We show that GSK-3beta enters the nucleus, forms a complex with beta-catenin and lowers the levels of beta-catenin/TCF-dependent transcription in a mechanism that involves GSK-3beta-Axin binding.
Asunto(s)
Núcleo Celular/metabolismo , Neoplasias Colorrectales/patología , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Cadherinas/metabolismo , Línea Celular , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3 beta , Humanos , Modelos Biológicos , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Transducción de Señal , Transcripción GenéticaRESUMEN
Adenomatous polyposis coli (APC) is mutated in most colorectal cancers. APC downregulates nuclear beta-catenin, which is thought to be critical for its tumour suppressor function. However, APC may have additional and separate functions at the cell periphery. Here, we examine polarized MDCK and WIF-B hepatoma cells and find that APC is associated with their lateral plasma membranes. This depends on the actin cytoskeleton but not on microtubules, and drug wash-out experiments suggest that APC is delivered continuously to the plasma membrane by a dynamic actin-dependent process. In polarized MDCK cells, APC also clusters at microtubule tips in their basal-most regions. Microtubule depolymerization causes APC to relocalize from these tips to the plasma membrane, indicating two distinct peripheral APC pools that are in equilibrium with each other in these cells. Truncations of APC such as those found in APC mutant cancer cells can neither associate with the plasma membrane nor with microtubule tips. The ability of APC to reach the cell periphery may thus contribute to its tumour suppressor function in the intestinal epithelium.
Asunto(s)
Actinas/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Células Epiteliales/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Carcinoma Hepatocelular/metabolismo , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular/fisiología , Citoesqueleto/metabolismo , Perros , Células Epiteliales/citología , Proteínas Fluorescentes Verdes , Humanos , Riñón/metabolismo , Riñón/ultraestructura , Proteínas Luminiscentes/genética , Sustancias Macromoleculares , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Mutagénesis Sitio-Dirigida , Nocodazol/farmacología , Unión Proteica/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tiazoles/farmacología , Tiazolidinas , TransfecciónRESUMEN
The adenomatous polpyposis coli (APC) protein is mutated in most colorectal tumours. Nearly all APC mutations are truncations, and many of these terminate in the mutation cluster region located halfway through the protein. In cancer cells expressing mutant APC, beta-catenin is stabilized and translocates into the nucleus to act as a transcriptional co-activator of T-cell factor. During normal development, APC also promotes the destabilization of beta-catenin and Drosophila Armadillo. It does so by binding to the Axin complex which earmarks beta-catenin/Armadillo for degradation by the proteasome pathway. APC has a regulatory role in this process, which is poorly understood. Here we show that APC contains highly conserved nuclear export signals 3' adjacent to the mutation cluster region that enable it to exit from the nucleus. This ability is lost in APC mutant cancer cells, and we provide evidence that beta-catenin accumulates in the nucleus as a result. Thus, the ability of APC to exit from the nucleus appears to be critical for its tumour suppressor function.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Transactivadores , Proteína de la Poliposis Adenomatosa del Colon , Secuencia de Aminoácidos , Animales , Transporte Biológico/efectos de los fármacos , Células COS , Núcleo Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Secuencia Conservada , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Drosophila , Ácidos Grasos Insaturados/farmacología , Genes Reporteros , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales Cultivadas , beta CateninaRESUMEN
The Rev protein of equine infectious anaemia virus (EIAV) was shown previously to stimulate the expression of a heterologous CAT reporter gene when the 3' half of the EIAV genome was present downstream in cis. However, computer analysis could not reveal the existence of a stable RNA secondary structure that could be analogous to the Rev-responsive element of other lentiviruses. In the present study, the inhibitory RNA element designated the cis-acting repressing sequence (CRS) has been localized to the centre of the EIAV genome. The inhibition exerted by this element could be overcome by supplying Rev in trans. The ability of the EIAV CRS to function in a heterologous context suggests that it does not require interactions with other viral proteins. Site-directed mutagenesis showed that the various centrally located suboptimal splice sites of the EIAV genome function as CRS and confer Rev-dependence on the CRS-containing transcripts. In addition, the data suggest that in canine Cf2Th cells, which are highly permissive for EIAV replication, CRS prevents nuclear export of CRS-containing transcripts and the supply of Rev relieves this suppression.
Asunto(s)
Productos del Gen rev/genética , Productos del Gen rev/metabolismo , Virus de la Anemia Infecciosa Equina/genética , Empalme del ARN , Elementos de Respuesta , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Perros , Regulación Viral de la Expresión Génica , Genes Reporteros , Virus de la Anemia Infecciosa Equina/fisiología , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Ribonucleasas/metabolismoRESUMEN
The Tat protein of equine infectious anemia virus, EIAV, was shown to augment viral gene expression, presumably through interaction with the Tat responsive element, TAR. Recently, cell-free polyadenylation assays suggested that perturbation of the EIAV TAR secondary structure diminished polyadenylation efficiency. The present study indicates that the EIAV TAR regulates the efficiency of the 3'-end processing of viral RNA also in transfected cells. Moreover, our data suggest that the provision of the EIAV Tat protein in trans potentiates read-through transcription through the 3' viral long terminal repeat (3' LTR), thus suggesting activation of downstream-located cellular genes.
Asunto(s)
Regulación de la Expresión Génica , Productos del Gen tat/metabolismo , ARN Viral/genética , Transcripción Genética , Animales , Sistema Libre de Células , Cartilla de ADN , Perros , Genes tat , Caballos , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , TransfecciónRESUMEN
The present study provides evidence on the occurrence of DNA rearrangement between the redundant 5' and 3' R domains of equine infectious anemia virus (EIAV) Tat cDNA. This was correlated with a gradual loss of cDNA copy number concomitantly with a decrease in gene expression. Removal of the 5' RU5 abolished rearrangement and stabilized Tat expression in EIAV tat cDNA transfectants. Our data suggest that prior removal of the 5' R from cloned retroviral cDNAs can impede DNA rearrangement, thus preventing cDNA excision that frequently occurs and hinders permanent expression of retroviral cDNAs in stable transfectants.
Asunto(s)
ADN Viral/genética , Expresión Génica , Reordenamiento Génico , Virus de la Anemia Infecciosa Equina/genética , ADN Complementario/genética , Regulación Viral de la Expresión Génica , TransfecciónRESUMEN
Trans-activator (tat) proteins are necessary components for the completion of the T replication cycle of lentiviruses. The three-dimensional structure of the equine infectious anemia virus (EIAV) tat protein (e-tat) was studied with CD spectroscopy, NMR spectroscopy, and restrained molecular-dynamics calculations. No stable elements of regular secondary structure were detected, but the sequence regions responsible for nucleic acid binding showed helix-forming tendency, e-tat exhibits a flexible tertiary structure, and only the amino acids comprising the core sequence region form a well-defined tertiary fold. The three-dimensional structure allows discussion of biochemical data as well as data from molecular biological investigations of lentiviral tat proteins.
Asunto(s)
Productos del Gen tat/química , Virus de la Anemia Infecciosa Equina/química , Conformación Proteica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Dicroismo Circular , Gráficos por Computador , Productos del Gen tat/biosíntesis , VIH/metabolismo , Caballos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10(11) bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by 'scrape loading'. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.
Asunto(s)
Productos del Gen tat/metabolismo , Virus de la Anemia Infecciosa Equina/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cartilla de ADN , Perros , Escherichia coli , Productos del Gen tat/biosíntesis , Productos del Gen tat/aislamiento & purificación , VIH-1/metabolismo , Virus de la Anemia Infecciosa Equina/genética , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Moldes Genéticos , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The solution structure of the 75-amino-acid trans-activator (Tat) protein of the equine infectious-anemia virus in trifluoroethanol-containing solution was determined by two-dimensional and three-dimensional nuclear magnetic resonance spectroscopy, resulting in a total of 838 nuclear-Over-hauser-enhancement distance restraints, and restrained molecular-dynamics simulations. In contrast to the recently determined structure of this protein in trifluoroethanol-free pH 6.3 solution, the hydrophobic core and the adjacent basic RNA-binding region of the protein showed well-defined alpha-helical secondary structure in trifluoroethanol-containing solution. The helical regions comprise those parts of the molecule whose helix-forming tendencies were noted earlier in trifluoroethanol-free solution. Two helices (Gln38-Arg43 and Asp48-Ala64) are connected by a tight type-II turn centered at the strictly conserved Gly46 leading to a helix-turn-helix motif in the core and basic region of the protein. A third helix (Thr9-Asn13) is located in the less well defined N-terminal part of the protein. These observations may support the notion that the protein adopts a helical structure in the RNA-binding region on complex formation. Although the secondary-structure elements become better defined in trifluoroethanol-containing solution, the opposite is true for the hydrophobically stabilized tertiary structure. This adds a caveat to studies of protein structures in trifluoroethanol-containing solution in general.
Asunto(s)
Productos del Gen tat/química , Virus de la Anemia Infecciosa Equina/química , Secuencia de Aminoácidos , Estabilidad de Medicamentos , Productos del Gen tat/genética , Secuencias Hélice-Asa-Hélice/genética , Virus de la Anemia Infecciosa Equina/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Soluciones , Termodinámica , TrifluoroetanolRESUMEN
Trans-activator (Tat) proteins regulate the transcription of lentiviral DNA in the host cell genome. These RNA binding proteins participate in the life cycle of all known lentiviruses, such as the human immunodeficiency viruses (HIV) or the equine infectious anemia virus (EIAV). The consensus RNA binding motifs [the trans-activation responsive element (TAR)] of HIV-1 as well as EIAV Tat proteins are well characterized. The structure of the 75-amino acid EIAV Tat protein in solution was determined by two- and three-dimensional nuclear magnetic resonance methods and molecular dynamics calculations. The protein structure exhibits a well-defined hydrophobic core of 15 amino acids that serves as a scaffold for two flexible domains corresponding to the NH2- and COOH-terminal regions. The core region is a strictly conserved sequence region among the known Tat proteins. The structural data can be used to explain several of the observed features of Tat proteins.
Asunto(s)
Productos del Gen tat/química , Virus de la Anemia Infecciosa Equina/química , Secuencia de Aminoácidos , Productos del Gen tat/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , ARN/metabolismo , Alineación de SecuenciaRESUMEN
Nuclear magnetic resonance (NMR) spectroscopy revealed features of the secondary structure of the equine infectious anemia virus (EIAV) Tat protein in solution. We could show that this protein, which is required in the replication cycle of lentiviruses, forms a predominantly helical structure in trifluoroethanol/water (40% by vol.) solution. In particular, the basic RNA-binding region and the adjacent core domain, which are highly conserved among lentiviral Tat proteins, show helix-type secondary structure under these conditions. Our observations, in concert with recent biochemical data from other laboratories, suggest that the core sequence region and the basic sequence region form interdependent structural domains, both possibly necessary for correct RNA binding.
Asunto(s)
Productos del Gen tat/química , Virus de la Anemia Infecciosa Equina/química , Secuencia de Aminoácidos , Secuencia de Bases , Productos del Gen tat/metabolismo , Virus de la Anemia Infecciosa Equina/metabolismo , Virus de la Anemia Infecciosa Equina/ultraestructura , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ARN/metabolismoRESUMEN
Three cDNA clones representing structurally distinct transcripts were isolated from a cDNA library prepared from cells infected with equine infectious anemia virus (EIAV) by using a probe representing the S3 open reading frame, which is thought to encode Rev. One species, designated p2/2, contained four exons and was identical to a previously described polycistronic mRNA that encodes Tat. This transcript was predicted to also direct the synthesis of a truncated form of the transmembrane protein and a putative Rev protein whose N-terminal 29 amino acids, derived from env, are linked to S3 sequences. The second cDNA, p176, also consisted of four exons which were generated by two of three of the same splicing events that occur with p2/2 but not with the Tat mRNA. The alternative splice site giving rise to the second exon of p176 results in a bicistronic message that would encode the same transmembrane and Rev proteins as p2/2. The first exon of the third transcript, p20, was identical to those of p2/2 and p176 but was spliced directly to S3. This monocistronic message could encode a second form of Rev that lacks env sequences, provided that Rev synthesis would initiate at a non-AUG codon. The coding capacity of each cDNA was assessed in a eukaryotic system using S3 antisera. Two putative Rev proteins with apparent molecular masses of 18 and 16 kDa were expressed by p2/2 and p176, while p20 expressed only a 16-kDa species. Analysis of EIAV-infected cells with S3 antisera revealed the presence of an 18-kDa protein. Surprisingly, the same protein was detected in purified virions. By using a reporter construct, the chloramphenicol acetyltransferase gene linked to EIAV env sequences, we were able to demonstrate greatly enhanced chloramphenicol acetyltransferase activity in cells cotransfected with this construct and any of the three cDNAs.
Asunto(s)
Expresión Génica , Productos del Gen rev/biosíntesis , Genes rev , Virus de la Anemia Infecciosa Equina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Clonación Molecular , Exones , Biblioteca de Genes , Productos del Gen rev/genética , Genes env , Genoma Viral , Virus de la Anemia Infecciosa Equina/metabolismo , Cinética , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , TransfecciónRESUMEN
The equine infectious anemia virus (EIAV) trans-activating (Tat) protein is a close homologue of the human immunodeficiency virus (HIV) Tat protein. Both of these proteins bind to an RNA trans-activation responsive element (TAR). We synthesized chemically a protein with the sequence of the 75 amino acid Tat protein from EIAV. The chemically synthesized protein was shown to be biologically active. Circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy were used to structurally characterize the synthetic protein. We obtained nearly complete resonance assignments in the 2D-NMR spectra of the protein at pH 3.0. There is at least some evidence from the experimental data that the basic TAR binding domain of the synthetic protein has a tendency to form a helix, but our experiments also indicate that the protein probably does not have an overall stable tertiary structure in aqueous solution at this pH. CD spectroscopy suggested that the protein adopts a more stable, predominantly alpha-helical structure in a trifluoroethanol/water solution.