RESUMEN
T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCRs), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high-avidity TCR with specificity for a HLA-B*07:02-(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity in vitro and in vivo. In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate, exemplarily for MPO, that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.
Asunto(s)
Antígenos HLA/inmunología , Leucemia Mieloide/genética , Leucemia Mieloide/inmunología , Péptidos/inmunología , Peroxidasa/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Modelos Animales de Enfermedad , Mapeo Epitopo , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Antígenos HLA/metabolismo , Antígeno HLA-B7/inmunología , Antígeno HLA-B7/metabolismo , Xenoinjertos , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/mortalidad , Ligandos , Ratones , Péptidos/metabolismo , Peroxidasa/química , Peroxidasa/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Transducción GenéticaRESUMEN
SETTING: A community health worker (CHW) program was established in Neno District, Malawi, in 2007 by Partners In Health in support of Ministry of Health activities. Routinely generated CHW data provide critical information for program monitoring and evaluation. Informal assessments of the CHW reports indicated poor quality, limiting the usefulness of the data. OBJECTIVES: 1) To establish the quality of aggregated measures contained in CHW reports; 2) to develop interventions to address poor data quality; and 3) to evaluate changes in data quality following the intervention. DESIGN: We developed a lot quality assurance sampling-based data quality assessment tool to identify sites with high or low reporting quality. Following the first assessment, we identified challenges and best practices and followed the interventions with two subsequent assessments. RESULTS: At baseline, four of five areas were classified as low data quality. After 8 months, all five areas had achieved high data quality, and the reports generated from our electronic database became consistent and plausible. CONCLUSION: Program changes included improving the usability of the reporting forms, shifting aggregation responsibility to designated assistants and providing aggregation support tools. Local quality assessments and targeted interventions resulted in immediate improvements in data quality.
RESUMEN
Placental tissue protein 13 (PP-13), one of the 56 known placental proteins identified till today, was purified from placentas obtained from women at delivery, and used to evoke antibodies against it. The purified PP-13 was lysed to peptides, which were sequenced, leading to the full-length cDNA sequencing and its expression in Escherichia coli. Sequence analysis in databases showed homology to the galectin family. Of the various antibody preparations developed, a pair of monoclonal antibodies (MAbs) coupled to the recombinant PP-13 (PP-13-R) was used for the immunodetection of PP-13 in pregnant women's serum with the solid-phase ELISA format. With a dynamic range of 25-500 pg/mL with no background in non-pregnant women's serum and men's serum, the ELISA test was suitable for the detection of PP-13 in the 1st, 2nd, and 3rd trimesters. PP-13 levels slowly increase during pregnancy. In the 1st trimester, lower than normal PP-13 levels were found in fetal growth restriction (IUGR), preeclampsia (PE), and particularly in early PE (<34 weeks of gestation). In the 2nd and 3rd trimesters, higher than normal concentrations were found in PE, IUGR and in preterm delivery (PTD). Application of PP-13 to cultured trophoblasts elicited depolarization carried by calcium ions, followed by liberation of linoleic and arachidonic acids from the trophoblast membrane, and a subsequent elevation of prostacyclin and thromboxane. These effects were negligible when PP-13 derived from the placentas of patients with IUGR, PE or PTD was used. The results are discussed in view of the potential utilization of PP-13 for early serum screening to assess the risk to develop placental insufficiency, coupled to a differential analysis of the various pathologies by analyzing cultured trophoblasts.
Asunto(s)
Líquidos Corporales/química , Complicaciones del Embarazo/metabolismo , Proteínas Gestacionales/análisis , Proteínas Gestacionales/farmacología , Trofoblastos/efectos de los fármacos , Secuencia de Aminoácidos , Líquido Amniótico/química , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Células Cultivadas , ADN Complementario/análisis , ADN Complementario/química , Ensayo de Inmunoadsorción Enzimática , Femenino , Retardo del Crecimiento Fetal/metabolismo , Galectinas , Edad Gestacional , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Trabajo de Parto Prematuro , Preeclampsia/metabolismo , Embarazo , Proteínas Gestacionales/genética , Radioinmunoensayo , Proteínas Recombinantes , Sensibilidad y Especificidad , Homología de SecuenciaRESUMEN
The goal of this study was to identify and characterise the major allergen(s) of sesame seed. Detection of specific IgE to sesame proteins was performed with Pharmacia CAP System and Western blotting, after separation of sesame proteins by SDS-PAGE, using sera from 28 subjects diagnosed as allergic to sesame. The major allergen was separated by gel filtration chromatography and identified by selective proteolysis followed by peptide sequence analyses, employing electrospray-ionization mass spectrometer. Twenty-four of the 28 subjects had sesame-specific IgE. A 14 kDa protein belonging to the 2S albumin family was recognised by 22 of the 24 sera used. Subjecting the 14 kDa after HPLC separation to proteolysis with Lys C yielded 3 peptides, but only one reacted positively in the dot blot test. This peptide, corresponds in the whole protein chain to residues 24-94. The reactivity of the 14 kDa protein with most of the sera indicates that this is the major sesame allergen, later identified as 2S albumin precursor; and its peptide which reacted positively in the dot blot test evidently contains an epitope(s). Some minor sesame allergens, of higher molecular weight, were also revealed.
Asunto(s)
Albúminas/análisis , Alérgenos/análisis , Hipersensibilidad a los Alimentos/inmunología , Inmunoglobulina E/análisis , Proteínas de Plantas/análisis , Semillas/inmunología , Sesamum/inmunología , Adolescente , Adulto , Albúminas/inmunología , Alérgenos/inmunología , Secuencia de Aminoácidos , Western Blotting , Niño , Preescolar , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Humanos , Inmunoglobulina E/inmunología , Lactante , Datos de Secuencia Molecular , Proteínas de Plantas/inmunología , Sesamum/químicaRESUMEN
DRP-1 is a pro-apoptotic Ca2+/calmodulin (CaM)-regulated serine/threonine kinase, recently isolated as a novel member of the DAP-kinase family of proteins. It contains a short extra-catalytic tail required for homodimerization. Here we identify a novel regulatory mechanism that controls its pro-apoptotic functions. It comprises a single autophosphorylation event mapped to Ser308 within the CaM regulatory domain. A negative charge at this site reduces both the binding to CaM and the formation of DRP-1 homodimers. Conversely, the dephosphorylation of Ser308, which takes place in response to activated Fas or tumour necrosis factor-alpha death receptors, increases the formation of DRP-1 dimers, facilitates the binding to CaM and activates the pro-apoptotic effects of the protein. Thus, the process of enzyme activation is controlled by two unlocking steps that must work in concert, i.e. dephosphorylation, which probably weakens the electrostatic interactions between the CaM regulatory domain and the catalytic cleft, and homodimerization. This mechanism of negative autophosphorylation provides a safety barrier that restrains the killing effects of DRP-1, and a target for efficient activation of the kinase by various apoptotic stimuli.
Asunto(s)
Apoptosis , Calmodulina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Cromatografía en Gel , Proteínas Quinasas Asociadas a Muerte Celular , Dimerización , Activación Enzimática , Humanos , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Alineación de Secuencia , Electricidad Estática , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/farmacologíaRESUMEN
The ABRF-98SEQ sample was the 11th in a series of amino acid sequencing studies performed by the Protein Sequence Research Group of the Association of Biomolecular Resource Facilities. This study was designed to aid participants' laboratories in determining their abilities to analyze the amino acid sequence of a peptide at high sensitivity using Edman degradation, mass spectrometry (MS), or both. ABRF-98SEQ is a 17-amino acid synthetic peptide (IFDDEIEEVQALYPTER) that resembles a typical tryptic peptide. It was distributed at the 2.8-pmol level. The sample was sent dried in a microfuge tube accompanied by instructions on solubilizing the sample and by a survey form. Including tentative calls, the correct sequence was obtained by 16% of the responding participants, compared with only 6% in the 1997 study when the low-level peptide was a minor component of a mixture. This increase probably reflects the purity of ABRF-98SEQ. A secondary factor in the increase in correct calls may be the larger number of respondents this year reporting that they perform sequence analysis at the 1- to 10-pmol level. Most respondents who obtained the correct sequence used a combination of Edman sequencing and molecular weight determination by MS. Overall, the accuracy and sensitivity of peptide sequencing by Edman degradation continue to improve and are clearly aided by the use of MS for molecular weight determination. Although peptide sequencing by MS is not yet routinely practiced by the participating laboratories, results of this study indicate that MS-derived sequence data, when properly interpreted, are valuable for correcting, completing, or corroborating sequence assignments derived by Edman.
RESUMEN
Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein-antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2K(b)-restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the K(b)-ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N-terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC-ova degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.
Asunto(s)
Epítopos/metabolismo , Antígenos H-2/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Sistema Libre de Células , Cromatografía Líquida de Alta Presión , Clonación Molecular , Ligandos , Ratones , Mutagénesis Insercional , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Ovalbúmina/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Conejos , Proteínas Recombinantes/metabolismo , Mapeo RestrictivoRESUMEN
The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation of a protein via this pathway involves selective modification of epsilon-NH2 groups of internal lysine residues by ubiquitination. To date, this amino group has been the only known target for ubiquitination. Here we report that the N-terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did not affect significantly its conjugation and degradation either in vivo or in vitro. In cells, degradation of the lysine-less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibition is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD. In striking contrast, wild-type MyoD, in which all the internal Lys residues have been retained but the N-terminus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell-free system, ATP and multiple ubiquitination are essential for degradation of the lysine-less protein. Specific chemical modifications have yielded similar results. Selective blocking of the alpha-NH2 group of wild-type protein renders it stable, while modification of the internal Lys residues with preservation of the free N-terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N-terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety.
Asunto(s)
Lisina/metabolismo , Proteína MioD/metabolismo , Ubiquitinas/metabolismo , Adenosina Trifosfato/fisiología , Animales , Células COS , Células Cultivadas , Proteína MioD/química , Proteína MioD/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Plásmidos/genética , Trazadores Radiactivos , Transfección , Ubiquitinas/fisiologíaRESUMEN
Heparan sulfate moieties of cell-surface proteoglycans modulate the biological responses to fibroblast growth factors (FGFs). We have reported previously that cell-associated heparan sulfates inhibit the binding of the keratinocyte growth factor (KGF), but enhance the binding of acidic FGF to the KGF receptor, both in keratinocytes, which naturally express this receptor, and in rat myoblasts, which ectopically express it (Reich-Slotky, R., Bonneh-Barkay, D., Shaoul, E., Berman, B., Svahn, C. M., and Ron, D. (1994) J. Biol. Chem. 269, 32279-32285). The proteoglycan bearing these modulatory heparan sulfates was purified to homogeneity from salt extracts of rat myoblasts by anion-exchange and FGF affinity chromatography and was identified as rat glypican. Affinity-purified glypican augmented the binding of acidic FGF and basic FGF to human FGF receptor-1 in a cell-free system. This effect was abolished following digestion of glypican by heparinase. Addition of purified soluble glypican effectively replaced heparin in supporting basic FGF-induced cellular proliferation of heparan sulfate-negative cells expressing recombinant FGF receptor-1. In keratinocytes, glypican strongly inhibited the mitogenic response to KGF while enhancing the response to acidic FGF. Taken together, these findings demonstrate that glypican plays an important role in regulating the biological activity of fibroblast growth factors and that, for different growth factors, glypican can either enhance or suppress cellular responsiveness.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Proteoglicanos/metabolismo , Receptores Virales/metabolismo , Animales , Línea Celular , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Sustancias de Crecimiento/metabolismo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/química , Heparitina Sulfato/aislamiento & purificación , Humanos , Proteoglicanos/química , Proteoglicanos/aislamiento & purificación , RatasRESUMEN
Transcription factor TFIID is a multiprotein complex composed of the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). TFIID plays an essential role in mediating transcriptional activation by gene-specific activators. Numerous transcriptional activators have been characterized from mammalian cells; however, molecular analysis of the components of mammalian TFIID has been incomplete. Here we describe isolation of cDNAs encoding two TAF subunits of the human transcription factor TFIID. The first cDNA is predicted to encode the C-terminal 947 residues of the 130-kDa human TAF subunit, hTAFII130. The second cDNA encodes the C-terminal 801 residues of the 100-kDa subunit, hTAFII100. Recombinant TAFs expressed in human cells by transient transfections are capable of associating with the endogenous TAFs and TBP to form a TFIID complex in vivo. Protein binding experiments demonstrate that hTAFII130, like its Drosophila homolog dTAFII110, interacts with the glutamine-rich activation domains of the human transcription factor Sp1. Furthermore, hTAFII130 shows reduced binding to the Sp1 mutants with impaired ability to activate transcription, suggesting a role for hTAFII130 as a direct coactivator target for Sp1.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Drosophila , Biblioteca de Genes , Glutamina , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Complejos Multiproteicos , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción TFIID , Factores de Transcripción/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
1. Recent examination of the hypothesis that distinctly phosphorylated NF-H isoforms exist in different types of neurons revealed that the extent of phosphorylation of the heavy neurofilament polypeptide of bovine ventral root motor neurons is markedly higher than that of dorsal root neurons. 2. In the present study we employed endoproteinase ASP-N for isolating the Lys-Ser-Pro (KSP)-rich domain of NF-H, which contains most of the NF-H phosphorylation sites. 3. Treatment of NF-H with ASP-N endoproteinase results in a cascade of products, the last of which is a polypeptide with apparent molecular weight of 120 kDa. Amino terminal sequence and amino acid composition analysis revealed that this fragment contains the KSP-rich domain of NF-H. 4. Treatment of ventral and dorsal root NF-H with ASP-N endoproteinase and analysis of the phosphoserine contents of the resulting 120 kDa fragments revealed that the 120 kDa fragment of ventral root NF-H is significantly more phosphorylated than that of dorsal root NF-H. 5. These findings show that the difference in extent of phosphorylation of ventral and dorsal root NF-H is due at least partly to the KSP-rich domain of NF-H.
Asunto(s)
Proteínas de Neurofilamentos/química , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Bovinos , Endopeptidasas/metabolismo , Metaloendopeptidasas , Datos de Secuencia Molecular , Peso Molecular , Neuronas Motoras/química , Proteínas de Neurofilamentos/metabolismo , Neuronas Aferentes/química , Fragmentos de Péptidos/aislamiento & purificación , Fosforilación , Procesamiento Proteico-Postraduccional , Raíces Nerviosas Espinales/químicaRESUMEN
Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin-selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. Here we report the purification and cloning of E2-C from clam oocytes. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties.
Asunto(s)
Proteínas Portadoras/metabolismo , Ciclinas/metabolismo , Ligasas , Enzimas Ubiquitina-Conjugadoras , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/aislamiento & purificación , Cromatografía de Afinidad , Cartilla de ADN , Humanos , Mitosis , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-stranded break repair and V(D)J recombination, comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit, DNA-PKcs. Here, we describe the cloning of the DNA-PKcs cDNA and show that DNA-PKcs falls into the phosphatidylinositol (PI) 3-kinase family. Biochemical assays, however, indicate that DNA-PK phosphorylates proteins but has no detectable activity toward lipids. Strikingly, DNA-PKcs is most similar to PI kinase family members involved in cell cycle control, DNA repair, and DNA damage responses. These include the FKBP12-rapamycin-binding proteins Tor1p, Tor2p, and FRAP, S. pombe rad3, and the product of the ataxia telangiectasia gene, mutations in which lead to genomic instability and predisposition to cancer. The relationship of these proteins to DNA-PKcs provides important clues to their mechanisms of action.
Asunto(s)
Ataxia Telangiectasia/genética , Proteínas de Unión al ADN , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Serina-Treonina Quinasas/genética , Homología de Secuencia de Aminoácido , Ataxia Telangiectasia/enzimología , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/aislamiento & purificación , Proteína Quinasa Activada por ADN , Humanos , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Proteínas Nucleares , Fosfatidilinositol 3-Quinasas , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/ultraestructura , Proteínas/fisiologíaRESUMEN
DDB is a damage-specific DNA binding protein whose binding activity is absent from a minority of cell strains from individuals with xeroderma pigmentosum Group E, a human hereditary disease characterized by defective nucleotide excision DNA repair and an increased incidence of skin cancer. The binding activity from HeLa cells is associated with polypeptides of M(r) 124,000 and 41,000 as determined by SDS-polyacrylamide gels. This report describes the isolation of full-length human cDNAs encoding each polypeptide of DDB. The predicted peptide molecular masses based on open reading frames are 127,000 and 48,000. When expressed in an in vitro rabbit reticulocyte system, the p48 subunit migrates with an M(r) of 41 kDa on SDS-polyacrylamide gels, similarly to the peptide purified from HeLa cells. There is no significant homology between the derived p48 peptide sequence and any proteins in current databases, and the derived peptide sequence of p127 has homology only with the monkey DDB p127 (98% nucleotide identity and only one conserved amino acid substitution). Using a fluorescence in situ hybridization technique, the DDB p127 locus (DDB1) was assigned to the chromosomal location 11q12-q13, and the DDB p48 locus (DDB2) to 11p11-p12.
Asunto(s)
Cromosomas Humanos Par 11 , Proteínas de Unión al ADN/genética , Hominidae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Daño del ADN , ADN Complementario , Células HeLa , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Conejos , Neoplasias Cutáneas/epidemiología , Neoplasias Cutáneas/genética , Transcripción Genética , Xerodermia Pigmentosa/genéticaRESUMEN
A human apurinic/apyrimidinic endonuclease activity, called AP endonuclease I, is missing from or altered specifically in cells cultured from Xeroderma pigmentosum group-D individuals (XP-D cells) (Kuhnlein, U., Lee, B., Penhoet, E. E., and Linn, S. (1978) Nucleic Acids Res. 5,951-960). We have now observed that another nuclease activity, UV endonuclease III, is similarly not detected in XP-D cells and is inseparable from the AP endonuclease I activity. This activity preferentially cleaves the phosphodiester backbone of heavily ultraviolet-irradiated DNA at unknown lesions as well as at one of the phosphodiester bonds within a cyclobutane pyrimidine dimer. The nuclease activities have been purified from mouse cells to yield a peptide of M(r) = 32,000, whose sequence indicates identity with ribosomal protein S3. The nuclease activities all cross-react with immunopurified antibody directed against authentic rat ribosomal protein S3, and, upon expression in Escherichia coli of a cloned rat cDNA for ribosomal protein S3, each of the activities was recovered and was indistinguishable from those of the mammalian UV endonuclease III. Moreover, the protein expressed in E. coli and its activities cross-react with the rat protein antibody. Ribosomal protein S3 contains a potential nuclear localization signal, and the protein isolated as a nuclease also has a glycosylation pattern consistent with a nuclear localization as determined by lectin binding. The unexpected role of a ribosomal protein in DNA damage processing and the unexplained inability to detect the nuclease activities in extracts from XP-D cells are discussed.
Asunto(s)
Daño del ADN , Proteínas de Escherichia coli , Proteínas Ribosómicas/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , ADN/efectos de la radiación , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/aislamiento & purificación , Endodesoxirribonucleasas/metabolismo , Humanos , Liasas/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , N-Glicosil Hidrolasas/aislamiento & purificación , N-Glicosil Hidrolasas/metabolismo , Dímeros de Pirimidina/metabolismo , Especificidad por Sustrato , Xerodermia Pigmentosa/enzimologíaRESUMEN
The small GTP-binding protein ARF plays an established role in the control of vesicular traffic and in the regulation of phospholipase D activity. Like other GTP binding proteins, ARF becomes activated upon the binding of GTP, whereas GTP hydrolysis acts as a turn-off signal. The fact that purified ARF proteins have negligible GTPase activity has suggested that GTP hydrolysis by ARFs is dependent on a GTPase-activating protein (GAP). Here we report the complete purification of an ARF GAP from rat liver cytosol. Advanced stages in the purification were carried out in the presence of denaturing agents, making use of an unusual conformational stability, or refolding capacity, of the GAP. The GAP was purified about 15,000-fold and was identified as a protein of 49 kDa. Partial amino acid sequence analysis showed that the GAP is a previously uncharacterized protein. Both crude and purified GAP migrated on a Superdex 200 column as a 200-kDa complex, suggesting a tetrameric structure. The purified ARF GAP was stimulated by phosphoinositides and was inhibited by phosphatidylcholine, similar to the results previously reported for a preparation from brain (Randazzo, P. A., and Kahn, R. A. (1994) J. Biol. Chem. 269, 10758). The availability of the ARF GAP molecule will advance the understanding of the regulation of the cellular processes in which ARF proteins participate.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Hígado/metabolismo , Proteínas/metabolismo , Factores de Ribosilacion-ADP , Animales , Encéfalo/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Proteínas de Unión al GTP/aislamiento & purificación , Proteínas Activadoras de GTPasa , Guanosina Trifosfato/metabolismo , Cinética , Fosfatidilcolinas/farmacología , Fosfatidilinositoles/farmacología , Proteínas/aislamiento & purificación , RatasRESUMEN
PSI is an RNA-binding protein involved in repressing splicing of the P element third intron in Drosophila somatic cell extracts. PSI produced in bacteria restores splicing inhibition to an extract relieved of inhibitory activity, indicating that PSI plays a direct role in somatic inhibition. Sequence analysis of cDNAs encoding PSI reveals three KH RNA-binding domains, a conserved motif also found in the yeast splicing regulator MER1. Notably, PSI is expressed highly in somatic embryonic nuclei but is undetectable in germ-line cells. In contrast, hrp48, another protein implicated in somatic inhibition, is found in the nucleus and cytoplasm of both tissues. The splicing inhibitory properties and soma-specific expression of PSI may be sufficient to explain the germ-line-specific transposition of P elements.
Asunto(s)
Empalme Alternativo/genética , Proteínas de Drosophila , Drosophila/genética , Proteínas Nucleares , Precursores del ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism. This mode of transposition requires repair of both a double-strand break at the donor DNA site and gapped DNA at the target site. Biochemical studies have identified a cellular non-P element-encoded DNA binding protein, termed the inverted repeat binding protein (IRBP), that specifically interacts with the outer half of the 31-bp terminal inverted repeats. Protein sequence information was used to isolate cDNA clones encoding IRBP. Sequence analysis shows that IRBP is related to the 70-kDa subunit of the human Ku autoimmune antigen. The mammalian Ku antigen binds free DNA termini and has been implicated in immunoglobulin VDJ recombination, DNA repair, and transcription. In addition, Ku is the DNA binding subunit of the double-strand DNA-dependent protein kinase. Cytogenetic mapping indicates that the IRBP gene maps to chromosomal position 86E on the right arm of the third chromosome.
Asunto(s)
Antígenos Nucleares , ADN Helicasas , Elementos Transponibles de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto , Proteínas Nucleares/química , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Hibridación in Situ , Autoantígeno Ku , Datos de Secuencia Molecular , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
RNA polymerase I and II transcription factors SL1 and TFIID, respectively, are composed of the TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs) responsible for promoter recognition. How the universal transcription factor TBP becomes committed to a TFIID or SL1 complex has not been known. Complementary DNAs encoding each of the three TAFIs that are integral components of SL1 have not been isolated. Analysis of subunit interactions indicated that the three TAFIs can bind individually and specifically to TBP. In addition, these TAFIs interact with each other to form a stable TBP-TAF complex. When TBP was bound first by either TAFI110, 63, or 48, subunits of TFIID such as TAFII250 and 150 did not bind TBP. Conversely, if TBP first formed a complex with TAFII250 or 150, the subunits of SL1 did not bind TBP. These results suggest that a mutually exclusive binding specificity for TBP intrinsic to SL1 and TFIID subunits directs the formation of promoter- and RNA polymerase-selective TBP-TAF complexes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1 , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Unión Competitiva , Clonación Molecular , ADN Complementario/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Células HeLa , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa I/metabolismo , TATA Box , Proteína de Unión a TATA-Box , Factor de Transcripción TFIID , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Transcripción GenéticaRESUMEN
The NF-AT transcription complex is required for the expression of a group of proteins that collectively coordinate the immune response. Here we purify two proteins encoded by separate genes that represent the pre-existing (p) and cytosolic (c) components of NF-AT. Expression of the full-length complementary DNA encoding NF-ATc activates the interleukin (IL-2) promoter in non-T lymphocytes, whereas a dominant negative of NF-ATc specifically blocks activation of the IL-2 promoter in T lymphocytes, indicating that NF-ATc is required for IL-2 gene expression. NF-ATc RNA expression is largely restricted to lymphoid tissues and is induced upon T-cell activation. The other protein, NF-ATp, is highly homologous to NF-ATc over a limited domain which shows similarity to the Dorsal/Rel family, but has a wider tissue distribution. Agents that increase intracellular Ca2+ or activate protein kinase C independently modify NF-ATc, indicating that distinct signalling pathways converge on NF-ATc to regulate its function.