RESUMEN
1AD9 is a murine monoclonal antibody (MAb) that was produced against the recombinant human alpha-subunit of protein kinase CK2, a pleiotropic and constitutively active serine/threonine protein kinase. We have further characterized this antibody, which is suitable for Western blot, immunoprecipitation and enzyme-linked immunosorbant assay tests. Using an overlapping peptide library, we have identified the epitope targeted by MAb1AD9 characterized by the following sequence: (319)MEHPYF(324).
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/química , Proteínas Serina-Treonina Quinasas/inmunología , Subunidades de Proteína/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión de Anticuerpos , Quinasa de la Caseína II , Dominio Catalítico , Bovinos , Línea Celular , Mapeo Epitopo , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/química , Subunidades de Proteína/químicaRESUMEN
From the venom of the American scorpion Centruroides sculpturatus Ewing we have isolated a minute peptide fraction (named CsEKerg1) which reversibly inhibits the current through ERG (ether-à-go-go-related gene) K(+) channels. Isolation was done by CM-cellulose column chromatography and reversed phase high-performance liquid chromatography. To test for an effect on ERG channels we used NG108-15 neuroblastomaxglioma hybrid cells voltage-clamped in the whole-cell mode. CsEKerg1 contains 43 amino acids and has a molecular weight of 4833. Its amino acid sequence is similar but not identical to that of ergtoxin, a peptide isolated recently from the venom of the Mexican scorpion Centruroides noxius [FASEB J. 13 (1999) 953]. Half inhibition of ERG current occurs at a peptide concentration of 1.12microg/ml.
Asunto(s)
Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Escorpiones/fisiología , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Canales de Potasio Éter-A-Go-Go , Glioma , Potenciales de la Membrana/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Neuroblastoma , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Ratas , Venenos de Escorpión/aislamiento & purificación , Análisis de Secuencia de Proteína , Espectrometría de Masa por Ionización de Electrospray , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/fisiologíaRESUMEN
The glioma amplified sequence 41 (GAS41) was previously isolated by microdissection mediated cDNA capture from the glioblastoma multiforme cell line TX3868 and shown to be frequently amplified in human gliomas. We determined the complete cDNA sequence of the GAS41 gene, demonstrated that the GAS41 protein is evolutionarily conserved, specifically at the N-terminus, and identified the yeast transcription factor tf2f domain within the GAS41 sequence. A human multiple-tissue Northern blot revealed ubiquitous expression of GAS41 with the highest expression in human brain. After generating polyclonal antibodies we found GAS41 protein expression in the nucleus of the TX3868 cell line by Western blot analysis and immunofluorescence microscopy. The nuclear localization was confirmed for several human tumors including gliomas of different grades of malignancy. In neuroblastoma however, GAS41 was found in the nucleoli but not in the nucleoplasm. Yeast two-hybrid screening of the TX3868 cell line identified the nuclear mitotic apparatus protein (NuMA), the KIAA1009 protein, and prefoldin subunit 1 (PFDN1) as potential interacting partners of GAS41. We generated a polyclonal antibody against the KIAA1009 protein and we demonstrated that the KIAA1009 protein is a nuclear protein, which appears to be co-localized with the GAS41 protein and NuMA.
Asunto(s)
Factores de Transcripción/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/química , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
PEX5 functions as a mobile import receptor for peroxisomal matrix proteins with a peroxisomal targeting signal 1 (PTS1). A critical step within the PTS1-import pathway is the interaction between PEX5 and the peroxisome membrane-associated protein PEX14. Based on two-hybrid analyses in mammalian cells and complementary in vitro binding assays, we demonstrate that the evolutionarily conserved pentapeptide repeat motifs, WX(E/D/Q/A/S)(E/D/Q)(F/Y), in PEX5 bind to PEX14 with high affinity. The results obtained indicate that each of the seven di-aromatic pentapeptides of human PEX5 interacts separately at the same binding site in the N terminus of PEX14 with equilibrium dissociation constants in the low nanomolar range. Mutational analysis of the PEX14-binding motifs reveals that the conserved aromatic amino acids at position 1 or 5 are essential for high affinity binding. We propose that the side chains of the aromatic amino acids are in close proximity as part of an amphipathic alpha-helix and together form hydrophobic anchors for binding PEX5 to individual PEX14 molecules.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Proteínas Represoras , Secuencias de Aminoácidos , Sitios de Unión , Humanos , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencias Repetitivas de AminoácidoRESUMEN
Glioblastoma multiforme (GBM), a malignant astrocytic tumour, represents the most frequent tumour of the human brain. Nevertheless, its molecular pathology is not well understood. We utilized the immune system, which contributes to cancer protection, to help identify new GBM-related genes. By screening a human GBM cDNA library with autologous patient serum (SEREX-approach), we isolated a gene termed PHF3 (PHD finger protein 3). The gene product of PHF3 is immunogenic in GBM as tested in an allogenic patient serum screening demonstrating antibodies in 24 of 39 (61.53%) sera, whereas none of the 14 healthy persons had antibodies against PHF3. While previous SEREX studies revealed allogenic antibody responses up to 40%, our results for PHF3 represent the highest reported rate for a specific antibody response. We show that GBM patients with an antibody response against PHF3 show significant better survival than patients without PHF3-specific antibodies. Because the amino acid sequence of PHF3 contains a PHD finger (also termed LAP motif), a TFIIS homology, a proline rich region and nuclear localization signals, it supposedly functions as a transcription factor. A polyclonal antibody generated against PHF3 shows nuclear expression in most investigated formalin-fixed, paraffin embedded tissues. In GBM, PHF3 expression is concentrated in cells surrounding necroses.
Asunto(s)
Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Adulto , Anciano , Secuencia de Bases , Western Blotting , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/genética , Cartilla de ADN , Femenino , Glioblastoma/sangre , Glioblastoma/genética , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70. Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70. Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered. Here we determined the concentrations of these two membrane proteins in dog pancreas microsomes and observed that the canine homologs of yeast proteins Sec62p and Sec63p are abundant proteins, present in almost equimolar concentrations as compared with Sec61alphap monomers. Furthermore, we detected fractions of these two proteins in association with each other as well as with the Sec61p complex. The J domain of the human Sec63p was shown to interact with immunoglobulin heavy chain binding protein. Thus, the membrane of the mammalian ER contains components, known from the posttranslationally operating protein translocase in yeast. We suggest that these components are required for efficient cotranslational protein transport into the mammalian ER as well as for other transport processes.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Microsomas/metabolismo , Páncreas/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Perros , Humanos , Datos de Secuencia Molecular , Páncreas/ultraestructura , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
The calcium channel alpha1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of alpha1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac alpha1E was determined by SDS-PAGE and immunoblotting (218 +/- 6 kD; n = 3). Compared to alpha1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of alpha1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of alpha1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of alpha1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of alpha1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca(2+)-dependent secretion of peptide hormones such as ANP and urodilatin.
Asunto(s)
Canales de Calcio Tipo T/análisis , Cromograninas/metabolismo , Activación del Canal Iónico , Túbulos Renales Distales/química , Miocardio/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Encéfalo/metabolismo , Encéfalo/patología , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/inmunología , Línea Celular Transformada , Femenino , Cobayas , Humanos , Immunoblotting/métodos , Inmunohistoquímica/métodos , Membranas Intracelulares/química , Túbulos Renales Distales/citología , Proteínas de la Membrana/análisis , Microsomas/química , Datos de Secuencia Molecular , Miocardio/citología , Ratas , Ratas Wistar , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca(2+) entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry that TRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only other TRP transcript found at considerable levels was TRP1, whereas TRP2, TRP3, TRP5(CCE2), and TRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca(2+) (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.
Asunto(s)
Corteza Suprarrenal/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión , Activación del Canal Iónico , Receptores de Superficie Celular/metabolismo , Corteza Suprarrenal/citología , Animales , Canales de Calcio/genética , Bovinos , ADN sin Sentido/farmacología , Conductividad Eléctrica , Hibridación in Situ , Inositol 1,4,5-Trifosfato/farmacología , Datos de Secuencia Molecular , ARN Mensajero/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Canales Catiónicos TRPC , Tapsigargina/farmacología , Distribución TisularRESUMEN
Dog pancreas microsomes represent the key components of the established model system for the analysis of protein transport into the mammalian endoplasmic reticulum. More recently, these microsomes were also employed in cell-free systems which address questions related to protein folding and protein degradation in the mammalian endoplasmic reticulum. In order to get at a complete picture of these undoubtedly related processes in the in vitro system we need to know all the proteins we are dealing with, and their respective stoichiometries. Here we give a progress report on our attempts to identify and to quantify the soluble molecular chaperones and folding catalysts which are present in the lumen of dog pancreas microsomes. Eventually, we will need to know how the in vitro system compares with the situation in intact pancreatic cells as well as in other cells.
Asunto(s)
Proteínas Fúngicas/química , Proteínas de Choque Térmico/química , Microsomas/metabolismo , Chaperonas Moleculares/química , Páncreas/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Perros , Escherichia coli , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/aislamiento & purificación , Proteínas de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Pliegue de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Mdm2 is a cellular oncoprotein the most obvious function of which is the down-regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro. Computer analysis of the primary amino acid sequence of mdm2 revealed 19 putative CK2 phosphorylation sites. By using deletion mutants of mdm2 and a peptide library we identified the serine residue at position 269 which lies within a canonical CK2 consensus sequence (EGQELSDEDDE) as the most important CK2 phosphorylation site. Moreover, by using the mdm2 S269A mutant for in vitro phosphorylation assays this site was shown to be phosphorylated by CK2. Binding studies revealed that phosphorylation of mdm2 at S269 does not have any influence on the binding of p53 to mdm2.
Asunto(s)
Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Quinasa de la Caseína II , Línea Celular , Codón , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Insectos , Datos de Secuencia Molecular , Mutación , Péptidos/química , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Tmp21 (p23) is involved in biosynthetic transport from the endoplasmic reticulum to the Golgi complex. We have recently characterized two cDNA-variants of human Tmp21, the Tmp21-isoforms-I and -II. Because of the lack of cDNA sequence data and protein expression data, it was not clear if Tmp21-II encodes a functional Tmp21-protein. Here we describe the cloning of the full length human Tmp21-II transcript. The putative open reading frame of Tmp21-II contains a reading frame jump and a nonsense mutation in comparison to all other Tmp21-I (p23) members. Our data indicate that hum-Tmp21-II is transcribed, but not translated. We conclude that Tmp21-II cDNA derives from a neutral pseudogene, which originates from a duplication event of the human Tmp21-isoform-I.
Asunto(s)
Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cricetinae , ADN Complementario , Humanos , Datos de Secuencia Molecular , Proteínas de Transporte Nucleocitoplasmático , Isoformas de Proteínas/genética , RatasRESUMEN
p53 is one of the most powerful negative regulators of growth. To manage this in an efficient way it has to interact with a set of different cellular proteins. Most contacts with the cellular environment occur in the N- or the C-terminal domain of the protein. Since we previously found that p53 binds to the regulatory beta-subunit of CK2 we now analyzed N- and C-terminal domains of p53 separately for the binding of protein kinase CK2, an enzyme which seems to have a certain importance for proliferation processes. With different overlay assays we could map the binding domain of protein kinase CK2 to a sequence between amino acids 325-344, a region which coincides with the interaction domain of some other p53 binding proteins. We also found that the regulatory beta-subunit of protein kinase CK2 binds independent of the catalytic alpha-subunit to this C-terminal domain of p53.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Quinasa de la Caseína II , Dominio Catalítico , Humanos , Datos de Secuencia Molecular , Mapeo Peptídico , Unión Proteica , Proteína p53 Supresora de Tumor/químicaRESUMEN
Recently, p24A and p23 (also termed Tmp21), two members of the p24 protein family, have been proposed to function as integral receptors for the COPI-vesicle coat. This study describes the intracellular localization and trafficking of p24A in comparison to p23. For immunolocalization of p24A and p23, strong reduction and denaturation conditions were necessary to allow antibody interaction. Both p24A and p23 cycle continuously between intermediate compartment (IC) elements and the cis-Golgi network. In vivo trafficking of p24A and p23 tagged to green fluorescent protein (GFP) revealed that both proteins travel by large (up to 1 micrometer in length) microtubule-dependent pre-Golgi carriers with a maximum speed of up to 1.6 micrometer s-1 from the IC to the Golgi cisternae. Aluminum fluoride, a general activator of heterotrimeric G-proteins, blocked peripheral pre-Golgi movements of GFP-p24A/p23 and inhibited fluorescence recovery after photobleaching in the perinuclear Golgi area. p24A and p23 are predominantly colocalized. Overexpression of GFP-p24A, to an extent which did not destroy the Golgi complex, induced delocalization of part of the proteins into ER elements. This study therefore gives new insights into the localization and trafficking behavior of the two COPI-binding proteins p24A and p23.
Asunto(s)
Membranas Intracelulares/metabolismo , Lectinas de Unión a Manosa , Proteínas de la Membrana/metabolismo , Compuestos de Aluminio/farmacología , Animales , Brefeldino A/farmacología , Células COS , Frío , Retículo Endoplásmico/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Fluoruros/farmacología , Expresión Génica , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Microtúbulos/metabolismo , Nocodazol/farmacología , Proteínas de Transporte Nucleocitoplasmático , Desnaturalización Proteica , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Sustancias Reductoras/farmacología , TransfecciónRESUMEN
We produced a rabbit monoclonal antibody (MAb) against human CDC25C phosphatase. The antibody reacts with a minimal epitope between amino acids 291-295 in the highly conserved C-terminal region of CDC25C. The antibody recognizes denatured CDC25C of recombinant and mammalian origin in Western blot analysis. The corresponding rabbit polyclonal serum is able to immunoprecipitate the native protein, but this ability has been lost during the selection procedure. Although the production of the rabbit MAb requires more effort and patience than the mouse MAb technology, it offers a true alternative in case of antigens that are not immunogenic in mice.
Asunto(s)
Proteínas de Ciclo Celular/inmunología , Fosfoproteínas Fosfatasas/inmunología , Fosfatasas cdc25 , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Proteínas de Ciclo Celular/química , Epítopos , Humanos , Isoenzimas/inmunología , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Homología de Secuencia de AminoácidoRESUMEN
In vivo p53 is multiply phosphorylated by different protein kinases suggesting a central role for phosphorylation in modulating p53 function. In addition, p53 was found to be associated with two protein kinases, p34cdc2 and protein kinase CK2. Here we report the precise mapping of the interaction sites of p53-p34cdc2 complexes. The p34cdc2 binding site on human p53 maps to one distinct C-terminal site LQIRGRERFE (aa 330-339) close to the corresponding phosphorylation site at serine 315. In order to test whether phosphorylation of p53 might influence the binding of p53 to p34cdc2 phosphorylation mutants of the C-terminus of p53, which mimick permanent phosphorylation, were tested on their ability to bind to p34cdc2 in vitro. Substitution of serine 315 (the p34cdc2 phosphorylation site) with aspartic acid had only little effect on complex formation whereas an exchange of serine 392 (the protein kinase CK2 phosphorylation site) to aspartic acid resulted in a significant reduced relative binding affinity of p53 to p34cdc2. The same result was obtained when the C-terminus of p53 was phosphorylated by purified protein kinase CK2 prior to examination of complex formation. In addition, the specificity of the complex formation has been checked by competition experiments with full length p53 proteins and the influence of cyclin B on complex formation was examined.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Proteína Quinasa CDC2/química , Proteína Quinasa CDC2/genética , Electroforesis en Gel de Poliacrilamida , Mapeo Peptídico , Fosforilación , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína p53 Supresora de Tumor/químicaRESUMEN
Using antibodies against the 31-kD and 70-kD subunits of vacuolar type H+-ATPase (V-ATPase) and light microscopic immunocytochemistry, we have demonstrated the presence of this V-ATPase in rat submandibular gland. We have also investigated the adaptive changes of this transporter during acid-base disturbances such as acute and chronic metabolic acidosis or alkalosis. Our results show intracellularly distributed V-ATPase in striated, granular, and main excretory duct cells in controls, but no V-ATPase immunoreaction in acinar cells. Both acute and chronic metabolic acidosis caused a shift in V-ATPase away from diffuse distribution towards apical localization in striated and granular duct cells, suggesting that a V-ATPase could be involved in the regulation of acid-base homeostasis. In contrast, during acidosis the main excretory duct cells showed no changes in the V-ATPase distribution compared to controls. With acute and chronic metabolic alkalosis, no changes in the V-ATPase distribution occurred. (J Histochem Cytochem 46:91-100, 1998)
Asunto(s)
Desequilibrio Ácido-Base , Adaptación Fisiológica , ATPasas de Translocación de Protón/metabolismo , Glándula Submandibular/enzimología , ATPasas de Translocación de Protón Vacuolares , Desequilibrio Ácido-Base/inducido químicamente , Acidosis/inducido químicamente , Acidosis/enzimología , Adaptación Fisiológica/efectos de los fármacos , Alcalosis/inducido químicamente , Alcalosis/enzimología , Cloruro de Amonio/farmacología , Animales , Western Blotting , Inmunohistoquímica , Masculino , Ratas , Ratas Wistar , Conductos Salivales/citología , Conductos Salivales/efectos de los fármacos , Conductos Salivales/enzimología , Bicarbonato de Sodio/farmacología , Glándula Submandibular/citología , Glándula Submandibular/efectos de los fármacosRESUMEN
Protein disulfide isomerase (PDI) and an additional lumenal protein of dog pancreas microsomes were previously observed to be in transient contact with secretory proteins during late stages of their co- or posttranslational translocation into these mammalian microsomes. The second protein was characterized as a 57 kDa glycoprotein. Here we identified this glycoprotein as the canine equivalent of human PDIp, a protein which was recently described as a new protein disulfide isomerase which is highly expressed in human pancreas. Canine PDIp is also a very abundant protein, its concentration in pancreatic microsomes approaches the concentration of PDI and of the major microsomal molecular chaperones. Apparently, PDIp shares with PDI not just the enzymatic but also the polypeptide binding or chaperoning activity. Furthermore, we suggest that PDIp, too, can be involved in completion of cotranslational as well as posttranslational translocation of proteins into mammalian microsomes.
Asunto(s)
Proteínas de Insectos , Isomerasas/metabolismo , Chaperonas Moleculares/metabolismo , Páncreas/enzimología , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Perros , Retículo Endoplásmico/metabolismo , Humanos , Hormonas de Insectos/metabolismo , Isomerasas/química , Microsomas/enzimología , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Peso Molecular , Páncreas/metabolismo , Prolactina/metabolismo , Biosíntesis de Proteínas , Proteína Disulfuro Isomerasas , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
We report here on the isolation, cloning, and expression of two Mr 21,000 proteins from rat pancreatic acinar cells, the rat-Tmp21 (transmembrane protein, Mr 21,000) and the rat-p24A. Both proteins are transmembrane proteins with type I topology and share weak but significant homology to one another (23% identity). We further show the cloning and characterization of the human homologs, hum-Tmp21, which is expressed in two variants (Tmp21-I and Tmp21-II), and hum-p24A. Tmp21 proteins and p24A have highly conserved COOH-terminal tails, which contain motifs related to the endoplasmic reticulum retention and retrieval consensus sequence KKXX. The rat-p24 sequence is identical to the hamster CHOp24, a recently characterized component of coatomer-coated transport vesicles, which defines a family of proteins (called the p24 family) proposed to be involved in vesicular transport processes (Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P., and Rothman, J. E.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8011-8015). Sequence alignment and structural features identify the Tmp21 protein as a new member of this p24 family. Northern analysis of various tissues indicates that the Tmp21 proteins and the p24A protein are ubiquitously expressed. The integral membrane components Tmp21 and p24A are localized in microsomal membranes, zymogen granule membranes, and the plasma membrane and are absent from the cytosol. Both p24A and Tmp21 show weak homology to the yeast protein Emp24p, which recently has been shown to be involved in secretory protein transport from the endoplasmic reticulum to the Golgi apparatus. This leads us to conclude that the receptor-like Tmp21 and p24A are involved in vesicular targeting and protein transport.
Asunto(s)
Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Microsomas/metabolismo , Páncreas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Clonación Molecular , Cricetinae , Cartilla de ADN , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas de Transporte Nucleocitoplasmático , Reacción en Cadena de la Polimerasa , Ratas , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
Monoclonal antibodies were produced against recombinant human RAD51 recombination protein. The antibodies of IgG subclasses were isolated from serum-free cell culture medium and purified by affinity chromatography on protein A-Sepharose. The antibodies can be used to detect specifically RAD51 protein on immunoblots of total cell lysates. Native RAD51 protein is specifically precipitated from lysates of human cells. In addition, these antibodies readily detect RAD51 in the cell nucleus by immunofluorescence staining. Epitope mapping on overlapping peptides spanning the complete primary amino acid sequence of human RAD51 revealed that three monoclonals recognize an epitope on RAD51 very close to the N-terminus of the protein (amino acids 16 to 20); the other three monoclonals interact with amino acids 85 to 95 of human RAD51.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Proteínas de Unión al ADN/inmunología , Secuencia de Aminoácidos , Animales , Células COS , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Conformación Proteica , Recombinasa Rad51 , Proteínas Recombinantes/inmunologíaRESUMEN
Originally identified as multicopy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast the tms1 gene product was found to form stable complexes with p53 in yeast. By using purified recombinant proteins multimeric complexes of tms1 and p53 could be demonstrated and recently the p53 binding site on the tms1 protein was established to the sequence YYITTEDFCT (aa 116-125) in the vicinity of a well conserved cell division motif. Here we report the precise mapping of the tms1 binding site on the p53 protein to the sequence LQIRGRERFE (aa 330-339) which defines a new functional domain on the p53 protein.