RESUMEN
Regulating the transition of cells such as T lymphocytes from quiescence (G(0)) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G(0). We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G(0) into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle.
Asunto(s)
Ciclo Celular/fisiología , Mapas de Interacción de Proteínas , Proteómica , Linfocitos T/metabolismo , Linfocitos T/fisiología , Ciclo Celular/genética , Núcleo Celular/metabolismo , Proliferación Celular , Cromatina/metabolismo , Análisis por Conglomerados , Proteínas de Unión al ADN/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/fisiología , Fase G1/fisiología , Humanos , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Factores de Empalme de ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Fase de Descanso del Ciclo Celular/fisiologíaRESUMEN
The nuclear matrix has classically been assumed to be a solid structure coherently aligning nuclear components, but its real nature remains obscure. We separated the proteins in a ribonucleoprotein-containing nuclear matrix fraction of HeLa cells by reversed-phase HPLC followed by SDS-PAGE, and identified 83 proteins through peptide mass fingerprint (PMF) analysis. Many nucleolar proteins, classical nuclear matrix proteins, RNA binding proteins, cytoskeletal proteins and five uncharacterized proteins were identified in this fraction. Four of the latter proteins were localized to the cell nucleus, BXDC1 and EBNA1BP2 being especially localized to the nucleolus. Fluorescence recovery after photobleaching and RNAi knockdown analyses suggested that BXDC1 and EBNA1BP2 function in a dynamic scaffold for ribosome biogenesis.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Nucléolo Celular/metabolismo , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Proteómica/métodos , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Nucléolo Celular/efectos de los fármacos , Análisis por Conglomerados , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Modelos Biológicos , Matriz Nuclear/química , Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteoma/análisis , ARN/fisiología , ARN Interferente Pequeño/farmacología , Proteínas de Unión al ARN , Ribonucleoproteínas Nucleares Pequeñas/metabolismoRESUMEN
Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.
Asunto(s)
Antígenos Nucleares/metabolismo , Polaridad Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Huso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/aislamiento & purificación , Células CACO-2 , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Complejo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Microtúbulos/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Unión Proteica/fisiología , Multimerización de Proteína/fisiología , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide (HMBA). Their nuclear matrix proteins (NMPs) were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.
Asunto(s)
Diferenciación Celular/fisiología , Núcleo Celular/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Osteosarcoma/metabolismo , Línea Celular Tumoral , Núcleo Celular/química , Electroforesis en Gel Bidimensional , Humanos , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Osteosarcoma/química , Osteosarcoma/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
AIM: To find and identify specific nuclear matrix proteins associated with proliferation and differentiation of carcinoma cells, which will be potential markers for cancer diagnosis and targets in cancer therapy. METHODS: Nuclear matrix proteins were selectively extracted from MGc80-3 cells treated with or without hexamethylamine bisacetamide (HMBA), and subjected to 2-D gel electrophoresis. The resulted protein patterns were analyzed by Melanie software. Spots of nuclear matrix proteins differentially expressed were excised and subjected to in situ digestion with trypsin. Peptide masses were obtained by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis and submitted for database searching using Mascot tool. RESULTS: The MGc80-3 cells were induced into differentiation by HMBA. There were 22 protein spots which changed remarkably in the nuclear matrix, from differentiation of MGc80-3 cells compared to control. Eleven of which were identified. Seven proteins--actin, prohibitin, porin 31HL, heterogeneous nuclear ribonucleoprotein A2/B1, vimentin, ATP synthase, and heat shock protein 60 were downregulated, whereas three proteins--heat shock protein gp96, heat shock protein 90-beta, and valosin-containing protein were upregulated, and the oxygen-regulated protein was only found in the differentiated MGc80-3 cells. CONCLUSION: The induced differentiation of carcinoma cells is accompanied by the changes of nuclear matrix proteins. Further characterization of those proteins will show the mechanism of cellular proliferation and differentiation, as well as cancer differentiation.
Asunto(s)
Adenocarcinoma Mucinoso/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma Mucinoso/patología , Diferenciación Celular , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Humanos , Proteínas de Neoplasias/aislamiento & purificación , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Mapeo Peptídico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Neoplasias Gástricas/patologíaRESUMEN
AIM: To separate and identify differentially expressed nuclear matrix proteins (NMPs) between the immortalized human esophageal epithelial cell line (SHEE) and the malignantly transformed esophageal carcinoma cell line (SHEEC), and to provide new ways for finding specific markers and the pathogenesis of esophageal carcinoma. METHODS: SHEE and SHEEC cell lines were used to extract NMPs. The quality of NMPs was monitored by Western blot analysis including DNA topoisomerase IIalpha, proliferation cell nuclear antigen (PCNA) and histone. NMPs of SHEE and SHEEC were analyzed by two-dimensional electrophoresis (2-DE), silver staining and PDQuest6.2 image analysis software. Three spots in which the differentially expressed NMPs were more obvious, were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI- TOF-MS) and database search. RESULTS: Western blot analysis revealed that DNA topoisomerase IIalpha and PCNA were detected, and the majority of histones were deleted in NMPs of SHEE and SHEEC. After 2-DE image analysis by PDQuest6.2 software, the 2-DE maps were detected with an average of 106+/-7.1 spots in SHEE and 132+/-5.0 spots in SHEEC. Most of them were matched one another (r=0.72), only 16 protein spots were found differing in intensity. Three NMPs including cytoskeletal tropomyosin, FK506-binding protein 6, similar to retinoblastoma binding protein 8 were preliminarily identified by MALDI- TOF-MS. CONCLUSION: These differentially expressed NMPs may play an important role during malignant transformation from SHEE to SHEEC. Their separation and identification will contribute to searching for specific markers and probing into the pathogenesis of esophageal carcinoma.
Asunto(s)
Neoplasias Esofágicas , Esófago/química , Esófago/citología , Proteínas Asociadas a Matriz Nuclear/análisis , Western Blotting , Línea Celular Transformada , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Humanos , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
UNLABELLED: Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA even after harsh deproteinization procedures. The amount of these proteins is 20-100 µg for mg of DNA depending on eukaryotic source. This experimental paper examines the possibility to use some TBP for clinical biomarker discovery, e.g. for identification of prognostic and diagnostic cancer markers. The main aim of this study was to designate differences between tightly DNA binding protein patterns extracted from rat liver and rat experimental hepatomas (Zajdela ascites hepatoma and hepatoma G-27) and to evaluate possibility that some of these proteins may be used as biomarkers for cell cancer transformation. METHODS: We used proteomics aproach as a tool for comparison of pattern of TBP from rat experimental hepatomas and normal liver cells. Combination of 2DE fractionation with mass spectrometry (MALDI TOF-MS) suitable for parallel profiling of complex TBP mixtures. RESULTS: Intriguingly 2DE protein maps of TBP from rat liver and rat experimental hepatomas (Zajdela acites hepatoma and hepatoma G-27) were quite different. We identified 9 proteins, some of them shared in all TBP patterns. Among identified tightly bound to DNA proteins there were three proteins considered as nuclear matrix proteins (lamin B1, scaffold attachment factor B1, heterogeneous nuclear ribonucleoprotein). Also we identified DNA repair protein RAD50, coiled-coil domain-containing protein 41, structural maintenance of chromosomes protein1A and some ATP -dependent RNA helicases indicating that TBP are of interest with respect to their potential involvement in the topological organization and/ or function of genomic DNA. CONCLUSIONS: We suppose that proteomic approach for TBP identification may be promising in development of biomarkers, also obtained results may be valuable for further understanding TBP functions in genome.
Asunto(s)
Biomarcadores de Tumor/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Enzimas Reparadoras del ADN/aislamiento & purificación , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/genética , Matriz Nuclear , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Proteínas Asociadas a Matriz Nuclear/metabolismo , Pronóstico , Unión Proteica , Proteómica , ARN Helicasas/aislamiento & purificación , ARN Helicasas/metabolismo , Ratas , Ratas Wistar , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Genetic experiments have established that IscU is involved in maturation of [Fe-S] proteins that require either [2Fe-2S] or [4Fe-4S] clusters for their biological activities. Biochemical studies have also shown that one [2Fe-2S] cluster can be assembled in vitro within each subunit of the IscU homodimer and that these clusters can be reductively coupled to form a single [4Fe-4S] cluster. In the present work, it is shown that the [4Fe-4S] cluster-loaded form of A. vinelandii IscU, but not the [2Fe-2S] cluster-loaded form, can be used for intact cluster transfer to an apo form of A. vinelandii aconitase A, a member of the monomeric dehydratase family of proteins that requires a [4Fe-4S] cluster for enzymatic activity. The rate of [4Fe-4S] cluster transfer from IscU to apo-aconitase A was not affected by the presence of the HscA/HscB co-chaperone system and MgATP. However, an altered form of a [4Fe-4S] cluster-containing IscU, having the highly conserved aspartate-39 residue substituted with alanine, is an effective inhibitor of wild-type [4Fe-4S] cluster-loaded IscU-directed activation of apo-aconitase A. In contrast, neither the clusterless form of IscU nor the [2Fe-2S] cluster-loaded form of IscU is an effective inhibitor of IscU-directed apo-aconitase A activation. These results are interpreted to indicate that the [2Fe-2S] and [4Fe-4S] cluster-loaded forms of IscU adopt different conformations that provide specificity with respect to the maturation of [2Fe-2S] and [4Fe-4S] centers in proteins.
Asunto(s)
Aconitato Hidratasa/metabolismo , Apoproteínas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Apoproteínas/química , Apoproteínas/aislamiento & purificación , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Clonación Molecular , Escherichia coli/genética , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/aislamiento & purificación , Cinética , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometría , TermodinámicaRESUMEN
Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.
Asunto(s)
Hemoglobina Fetal/genética , Regulación del Desarrollo de la Expresión Génica , Globinas/genética , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción/metabolismo , Adulto , Animales , Especificidad de Anticuerpos , Western Blotting , Fraccionamiento Celular , Línea Celular Tumoral , Cromatografía de Afinidad , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/inmunología , ADN (Citosina-5-)-Metiltransferasas/aislamiento & purificación , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Humanos , Leucemia Eritroblástica Aguda , Espectrometría de Masas , Ratones , Ratones Transgénicos , Proteínas Asociadas a Matriz Nuclear/inmunología , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Proteínas Asociadas a Matriz Nuclear/metabolismo , Mutación Puntual , Factor de Transcripción Sp1/inmunología , Factor de Transcripción Sp1/aislamiento & purificación , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/inmunología , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción TFII/inmunología , Factores de Transcripción TFII/aislamiento & purificación , Factores de Transcripción TFII/metabolismo , Activación TranscripcionalRESUMEN
Matrin 3 is a nuclear matrix protein that has been implicated in interacting with other nuclear proteins to anchor hyperedited RNAs to the nuclear matrix, in modulating the activity of proximal promoters, and as the main PKA substrate following NMDA receptor activation. In our proteome-wide selections for calmodulin (CaM) binding proteins and for caspase substrates using mRNA-displayed human proteome libraries, matrin 3 was identified as both a Ca(2+)-dependent CaM-binding protein and a downstream substrate of caspases. We report here, the in vitro characterization of the CaM-binding motif and the caspase cleavage site on matrin 3. Significantly, the Ca(2+)/CaM-binding motif is partially overlapped by the RRM of matrin 3 and is also very close to the bipartite NLS that is essential for its nuclear localization. The caspase cleavage site is downstream of the NLS but upstream of the second U1-like zinc finger. Our results suggest that the functions of matrin 3 could be regulated by both Ca(2+)-dependent interaction with CaM and caspase-mediated cleavage.
Asunto(s)
Calcio/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Caspasas/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biotinilación , Señalización del Calcio , Caspasa 3/metabolismo , Secuencia Conservada , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Proteoma/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificación , Sefarosa/metabolismo , Especificidad por Sustrato , Factores de TiempoRESUMEN
We tested whether the mechanisms of chromosome movement during anaphase in locust (Locusta migratoria L.) spermatocytes might be similar to those described for crane-fly spermatocytes. Actin and myosin have been implicated in anaphase chromosome movements in crane-fly spermatocytes, as indicated by the effects of inhibitors and by the localisations of actin and myosin in spindles. In this study, we tested whether locust spermatocyte spindles also utilise actin and myosin, and whether actin is involved in microtubule flux. Living locust spermatocytes were treated with inhibitors of actin (latrunculin B and cytochalasin D), myosin (BDM), or myosin phosphorylation (Y-27632 and ML-7). We added drugs (individually) during anaphase. Actin inhibitors alter anaphase: chromosomes either completely stop moving, slow, or sometimes accelerate. The myosin inhibitor, BDM, also alters anaphase: in most cases, the chromosomes drastically slow or stop. ML-7, an inhibitor of MLCK, causes chromosomes to stop, slow, or sometimes accelerate, similar to actin inhibitors. Y-27632, an inhibitor of Rho-kinase, drastically slows or stops anaphase chromosome movements. The effects of the drugs on anaphase movement are reversible: most of the half-bivalents resumed movement at normal speed after these drugs were washed out. Actin and myosin were present in the spindles in locations consistent with their possible involvement in force production. Microtubule flux along kinetochore fibres is an actin-dependent process, since LatB completely removes or drastically reduces the gap in microtubule acetylation at the kinetochore. These results suggest that actin and myosin are involved in anaphase chromosome movements in locust spermatocytes.
Asunto(s)
Actinas/fisiología , Anafase , Segregación Cromosómica , Cromosomas/fisiología , Saltamontes/citología , Miosinas/fisiología , Espermatocitos/citología , Actinas/análisis , Actinas/antagonistas & inhibidores , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cromosomas/efectos de los fármacos , Citocalasina D/farmacología , Citocinesis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Masculino , Meiosis , Movimiento/efectos de los fármacos , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/metabolismo , Miosinas/análisis , Miosinas/antagonistas & inhibidores , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Proteínas Asociadas a Matriz Nuclear/metabolismo , Espermatocitos/fisiología , Espermatocitos/ultraestructura , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Tiazolidinas/farmacologíaRESUMEN
This article describes a novel technique whereby fully functional proteins or multiprotein complexes are efficiently extracted from biological samples to chemically derivatized walls of fused-silica open-tube capillary columns. Proteins are eluted with very high yields into elution volumes that are smaller in volume than the internal volume of the open-tube capillary column itself, thereby achieving 100-fold increases in target protein concentrations from starting samples of less than 1 ml. The open-tube capillary columns are designed for single use; combined with the physical and chemical characteristics of the open-tube capillary column, this provides exceptional purity to the eluted proteins. Affinity-based open-tube capillary columns are demonstrated here to purify, enrich, and maintain functionality for a monomeric and dimeric enzyme, a low-abundance HeLa nuclear complex, and a light-harvesting octadecameric membrane protein complex. The design of the open-tube capillary column allows for facile direction of the processed protein sample to any number of final detection techniques and is capable of generating final protein concentrations required for many structural biology experiments. The open-tube capillary columns are also characterized by exceptional ease of use. Current designs allow for up to 10 open-tube capillary columns to be applied simultaneously with no fundamental impediments to even greater parallel operation.
Asunto(s)
Fraccionamiento Químico/instrumentación , Fraccionamiento Químico/métodos , Cromatografía de Afinidad/instrumentación , Complejos Multiproteicos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Cromatografía de Afinidad/métodos , Proteínas de Unión al ADN , Glutatión Transferasa/aislamiento & purificación , Células HeLa , Ribonucleoproteínas Nucleares Heterogéneas/aislamiento & purificación , Humanos , Complejos de Proteína Captadores de Luz/aislamiento & purificación , Microquímica/instrumentación , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Factores de Transcripción de Octámeros/aislamiento & purificación , Proteína de Unión al Tracto de Polipirimidina/aislamiento & purificación , Proteínas de Unión al ARN/aislamiento & purificación , Rhodobacter sphaeroides/químicaRESUMEN
BACKGROUND & OBJECTIVE: Recently, changes in composition, structure, and function of nuclear matrix proteins (NMPs) in generation and development of tumors evoked more and more attention. Separation and identification of tumor-related NMPs is a new way to search for tumor specific biomarkers, and to study tumor pathogenesis. This study was to analyze differential expression of STRBP8, one of esophageal carcinoma specific NMPs, in cancerization of immortalized human esophageal epithelial cells. METHODS: NMPs were extracted from immortalized human esophageal epithelial cell line SHEE and malignantly transformed esophageal carcinoma cell line SHEEC. Differential expression of STRBP8 was detected by two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS), and reverse transcription-polymerase chain reaction (RT-PCR). STRBP8 cDNA obtained by RT-PCR was linked to pGEM-T easy vector, and introduced into TOP10F' E.coli competent cells. Positive clones were sequenced and analyzed with BLAST. RESULTS: STRBP8 was only detected in SHEEC cells by 2-DE, MALDI-TOF-MS, and RT-PCR. The sequence of positive clones contained STRBP8 cDNA was identical to that in GenBank database. CONCLUSION: STRBP8, as a candidate oncogene, might relate to cancerization of esophageal epithelial cells, which might be a specific biomarker of esophageal carcinoma, and probe into the pathogenesis of esophageal carcinoma.
Asunto(s)
Proteínas Portadoras/metabolismo , Neoplasias Esofágicas/metabolismo , Esófago/metabolismo , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Proteínas Nucleares/metabolismo , Secuencia de Bases , Biomarcadores de Tumor , Proteínas Portadoras/genética , Línea Celular Transformada , Línea Celular Tumoral , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel Bidimensional , Endodesoxirribonucleasas , Células Epiteliales/citología , Neoplasias Esofágicas/patología , Esófago/citología , Regulación Neoplásica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The biological effects of ionizing radiation are attributable, in large part, to induction of DNA double-strand breaks. We report here the identification of a new protein factor that reconstitutes efficient double-strand break rejoining when it is added to a reaction containing the five other polypeptides known to participate in the human nonhomologous end-joining pathway. The factor is a stable heteromeric complex of polypyrimidine tract-binding protein-associated splicing factor (PSF) and a 54-kDa nuclear RNA-binding protein (p54(nrb)). These polypeptides, to which a variety of functions have previously been attributed, share extensive homology, including tandem RNA recognition motif domains. The PSF.p54(nrb) complex cooperates with Ku protein to form a functional preligation complex with substrate DNA. Based on structural comparison with related proteins, we propose a model where the four RNA recognition motif domains in the heteromeric PSF.p54(nrb) complex cooperate to align separate DNA molecules.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Antígenos Nucleares/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Autoantígeno Ku , Modelos Biológicos , Peso Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Factores de Transcripción de Octámeros , Factor de Empalme Asociado a PTB , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Estructura Terciaria de Proteína , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificación , Especificidad por SustratoRESUMEN
Nuclear shape and the underlying nuclear structure, the nuclear matrix in cancer cells. Since the NM composition is considered to maintain nuclear shape and architecture, nuclear matrix proteins (NMPs) may be involved in transformation. Our laboratory has recently characterized a subset of NMPs that are associated with prostate cancer development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. One of the identified NMPs, E3E, has a similar molecular weight (22 kDa) with a protein known as HMGI(Y). HMGI(Y) belongs to a group of non-histone and chromatin-associated proteins, high-mobility-group (HMG) proteins, and it has been shown to associate with the NM. HMGI(Y) has been reported to be elevated in different types of cancer including prostate cancer. In this study, we examined the expression of HMGI(Y) protein in the NMP composition of the TRAMP model during the progression from normal to neoplasia. The expression of HMGI(Y) in the NMP extracts of three prostatic epithelial cell lines derived from a 32-week TRAMP mouse: TRAMP-C1, TRAMP-C2, and TRAMP-C3 was also examined. Using both one-dimensional and high-resolution two-dimensional immunoblot analyses, we found that: (i) HMGI(Y) is a nuclear matrix protein expressed as two protein bands with MW of 22-24 kDa and (ii) HMGI(Y) expression is correlated with neoplastic and malignant properties in late stage TRAMP prostate tumors. Overall, these findings support the evidence that HMGI(Y) can be utilized as a marker and prognostic tool for prostate cancer.
Asunto(s)
Adenocarcinoma/metabolismo , Proteína HMGA1a/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Matriz Nuclear/metabolismo , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Línea Celular , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Transgénicos , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
Megalin is a member of the LDL receptor gene family that plays an important role in forebrain development and in cellular vitamin D metabolism through endocytic uptake of vitamin D metabolites. Similar to other receptors in this gene family, megalin is believed to functionally interact with intracellular proteins through adaptors that bind to the receptor tail and regulate its endocytic and signal transducing activities. Using yeast two-hybrid screens, we identified a novel scaffold protein with tetratrico peptide repeats, the megalin-binding protein (MegBP) that associates with the receptor. The binding site of MegBP was mapped to an N-terminal region on the receptor tail harboring a proline-rich peptide element. MegBP binding did not block the endocytic activity of the receptor; however, overexpression resulted in cellular lethality. In further screens, we identified proteins that bound to MegBP and thus might be recruited to the megalin tail. MegBP-interacting partners included several transcriptional regulators such as the SKI-interacting protein (SKIP), a co-activator of the vitamin D receptor. These finding suggest a model whereby megalin directly participates in transcriptional regulation through controlled sequestration or release of transcription factors via MegBP.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/aislamiento & purificación , Proteínas Portadoras/aislamiento & purificación , Membrana Celular/metabolismo , Endocitosis/fisiología , Células Eucariotas/metabolismo , Genes Reguladores/fisiología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/aislamiento & purificación , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Sitios de Unión/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , ADN Complementario/análisis , ADN Complementario/genética , Humanos , Ligandos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Datos de Secuencia Molecular , Proteínas Asociadas a Matriz Nuclear/genética , Péptidos/genética , Péptidos/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiologíaRESUMEN
In a screen for binding partners of the Epstein-Barr virus transformation-related protein EBNA2, we cloned a novel, evolutionarily conserved protein showing similarity to the Drosophila Parallel Sister Chromatids Protein (PASC). We have named this protein "Friend of EBNA2" (FOE). Human FOE encodes a protein of 1227 amino acids with a functional bipartite nuclear localization signal, an arginine-rich motif, a putative nuclear export signal as well as with three highly acidic regions and a predicted coiled-coil domain. FOE and EBNA2 coimmunoprecipitate from lymphocyte nuclear extracts. RNA and protein blots show that FOE is expressed in all human tissues. FOE is a nuclear protein with the bulk of the protein associated with the insoluble nuclear fraction biochemically defined as the nuclear matrix. Indirect immunofluorescence and dynamic imaging studies suggest that FOE associates with transcriptionally active nuclear subregions in interphase cells and concentrates at the ends of formed chromosomes during mitosis.
Asunto(s)
Proteínas Portadoras/metabolismo , Cromatina/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/inmunología , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Proteínas Oncogénicas/metabolismo , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Células COS , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Células HeLa , Herpesvirus Humano 4/genética , Humanos , Células Jurkat , Linfocitos/inmunología , Mitosis/genética , Datos de Secuencia Molecular , Matriz Nuclear/genética , Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/aislamiento & purificación , Estructura Terciaria de Proteína/genética , Proteínas Proto-Oncogénicas , ARN Mensajero/metabolismo , Transducción de Señal/genética , Activación Transcripcional/genética , Proteínas ViralesRESUMEN
Toll-like receptors and the IL-1R are part of the innate immune response aimed at mobilizing defense mechanisms in response to infections or injury. These receptors can initiate common intracellular signaling cascades. One intermediate component in these signaling cascades is Pellino, which was first identified in Drosophila and shown to interact with IL-1R-associated kinase. Two homologues, Pellino1 and Pellino2, have been identified in mammals. A novel member of the Pellino protein family has been identified and named Pellino3. Pellino3 shares 84 and 85% amino acid identity with Pellino1 and Pellino2, respectively. Two alternatively spliced Pellino3 mRNAs, Pellino3a and Pellino3b, are widely expressed. Pellino3 physically interacts with IL-1R-associated kinase-1, TNF receptor-associated factor-6, TGF-beta-activated kinase-1, and NF-kappaB-inducing kinase in an IL-1-dependent manner, suggesting that it plays a role as a scaffolding protein. In reporter assays Pellino3 leads to activation of c-Jun and Elk-1, but not NF-kappaB. Pellino3 also leads to activation of c-Jun N-terminal kinase. These data suggest that Pellino3 plays an important role in the innate immune response.