RESUMO
OBJECTIVE: To monitor the phosphorylation modifications and cellular localization of poly(rC)-binding protein-1 (PCBP1) during the cell cycle progression of Hela cells. RESULT: Hela cells highly synchronized at five different phases from interphase to mitosis were obtained. Using mitotic phosphoprotein-specific monoclonal antibody MPM-2, the exclusive occurrences of multiphosphorylation statuses of PCBP1 in mitosis were confirmed by a series of spots with increasing acidic pI (isoelectric point) in two rounds of 2D western blotting on the same membrane, and a visible molecular mass shift that can be eliminated by the treatment with λ phosphatase in 1D western blotting. Immnuofluorescence revealed the localization shift of PCBP1 during cell cycle, with accumulations in nucleus as a patch pattern in interphase, and a dispersive distribution without the area of the condensed chromosomes during mitosis. CONCLUSIONS: These observations of mitosis-specific multiphosphorylations and localization shifts of PCBP1 suggest that the versatile PCBP1 was regulatable in a phosphorylation modification- and temporospatial-dependent manner in mitotic regulatory networks.
Assuntos
Núcleo Celular/química , Células Epiteliais/química , Células Epiteliais/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Mitose , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a DNA , Células HeLa , Humanos , Fosforilação , Proteínas de Ligação a RNARESUMO
PURPOSE OF INVESTIGATION: Heterogeneous nuclear ribonucleoprotein (hnRNP) family possesses decreasing effect towards endometrial cancer (EC) and human transformer-2-betal (hTra2-betal) performs an intimate relationship with EC, either. Recent study shows that hnRNPs and hTra2-betal regulate the genetic expression, which is concerned with estrogen receptor (ER). MATERIALS AND METHODS: The present study was designed to investigate the link between ER and hnRNPs or hTra2-betal in the prognosis of EC patients by Real-time PCR and immunohistochemisty (IHC). RESULTS: Results showed that ER protein expression presented a significant change in the recurrence and outcome of EC patients, and the nucleus hTra2-betal protein expression was also increased in the recurrent patients, indicating that the three might be important in ER expression in the prognosis therapy of EC patients. CONCLUSION: The present findings provide an insight of pharmaceutical targeting therapy and prognosis of EC.
Assuntos
Neoplasias do Endométrio/genética , Receptor alfa de Estrogênio/genética , Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Ligação a RNA/fisiologia , Neoplasias do Endométrio/patologia , Receptor alfa de Estrogênio/análise , Receptor alfa de Estrogênio/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Recidiva Local de Neoplasia/epidemiologia , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Prognóstico , RNA Mensageiro/análise , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Processamento de Serina-ArgininaRESUMO
Labeled with pluripotent potential, the transplantation of bone marrow mesenchymal stem cells (BMSCs) is considered as a promising strategy for treating osteoporosis (OP). Melatonin (MEL) has been investigated to be an essential regulator involved in bone metabolism, as well as BMSCs differentiation. Circular RNAs (circRNAs) are a unique kind of non-coding RNA and play an important regulatory role in OP. However, whether circRNAs are implicated in the effects of MEL on BMSCs osteogenic differentiation remains largely indeterminate. Expression of circ_0005753 in human BMSCs with MEL treatment, clinical specimens diagnosed with OP, either with ovariectomy (OVX)-induced mice, was measured by RT-qPCR. Western blot was conducted to analyze protein levels of osteogenesis-related molecules (Opg, RUNX2, ALP, BMP4) and TXNIP. RNA immunoprecipitation (RIP) and RNA pull-down assays were performed to validate the binding relationship among circ_0005753, PTBP1, and TXNIP. Alkaline phosphatase (ALP) and alizarin red staining (ARS) were performed to evaluate osteogenic capacity of BMSCs. OP mouse model was established by ovariectomy, as evaluated pathologic changes via hematoxylin-eosin (HE), Masson, and Immunohistochemistry (IHC) staining. Expression of circ_0005753 was remarkably decreased during MEL-induced osteogenic differentiation of BMSCs. Interestingly, not only circ_0005753 knockdown significantly promoted osteogenic differentiation of BMSCs, but circ_0005753 overexpression also weakened osteogenic differentiation induced by MEL treatment. Mechanistically, circ_0005753 maintained the stabilization of TXNIP mRNA via recruiting PTBP1. Additionally, reinforced circ_0005753 abrogated MEL-mediated protective effects on OP pathogenesis in a mouse model. This work shows that MEL facilitates osteogenic differentiation of BMSCs via the circ_0005753/PTBP1/TXNIP axis, which may shed light on the development of a novel therapeutic strategy to prevent OP.
Assuntos
Melatonina , Células-Tronco Mesenquimais , MicroRNAs , Osteoporose , Feminino , Camundongos , Humanos , Animais , Osteogênese , Melatonina/farmacologia , RNA Circular/genética , RNA Circular/análise , RNA Circular/metabolismo , Células Cultivadas , Osteoporose/tratamento farmacológico , Osteoporose/genética , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Modelos Animais de Doenças , MicroRNAs/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/análise , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/farmacologia , Proteínas de Transporte/metabolismoRESUMO
The hs-GAL4(t)-driven expression of the hsrω-RNAi transgene or EP93D allele of the noncoding hsrω resulted in global down- or upregulation, respectively, of the large hsrω-n transcripts following heat shock. Subsequent to temperature shock, hsrω-null or those expressing hsrω-RNAi or the EP93D allele displayed delayed lethality of most embryos, first or third instar larvae. Three-day-old hsrω-null flies mostly died immediately or within a day after heat shock. Heat-shock-induced RNAi or EP expression in flies caused only a marginal lethality but severely affected oogenesis. EP allele or hsrω-RNAi expression after heat shock did not affect heat shock puffs and Hsp70 synthesis. Both down- and upregulation of hsrω-n transcripts suppressed reappearance of the hsrω-n transcript-dependent nucleoplasmic omega speckles during recovery from heat shock. Hrp36, heterochromatin protein 1, and active RNA pol II in unstressed or heat-shocked wild-type or hsrω-null larvae or those expressing the hs-GAL4(t)-driven hsrω-RNAi or the EP93D allele were comparably distributed on polytene chromosomes. Redistribution of these proteins to pre-stress locations after a 1- or 2-h recovery was severely compromised in glands with down- or upregulated levels of hsrω-n transcripts after heat shock. The hsrω-null unstressed cells always lacked omega speckles and little Hrp36 moved to any chromosome region following heat shock, and its relocation to chromosome regions during recovery was also incomplete. This present study reveals for the first time that the spatial restoration of key regulatory factors like hnRNPs, HP1, or RNA pol II to their pre-stress nuclear targets in cells recovering from thermal stress is dependent upon critical level of the large hsrω-n noncoding RNA. In the absence of their relocation to pre-stress chromosome sites, normal developmental gene activity fails to be restored, which finally results in delayed organismal death.
Assuntos
Adaptação Fisiológica/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila , Resposta ao Choque Térmico/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , RNA Polimerase II/metabolismo , RNA não Traduzido/fisiologia , Adaptação Fisiológica/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Proteínas Nucleares , Transporte Proteico/genética , Transporte Proteico/fisiologia , RNA Polimerase II/genética , RNA Polimerase II/fisiologia , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , TemperaturaRESUMO
This work presents the proteome profile of cultured human skin fibroblasts established from a patient affected by DNA ligase I (Lig I) deficiency syndrome, a rare disorder characterized by immunodeficiency, growth retardation and sun sensitivity. 2-DE (in the 3-10 and 4-7 pH ranges) was the separation technique used for the production of maps. MALDI-TOF/MS and LC-MS/MS were the mass spectrometry platforms applied for the identification of proteins in gel spots. A total of 154 proteins, including 41 never detected before in skin fibroblasts with this approach, were identified in gel spots analyzed. This newly generated extensive database provides for the first time a global picture of abundant proteins in 46BR.1G1 skin fibroblasts. While being relevant to the particular disorder considered, these results may be regarded as an intriguing starting point on the way to achieve a reference map of the proteins highly expressed in an inherited syndrome with defect in DNA replication and repair pathways.
Assuntos
DNA Ligases/deficiência , Fibroblastos/metabolismo , Proteoma/análise , Linhagem Celular Transformada , Cromatografia Líquida , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/química , DNA Ligase Dependente de ATP , Eletroforese em Gel Bidimensional , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/química , Humanos , Concentração de Íons de Hidrogênio , Dobramento de Proteína , Proteínas/análise , Proteínas/química , Proteínas/classificação , Proteoma/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.
Assuntos
Neoplasias Hepáticas/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Matriz Nuclear/metabolismo , Proteômica/métodos , Animais , Western Blotting , Proteínas de Ciclo Celular , Eletroforese em Gel Bidimensional , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Queratinas Tipo II/análise , Queratinas Tipo II/metabolismo , Laminas/análise , Laminas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/ultraestrutura , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/análise , Microscopia Eletrônica , Matriz Nuclear/química , Matriz Nuclear/ultraestrutura , Proteínas Associadas à Matriz Nuclear/análise , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Nuclear/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Endogâmicos F344 , Ribonucleosídeos/química , Ribonucleosídeos/metabolismo , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo , Vanadatos/química , Vanadatos/metabolismoRESUMO
Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.
Assuntos
Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/análise , Spliceossomos/química , Animais , Linhagem Celular , Galinhas/metabolismo , Ciclofilinas/análise , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas/análise , Humanos , Espectrometria de Massas , Proteínas Nucleares/análise , Peptídeos/análise , Peptídeos/isolamento & purificação , Proteômica , RNA Helicases/análise , Precursores de RNA/isolamento & purificação , RNA Mensageiro/isolamento & purificação , RNA Nuclear Pequeno/análise , RNA Nuclear Pequeno/isolamento & purificação , Proteínas de Ligação a RNA/análise , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas Nucleares Pequenas/análise , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Fatores de Processamento de Serina-ArgininaRESUMO
Thioredoxin (TRX) is a key protein of the cellular redox metabolism, which expression is increased in several tumors especially gastric tumors. Even though ultraviolet (UV) and hypoxia specifically induce TRX, the mechanisms that lead to increased TRX levels are still ill defined. Here, we show that the heterogenous ribonucleoprotein A18 (hnRNP A18) RNA Binding Domain (RBD) and the arginine, glycine (RGG) rich domain can bind TRX 3'-untranslated region (3'-UTR) independently but both domains are required for maximal binding. Immunoprecipitation (IP) of hnRNP A18-mRNAs complexes and co-localization of hnRNP A18 and TRX transcripts on ribosomal fractions confirm the interaction of hnRNP A18 with TRX transcripts in cells. Moreover, down regulation of hnRNP A18 correlates with a significant reduction of TRX protein levels. In addition, hnRNP A18 increases TRX translation and interacts with the eukaryotic Initiation Factor 4G (eIF4G), a component of the general translational machinery. Furthermore, hnRNP A18 phosphorylation by the hypoxia inducible GSK3beta increases hnRNP A18 RNA binding activity in vitro and in RKO cells in response to UV radiation. These data support a regulatory role for hnRNP A18 in TRX post-transcriptional expression possibly through a kissing loop model bridging TRX 3'- and 5'-UTRs through eIF4G.
Assuntos
Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Biossíntese de Proteínas , Tiorredoxinas/genética , Regiões 3' não Traduzidas/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Fator de Iniciação Eucariótico 4G/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/química , Humanos , Fosforilação , Estrutura Terciária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ribossomos/química , Tiorredoxinas/biossíntese , Raios UltravioletaRESUMO
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of a disease spectrum with shared clinical, genetic and pathological features. These include near ubiquitous pathological inclusions of the RNA-binding protein (RBP) TDP-43, and often the presence of a GGGGCC expansion in the C9ORF72 (C9) gene. Previously, we reported that the sequestration of hnRNP H altered the splicing of target transcripts in C9ALS patients (Conlon et al., 2016). Here, we show that this signature also occurs in half of 50 postmortem sporadic, non-C9 ALS/FTD brains. Furthermore, and equally surprisingly, these 'like-C9' brains also contained correspondingly high amounts of insoluble TDP-43, as well as several other disease-related RBPs, and this correlates with widespread global splicing defects. Finally, we show that the like-C9 sporadic patients, like actual C9ALS patients, were much more likely to have developed FTD. We propose that these unexpected links between C9 and sporadic ALS/FTD define a common mechanism in this disease spectrum.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/genética , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Ribonucleoproteínas Nucleares Heterogêneas/análise , Proteína de Ligação a Regiões Ricas em Polipirimidinas/análise , Splicing de RNA , Encéfalo/patologia , Proteínas de Ligação a DNA/análise , Humanos , Mutagênese InsercionalRESUMO
Synaptotagmin 7 (SYT7) is ubiquitously expressed calcium sensor, involved in neuronal membrane trafficking. Immunoprecipitation experiments demonstrated that SYT7 interacts with Synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a component of mRNA granules, which are transported to dendrites and are prerequisite for synaptic plasticity. Given the potential significance of SYT7 regulation in processes of neurodegeneration, which are characterized by high level of synaptic vulnerability, we aimed to analyse and compare the distribution of SYT7 and SYNCRIP proteins in the adult rat striatum, hippocampus, cerebral and cerebellar cortex. We investigated the degree of SYT7-SYNCRIP co-localization in order to examine possible functional interaction of these two proteins. We found that SYT7 is abundantly distributed in neuropil of all examined anatomical areas of the brain, most prominently in axons. On the contrary, SYNCRIP had cytoplasmic somatodendritic pattern of expression, which was most prominent in the hippocampus and cerebellum. In the striatum, hippocampus and cerebral cortex SYT7 and SYNCRIP immunofluorescent signals were mutually excluded, thus diminishing the probability for their physiological interaction. In somata of Purkinje neurons in the cerebellar cortex, both SYT7 and SYNCRIP were expressed and partially co-localized suggesting possible functional connection between SYT7 and SYNCRIP proteins in Purkinje neurons.
Assuntos
Química Encefálica , Encéfalo/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/biossíntese , Células de Purkinje/metabolismo , Sinaptotagminas/biossíntese , Animais , Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/genética , Masculino , Células de Purkinje/química , Ratos , Ratos Wistar , Sinaptotagminas/análise , Sinaptotagminas/genéticaRESUMO
BACKGROUND: Depletion of Poly-C binding protein-1(PCBP1) is implicated in various human malignancies. However, the underlying biological effect of PCBP1 in cancers, including acute myeloid leukemia (AML), still remains elusive. The purpose of this study was to examine the expression and clinical outcome of PCBP1in acute myeloid leukemia. METHODS: Bone marrow fluids of 88 newly diagnosed AML patients were sampled, and the PCBP1 mRNA expression level was evaluated using quantitative RT-PCR. The association between PCBP1 expression and clinicopathological features or the survival status of the patients was assessed by Chi-square test and Kaplan-Meier method. RESULTS: Comparing newly diagnosed AML patients to normal healthy donors, PCBP1 expression was significantly decreased in AML patients (P < 0.001). Conversely, PCBP1 expression had accordingly recovered back to normal in patients with complete remission (P < 0.001). Clinical feature analyses showed that PCBP1 expression was negatively correlated with white blood cell count (P = 0.024). In addition, patients with low PCBP1 expression had poor disease-free survival (11.8% vs. 45.3%; P = 0.01) and overall survival (18.2% vs. 42.4%; P = 0.032), respectively. CONCLUSIONS: Taken together, our results showed for the first time that expression of PCBP1 was down-regulated in newly diagnosed AML patients and might be an independent prognostic marker in AML and should to be further investigated.
Assuntos
Biomarcadores Tumorais/análise , Ribonucleoproteínas Nucleares Heterogêneas/biossíntese , Leucemia Mieloide Aguda/patologia , Adolescente , Adulto , Idoso , Proteínas de Ligação a DNA , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Ribonucleoproteínas Nucleares Heterogêneas/análise , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Poly (C)-binding Protein 1 (PCBP1) is a 35 kDa protein involved in a number of biological processes. Recently, the research found that PCBP1 might be involved in epithelial-mesenchymal transition (EMT). However, the role of PCBP1 in non-small-cell lung cancer (NSCLC) metastasis needs further elucidation. The purpose of this study was to determine whether PCBP1 could serve as a biomarker for stratification and prediction of prognosis in NSCLC as a regulator of EMT formation. In this study, PCBP1 expression was evaluated by Western blot in 8 fresh lung cancer tissues and immunohistochemistry (IHC) on 145 paraffin-embedded slices. PCBP1 was highly expressed in non-metastatic NSCLC specimens and significantly correlated with lymph node status (P < 0.001), clinical stage (P = 0.001), vimentin expression (P = 0.033) and E-cadherin expression (P = 0.042). Our study showed that the low expression of PCBP1 was correlated with decreased expression of E-cadherin and elevated expression of vimentin, which were the markers of EMT. Besides, high expression of PCBP1 was correlated with better prognosis. These findings suggested that PCBP1 might play an important role in preventing the process of EMT in NSCLC, thus be a promising therapeutic target to inhibit NSCLC metastasis.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/química , Transição Epitelial-Mesenquimal , Ribonucleoproteínas Nucleares Heterogêneas/análise , Neoplasias Pulmonares/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD , Western Blotting , Caderinas/análise , Carcinoma Pulmonar de Células não Pequenas/secundário , Diferenciação Celular , Proteínas de Ligação a DNA , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Proteínas de Ligação a RNA , Vimentina/análiseRESUMO
AIMS: Folate receptors (FRs) mediate cellular uptake of folates in many cancer cells and in folate deficiency heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) mediates translational upregulation of FR in cultured cervical cancer cells. hnRNP-E1 can also interfere with human papillomavirus 16 (HPV-16) viral capsid protein synthesis (and thereby HPV proliferation) in vitro. This study aimed to evaluate prospectively the relevance of FR and hnRNP-E1 expression in the normal cervix, cervical dysplasia, and cancer. METHODS: Cervical tissues from 12 women with normal histology and 69 consecutive women with varying grades of cervical dysplasia and cancer were prospectively evaluated for immunohistochemical expression of FR, hnRNP-E1, proliferating cell nuclear antigen (PCNA), and HPV. There were 22 women with low grade squamous intraepithelial lesions (LGSIL), 22 with high grade squamous intraepithelial lesions (HGSIL), and 25 with invasive cervical carcinoma. RESULTS: Among normal subjects, 100% and 92% expressed hnRNP-E1 and FR, respectively. FR expression decreased from 91% in LGSIL to 68% and 64% in women with HGSIL and cancer, respectively. Similarly, hnRNP-E1 expression decreased from 86% in LGSIL to 68% and 40% in HGSIL and cancer, respectively. There was a highly significant positive correlation between the extent of FR and hnRNP-E1 expression, and an inverse correlation between HPV infection and hnRNP-E1 expression during progression of cervical dysplasia to cancer. CONCLUSION: These results are consistent with a hypothesis that reduced hnRNP-E1 expression may be permissive for HPV proliferation and progression to cervical cancer, and support the need for prospective longitudinal studies of hnRNP-E1 expression in HPV-16 infected women.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/química , Proteínas de Transporte/análise , Ribonucleoproteínas Nucleares Heterogêneas/análise , Papillomaviridae , Receptores de Superfície Celular , Proteínas Repressoras , Neoplasias do Colo do Útero/química , Biópsia , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/virologia , Estudos de Casos e Controles , Transformação Celular Viral , Colo do Útero/patologia , Colo do Útero/virologia , Distribuição de Qui-Quadrado , Proteínas de Ligação a DNA , Feminino , Receptores de Folato com Âncoras de GPI , Humanos , Imuno-Histoquímica/métodos , Proteínas Oncogênicas Virais/análise , Infecções por Papillomavirus/diagnóstico , Antígeno Nuclear de Célula em Proliferação/análise , Estudos Prospectivos , Proteínas de Ligação a RNA , Estatísticas não Paramétricas , Infecções Tumorais por Vírus/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/patologiaRESUMO
We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. Profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. However, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. However, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin.
Assuntos
Profilinas/análise , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Núcleo Celular/metabolismo , Chironomidae/crescimento & desenvolvimento , Chironomidae/metabolismo , Dactinomicina/farmacologia , Drosophila melanogaster/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Larva/metabolismo , Dados de Sequência Molecular , Cromossomos Politênicos/metabolismo , Profilinas/genética , Profilinas/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas/análise , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Glândulas Salivares/metabolismo , Alinhamento de Sequência , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND & OBJECTIVE: Proteasome inhibitor, which can induce apoptosis in various tumor cells, is a kind of potential antitumor drug. This study was to identify the proteins involved in G(2)/M arrest of leukemia cell line HL-60 exposed to proteasome inhibitor MG132 by proteomic techniques. METHODS: Flow cytometry was used to examine cell cycle of HL-60 cells exposed to 2.5 micromol/L MG132. Nuclear extracts of HL-60 cells were prepared, and the purity was detected by light microscopy and Western blot, and the differentially expressed protein spots were determined by two-dimensional gel electrophoresis and identified with MALDI-TOF-TOF/MS. RESULTS: There was a distinct G(2)/M phase arrest before the apoptosis of HL-60 cells induced by 2.5 micromol/L MG132. Twenty-three differentially expressed protein spots were found between MG132-treated and control HL-60 cells; 8 nuclear proteins were identified by MALDI-TOF-TOF/MS analysis. CONCLUSIONS: The detected proteins, such as eIF5A and splicing factor, may be involved in regulation of G(2)/M arrest of HL-60 cells. These findings will be helpful for revealing molecular mechanisms of proteasome inhibitor-induced G(2)/M phase arrest and apoptosis of leukemia cell line.
Assuntos
Ciclo Celular/efeitos dos fármacos , Leupeptinas/farmacologia , Proteínas Nucleares/análise , Proteômica/métodos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Fase G2/efeitos dos fármacos , Células HL-60 , Ribonucleoproteínas Nucleares Heterogêneas/análise , Humanos , Fatores de Iniciação de Peptídeos/análise , Proteínas de Ligação a RNA/análise , Fatores de Processamento de Serina-Arginina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Detailed knowledge of the composition and structure of the spliceosome and its assembly intermediates is a prerequisite for understanding the complex process of pre-mRNA splicing. To this end, we have developed a tobramycin affinity-selection method that is generally applicable for the purification of native RNP complexes. By using this method, we have isolated human prespliceosomes that are ideally suited for both biochemical and structural studies. MS identified >70 prespliceosome-associated proteins, including nearly all known U1 and U2 snRNP proteins, and expected non-snRNP splicing factors. In addition, the DEAD-box protein p68, RNA helicase A, and a number of proteins that appear to perform multiple functions in the cell, such as YB-1 and TLS, were detected. Several previously uncharacterized proteins of unknown function were also identified, suggesting that they play a role in splicing and potentially act during prespliceosome assembly. These data provide insight into the complexity of the splicing machinery at an early stage of its assembly.
Assuntos
Proteínas Nucleares/análise , Spliceossomos/química , Tobramicina/química , Centrifugação com Gradiente de Concentração , Ribonucleoproteínas Nucleares Heterogêneas/análise , Humanos , Espectrometria de Massas , Precursores de RNA/análise , Ribonucleoproteínas Nucleares Pequenas/análise , Tobramicina/metabolismoRESUMO
All-trans-retinoic acid (ATRA) induces growth inhibition, differentiation, and apoptosis in cancer cells, including acute promyelocytic leukemia (APL). In APL, expression of promyelocytic leukemia protein retinoic acid receptor-alpha (PML-RARalpha) fusion protein, owing to the t(15; 17) reciprocal translocation, leads to a block in the promyelocytic stage of differentiation. Here, we studied molecular mechanisms involved in ATRA-induced growth inhibition and myeloid cell differentiation in APL. By employing comprehensive high-throughput proteomic methods of 2-dimensional (2-D) gel electrophoresis and amino acid-coded mass tagging coupled with electrospray ionization (ESI) mass spectrometry, we systematically identified a total of 59 differentially expressed proteins that were consistently modulated in response to ATRA treatment. The data revealed significant down-regulation of eukaryotic initiation and elongation factors, initiation factor 2 (IF2), eukaryotic initiation factor 4AI (eIF4AI), eIF4G, eIF5, eIF6, eukaryotic elongation factor 1A-1 (eEF1A-1), EF-1-delta, eEF1gamma, 14-3-3epsilon, and 14-3-3zeta/delta (P <.05). The translational inhibitor DAP5/p97/NAT1 (death-associated protein 5) and PML isoform-1 were found to be up-regulated (P <.05). Additionally, the down-regulation of heterogeneous nuclear ribonucleoproteins (hnRNPs) C1/C2, UP2, K, and F; small nuclear RNPs (snRNPs) D3 and E; nucleoprotein tumor potentiating region (TPR); and protein phosphatase 2A (PP2A) were found (P <.05); these were found to function in pre-mRNA processing, splicing, and export events. Importantly, these proteomic findings were validated by Western blot analysis. Our data in comparison with previous cDNA microarray studies and our reverse transcription-polymerase chain reaction (RT-PCR) experiments demonstrate that broad networks of posttranscriptional suppressive pathways are activated during ATRA-induced growth inhibition processes in APL.
Assuntos
Antineoplásicos/farmacologia , Leucemia Promielocítica Aguda , Proteômica/métodos , Processamento Pós-Transcricional do RNA/fisiologia , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional/normas , Ribonucleoproteínas Nucleares Heterogêneas/análise , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Fatores de Alongamento de Peptídeos/análise , Fatores de Alongamento de Peptídeos/genética , Fatores de Iniciação de Peptídeos/análise , Fatores de Iniciação de Peptídeos/genética , Proteômica/normas , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hepatitis C virus (HCV) protein translation is mediated by a cis-acting RNA, an internal ribosomal entry site (IRES), located in the 5' nontranslated region of the viral RNA. To examine proteins bound to the IRES, which could include proteins important for its function as well as potential drug targets, we used shotgun peptide sequencing to identify proteins in quadruplicate protein affinity extracts of lysed Huh7 cells, obtained using a biotinylated IRES. Twenty-six proteins bound the HCV IRES but not a reversed complementary sequence RNA or vector RNA controls. These included five ribosomal subunits, nine eukaryotic initiation factor 3 subunits, and novel interacting proteins such as the cytoskeletal-related proteins actin, FHOS (formin homologue overexpressed in spleen) and MIP-T3 (microtubule interacting protein that associates with TRAF3). Other novel HCV IRES-binding proteins included UNR (upstream of N-ras), UNR-interacting protein, and the RNA-binding proteins PAI-1 (plasminogen activator inhibitor-1) mRNA binding protein and Ewing sarcoma breakpoint 1 region protein EWS. A large set of additional proteins bound both the HCV IRES and a reversed complementary IRES sequence control, including the known HCV interactors PTB (polypyrimidine tract binding protein), the La autoantigen, and nucleolin. The discovery of these novel HCV IRES-binding proteins suggests links between IRES biology and the cytoskeleton, signal transduction, and other cellular functions.