Assuntos
Autoanticorpos/imunologia , Encefalite Límbica/imunologia , Neoplasias Pulmonares/complicações , Carcinoma de Pequenas Células do Pulmão/complicações , Autoantígenos/imunologia , Proteínas ELAV/imunologia , Humanos , Encefalite Límbica/diagnóstico , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/imunologia , Fatores de Transcrição SOXB1/imunologia , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Pequenas Células do Pulmão/imunologiaRESUMO
Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3'-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3'-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein-protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.
Assuntos
Proteínas ELAV/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitógenos/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Regiões 3' não Traduzidas/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Proteínas ELAV/genética , Proteína Semelhante a ELAV 1 , Células HEK293 , Células HeLa , Humanos , Camundongos , Mutação , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/química , Transativadores/genética , Transativadores/metabolismoRESUMO
Human antigen R (HuR) is a ubiquitous protein that recognizes adenylate and uridylate-rich elements in mRNA, thereby interfering with the fate of protein translation. This protein plays a central role in the outcome of the inflammatory response as it may stabilize or silence mRNAs of key components of the immune system. HuR is able to interact with other RNA-binding proteins, reflecting a complex network that dictates mRNAs post-transcriptional control. HuR is composed of three functional domains, known as RNA-recognition motifs (RRM1, RRM2 and RRM3). It is known that RRM1 is the most important domain for mRNA-binding affinity. In this study, we completed the NMR chemical shift assignment of the RRM1 domain of HuR, as a first step to further establishing the structure, dynamics and function relationship for this protein.
Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Proteínas ELAV/química , Espectroscopia de Prótons por Ressonância Magnética , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Microarray data were collected from bile duct samples from subjects with malignant biliary strictures by endoscopic retrograde cholangiopancreatography to screen for key genes associated with this disease. A predicted interaction network was constructed for these genes to interpret their functions. The gene expression dataset GSE34166 (10 samples: 6 malignant and 4 benign control samples) was downloaded from the Gene Expression Omnibus database. R package scripts were used to process the data and screen for differentially expressed genes. Genes identified were uploaded to the analysis tool String 8.3 to generate a gene interaction network. A hub gene was identified by calculating the node degree. The interaction network of the hub gene with other genes in the human genome was constructed and screened (score >0.9), and pathway-enrichment analysis was performed to elucidate the hub gene function. In total, 377 differentially expressed genes were identified and a network comprising 209 pairs of interactions was constructed. The most critical hub gene was identified as GSTA1, and a GSTA1-based interaction network was constructed consisting of 25 genes (containing the differentially expressed gene GSTA3). The cytochrome P450 (CYP450)-metabolic pathway displayed the most significant enrichment. Additionally, 4 transcription factors and their binding sites were also identified. In conclusion, we have identified the differentially expressed genes GSTA1 (a hub gene) and GSTA3, which may cause abnormal gene expression and tumorigenesis through CYP450-metabolic pathways. The transcription factors and their binding sites in the promoter of the hub gene provide potential directions for future drug design.
Assuntos
Neoplasias dos Ductos Biliares/genética , Proteínas ELAV/biossíntese , Glutationa Transferase/biossíntese , Redes e Vias Metabólicas , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Biologia Computacional , Proteína Semelhante a ELAV 2 , Regulação Neoplásica da Expressão Gênica , Glutationa Transferase/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Mapas de Interação de Proteínas/genéticaRESUMO
Human Interleukin-3 (IL-3) is a lymphokine member of a class of transiently expressed mRNAs harboring Adenosine/Uridine-Rich Elements (ARE) in their 3' untranslated regions (3'-UTRs). The regulatory effects of AREs are often mediated by specific ARE-binding proteins (ARE-BPs). In this report, we show that the human IL-3 3'-UTR plays a post-transcriptional regulation role in two human transformed cell lines. More specifically, we demonstrate that the hIL-3 3'-UTR represses the translation of a luciferase reporter both in HeLa and Jurkat T-cells. These results also revealed that the hIL-3 3'-UTR-mediated translational repression is exerted by an 83 nt region comprised mainly by AREs and some non-ARE sequences. Moreover, electrophoretic mobility shift assays (EMSAs) and UV-crosslinking analysis show that this hIL-3 ARE-rich region recruits five specific protein complexes, including the ARE-BPs HuR and TIA-1. HuR binding to this ARE-rich region appears to be spatially modulated during T-cell activation. Together, these results suggest that HuR recognizes the ARE-rich region and plays a role in the IL-3 3'-UTR-mediated post-transcriptional control in T-cells.
Assuntos
Regiões 3' não Traduzidas , Proteínas ELAV/fisiologia , Interleucina-3/genética , Interleucina-3/metabolismo , Proteínas de Ligação a RNA/fisiologia , Proteína Semelhante a ELAV 1 , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Células Jurkat , Ativação Linfocitária , Proteínas de Ligação a Poli(A)/fisiologia , Antígeno-1 Intracelular de Células T , Transformação GenéticaRESUMO
AIM: To investigate the effect of quercetin supplementation on the myenteric neurons and glia in the cecum of diabetic rats. METHODS: Total preparations of the muscular tunic were prepared from the ceca of twenty-four rats divided into the following groups: control (C), control supplemented with quercetin (200 mg/kg quercetin body weight) (CQ), diabetic (D) and diabetic supplemented with quercetin (DQ). Immunohistochemical double staining technique was performed with HuC/D (general population)/nitric oxide synthase (nNOS), HuC-D/S-100 and VIP. Density analysis of the general neuronal population HuC/D-IR, the nNOS-IR (nitrergic subpopulation) and the enteric glial cells (S-100) was performed, and the morphometry and the reduction in varicosity population (VIP-IR) in these populations were analyzed. RESULTS: Diabetes promoted a significant reduction (25%) in the neuronal density of the HuC/D-IR (general population) and the nNOS-IR (nitrergic subpopulation) compared with the C group. Diabetes also significantly increased the areas of neurons, glial cells and VIP-IR varicosities. Supplementation with quercetin in the DQ group prevented neuronal loss in the general population and increased its area (P < 0.001) and the area of nitrergic subpopulation (P < 0.001), when compared to C group. Quercetin induced a VIP-IR and glial cells areas (P < 0.001) in DQ group when compared to C, CQ and D groups. CONCLUSION: In diabetes, quercetin exhibited a neuroprotective effect by maintaining the density of the general neuronal population but did not affect the density of the nNOS subpopulation.
Assuntos
Ceco/inervação , Nefropatias Diabéticas/tratamento farmacológico , Plexo Mientérico/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Quercetina/farmacologia , Animais , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 3 , Proteína Semelhante a ELAV 4 , Masculino , Plexo Mientérico/metabolismo , Plexo Mientérico/patologia , Plexo Mientérico/fisiopatologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios Nitrérgicos/efeitos dos fármacos , Neurônios Nitrérgicos/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ratos , Ratos Wistar , Proteínas S100/metabolismo , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
The HuR protein regulates the expression of thousands of cellular transcripts by modulating mRNA splicing, trafficking, translation, and stability. Although it serves as a model of RNA-protein interactions, many features of HuR's interactions with RNAs remain unknown. In this report, we deployed the cryogenic RNA immunoprecipitation technique to analyze HuR-interacting RNAs with the Affymetrix all-exon microarray platform. We revealed several thousand novel HuR-interacting RNAs, including hundreds of non-coding RNAs such as natural antisense transcripts from stress responsive loci. To gain insight into the mechanisms of specificity and sensitivity of HuR's interaction with its target RNAs, we searched HuR-interacting RNAs for composite patterns of primary sequence and secondary structure. We provide evidence that secondary structures of 66-75 nucleotides enhance HuR's recognition of its specific RNA targets composed of short primary sequence patterns. We validated thousands of these RNAs by analysis of overlap with recently published findings, including HuR's interaction with RNAs in the pathways of RNA splicing and stability. Finally, we observed a striking enrichment for members of ubiquitin ligase pathways among the HuR-interacting mRNAs, suggesting a new role for HuR in the regulation of protein degradation to mirror its known function in protein translation.
Assuntos
Proteínas ELAV/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ubiquitina/metabolismo , Proteínas ELAV/química , Humanos , Imunoprecipitação/métodos , RNA Antissenso/metabolismo , RNA Mensageiro/genética , TranscriptomaRESUMO
The human embryonic-lethal abnormal vision (ELAV)-like protein, HuR, has been recently found to be involved in the regulation of protein synthesis. In this study we show that HuR participates in the translational control of the HIV-1 and HCV IRES elements. HuR functions as a repressor of HIV-1 IRES activity and acts as an activator of the HCV IRES. The effect of HuR was evaluated in three independent experimental systems, rabbit reticulocyte lysate, HeLa cells, and Xenopus laevis oocytes, using both overexpression and knockdown approaches. Furthermore, results suggest that HuR mediated regulation of HIV-1 and HCV IRESes does not require direct binding of the protein to the RNA nor does it need the nuclear translocation of the IRES-containing RNAs. Finally, we show that HuR has a negative impact on post-integration steps of the HIV-1 replication cycle. Thus, our observations yield novel insights into the role of HuR in the post-transcriptional regulation of HCV and HIV-1 gene expression.
Assuntos
Antígenos de Superfície/metabolismo , HIV-1/metabolismo , Hepacivirus/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Animais , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação Viral da Expressão Gênica , HIV-1/fisiologia , Células HeLa , Hepacivirus/fisiologia , Humanos , Oócitos , RNA Viral/metabolismo , Coelhos , Ribossomos/metabolismo , Xenopus laevisRESUMO
The region that surrounds the central canal of the spinal cord derives from the neural tube and retains a substantial degree of plasticity. In turtles, this region is a neurogenic niche where newborn neurons coexist with precursors, a fact that may be related with the endogenous repair capabilities of low vertebrates. Immunohistochemical evidence suggests that the ependyma of the mammalian spinal cord may contain cells with similar properties, but their actual nature remains unsolved. Here, we combined immunohistochemistry for cell-specific markers with patch-clamp recordings to test the hypothesis that the ependyma of neonatal rats contains immature neurons similar to those in low vertebrates. We found that a subclass of cells expressed HuC/D neuronal proteins, doublecortin, and PSA-NCAM (polysialylated neural cell adhesion molecule) but did not express NeuN (anti-neuronal nuclei). These immature neurons displayed electrophysiological properties ranging from slow Ca(2+)-mediated responses to fast repetitive Na(+) spikes, suggesting different stages of maturation. These cells originated in the embryo, because we found colocalization of neuronal markers with 5-bromo-2'-deoxyuridine when injected during embryonic day 7-17 but not in postnatal day 0-5. Our findings represent the first evidence that the ependyma of the rat spinal cord contains cells with molecular and functional features similar to immature neurons in adult neurogenic niches. The fact that these cells retain the expression of molecules that participate in migration and neuronal differentiation raises the possibility that the ependyma of the rat spinal cord is a reservoir of immature neurons in "standby mode," which under some circumstances (e.g., injury) may complete their maturation to integrate spinal circuits.
Assuntos
Epêndima/citologia , Neurônios/citologia , Medula Espinal/citologia , Ácidos/metabolismo , Potenciais de Ação , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Bromodesoxiuridina , Cálcio/metabolismo , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 3 , Proteína Semelhante a ELAV 4 , Epêndima/crescimento & desenvolvimento , Epêndima/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Potenciais da Membrana , Proteínas Associadas aos Microtúbulos/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Neuropeptídeos/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Ácidos Siálicos/metabolismo , Sódio/metabolismo , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismoRESUMO
A small number of patients with carcinoma of the gallbladder can present with a variety of paraneoplastic syndromes, including the Cushing syndrome, hypercalcemia, acanthosis nigricans, bullous pemphigoid, dermatomyositis, and the Leser-Trélat sign. We report on what appears to be the first case of a patient, a 48-year-old woman, with anti-Hu paraneoplastic sensory neuropathy and small cell carcinoma of the gallbladder. The patient's neurologic symptoms preceded the diagnosis of small cell carcinoma by 11 months. These symptoms improved after surgical removal of the tumor and chemotherapy. The small cell carcinoma was relatively small and was not associated with gallstones. In spite of the small size of the tumor, it metastasized to a celiac lymph node and probably to the liver. Anti-Hu paraneoplastic sensory neuropathy should be added to the list of paraneoplastic syndromes associated with small cell carcinoma of the gallbladder.