RESUMO
Eukaryotic gene expression is tightly regulated post-transcriptionally by RNA-binding proteins (RBPs) and microRNAs. The RBP AU-rich-binding factor 1 (AUF1) isoform p37 was found to have high affinity for the microRNA let-7b in vitro (Kd = â¼ 6 nM) in cells. Ribonucleoprotein immunoprecipitation, in vitro association, and single-molecule-binding analyses revealed that AUF1 promoted let-7b loading onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2-let-7 triggered target mRNA decay. Our findings uncover a novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing.
Assuntos
Proteínas Argonautas/metabolismo , Inativação Gênica/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , MicroRNAs/metabolismo , Animais , Células Cultivadas , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , Camundongos , Ligação Proteica , Estabilidade de RNA/fisiologiaRESUMO
Atherosclerosis is a complex disease that involves alterations in lipoprotein metabolism and inflammation. Protein and lipid glycosylation events, such as sialylation, contribute to the development of atherosclerosis and are regulated by specific glycosidases, including sialidases. To evaluate the effect of the sialidase neuraminidase 1 (NEU1) on atherogenesis, here we generated apolipoprotein E (ApoE)-deficient mice that express hypomorphic levels of NEU1 (Neu1hypoApoe-/-). We found that the hypomorphic NEU1 expression in male Apoe-/- mice reduces serum levels of very-low-density lipoprotein (VLDL) and LDL cholesterol, diminishes infiltration of inflammatory cells into lesions, and decreases aortic sinus atherosclerosis. Transplantation of Apoe-/- bone marrow (BM) into Neu1hypoApoe-/- mice significantly increased atherosclerotic lesion development and had no effect on serum lipoprotein levels. Moreover, Neu1hypoApoe-/- mice exhibited a reduction in circulating monocyte and neutrophil levels and had reduced hyaluronic acid and P-selectin adhesion capability on monocytes/neutrophils and T cells. Consistent with these findings, administration of a sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, had a significant anti-atherogenic effect in the Apoe-/- mice. In summary, the reduction in NEU1 expression or function decreases atherosclerosis in mice via its significant effects on lipid metabolism and inflammatory processes. We conclude that NEU1 may represent a promising target for managing atherosclerosis.
Assuntos
Apolipoproteínas E/genética , Aterosclerose/metabolismo , Quimiotaxia de Leucócito , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Regulação para Baixo , Neuraminidase/metabolismo , Animais , Aorta/patologia , LDL-Colesterol/metabolismo , VLDL-Colesterol/metabolismo , Ácido Hialurônico/metabolismo , Fígado/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Knockout para ApoE , Músculo Liso Vascular/citologia , Selectina-P/metabolismo , Linfócitos T/citologia , Triglicerídeos/metabolismoRESUMO
Hsp70 is a protein chaperone that prevents protein aggregation and aids protein folding by binding to hydrophobic peptide domains through a reversible mechanism directed by an ATPase cycle. However, Hsp70 also binds U-rich RNA including some AU-rich elements (AREs) that regulate the decay kinetics of select mRNAs and has recently been shown to bind and stabilize some ARE-containing transcripts in cells. Previous studies indicated that both the ATP- and peptide-binding domains of Hsp70 contributed to the stability of Hsp70-RNA complexes and that ATP might inhibit RNA recruitment. This suggested the possibility that RNA binding by Hsp70 might mimic features of its peptide-directed chaperone activities. Here, using purified, cofactor-free preparations of recombinant human Hsp70 and quantitative biochemical approaches, we found that high-affinity RNA binding requires at least 30 nucleotides of RNA sequence but is independent of Hsp70's nucleotide-bound status, ATPase activity, or peptide-binding roles. Furthermore, although both the ATP- and peptide-binding domains of Hsp70 could form complexes with an ARE sequence from VEGFA mRNA in vitro, only the peptide-binding domain could recover cellular VEGFA mRNA in ribonucleoprotein immunoprecipitations. Finally, Hsp70-directed stabilization of VEGFA mRNA in cells was mediated exclusively by the protein's peptide-binding domain. Together, these findings indicate that the RNA-binding and mRNA-stabilizing functions of Hsp70 are independent of its protein chaperone cycle but also provide potential mechanical explanations for several well-established and recently discovered cytoprotective and RNA-based Hsp70 functions.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Elementos Ricos em Adenilato e Uridilato , Regulação Alostérica , Sítios de Ligação , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Células HeLa , Humanos , Imunoprecipitação , Cinética , Mutação , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , RNA/antagonistas & inibidores , RNA/metabolismo , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into multinucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes. In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Here, using ribonucleoprotein immunoprecipitation (RIP) analysis, we discovered a novel function for MYF5 as an RNA-binding protein which associated with a subset of myoblast mRNAs. One prominent MYF5 target was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking and gel shift experiments indicated that MYF5 was capable of binding the 3' untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. Silencing MYF5 expression in proliferating myoblasts revealed that MYF5 promoted CCND1 translation and modestly increased transcription of Ccnd1 mRNA. Accordingly, overexpressing MYF5 in C2C12 cells upregulated CCND1 expression while silencing MYF5 reduced myoblast proliferation as well as differentiation of myoblasts into myotubes. Moreover, MYF5 silencing reduced myogenesis, while ectopically restoring CCND1 abundance partially rescued the decrease in myogenesis seen after MYF5 silencing. We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation.
Assuntos
Ciclina D1/genética , Desenvolvimento Muscular/genética , Fator Regulador Miogênico 5/genética , RNA Mensageiro/genética , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Ciclina D1/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Análise em Microsséries , Mioblastos , Fator Regulador Miogênico 5/metabolismo , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Sandhoff disease (SD) is a lysosomal storage disorder caused by a lack of a functional ß-subunit of the ß-hexosaminidase A and B enzymes, leading to the accumulation of gangliosides in the central nervous system (CNS). The Hexb-/- mouse model of SD shows a progressive neurodegenerative phenotype similar to the human equivalent. Previous studies have revealed that Hexb-/- mice suffer from chronic neuroinflammation characterized by microglial activation and expansion. Tumor necrosis factor-α (TNFα), a key modulator of the CNS immune response in models of neurodegeneration, is a hallmark of this activation. In this study, we explore the role of TNFα in the development and progression of SD in mice, by creating a Hexb-/- Tnfα-/- double-knockout mouse. Our results revealed that the double-knockout mice have an ameliorated disease course, with an extended lifespan, enhanced sensorimotor coordination and improved neurological function. TNFα-deficient SD mice also show decreased levels of astrogliosis and reduced neuronal cell death, with no alterations in neuronal storage of gangliosides. Interestingly, temporal microglia activation appears similar between the Hexb-/- Tnfα-/- and SD mice. Evidence is provided for the TNFα activation of the JAK2/STAT3 pathway as a mechanism for astrocyte activation in the disease. Bone marrow transplantation experiments reveal that both CNS-derived and bone marrow-derived TNFα have a pathological effect in SD mouse models, with CNS-derived TNFα playing a larger role. This study reveals TNFα as a neurodegenerative cytokine mediating astrogliosis and neuronal cell death in SD and points to TNFα as a potential therapeutic target to attenuate neuropathogenesis.
Assuntos
Doença de Sandhoff/metabolismo , Doença de Sandhoff/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/fisiologia , Animais , Transplante de Medula Óssea , Encéfalo/metabolismo , Morte Celular , Modelos Animais de Doenças , Feminino , Gangliosídeos/metabolismo , Gliose/genética , Gliose/patologia , Humanos , Camundongos , Camundongos Knockout , Microglia/metabolismo , Doença de Sandhoff/genética , Doença de Sandhoff/terapia , Transdução de Sinais , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
AUF1 is a family of four proteins generated by alternative pre-mRNA splicing that form high affinity complexes with AU-rich, mRNA-destabilizing sequences located within the 3' untranslated regions of many labile mRNAs. While AUF1 binding is most frequently associated with accelerated mRNA decay, emerging examples have demonstrated roles as a mRNA stabilizer or even translational regulator for specific transcripts. In this review, we summarize recent advances in our understanding of mRNA recognition by AUF1 and the biochemical and functional consequences of these interactions. In addition, unique properties of individual AUF1 isoforms and the roles of these proteins in modulating expression of genes associated with inflammatory, neoplastic, and cardiac diseases are discussed. Finally, we describe mechanisms that regulate AUF1 expression in cells, and current knowledge of regulatory switches that modulate the cellular levels and/or activities of AUF1 isoforms through distinct protein post-translational modifications. This article is part of a Special Issue entitled: RNA Decay mechanisms.
Assuntos
Elementos Ricos em Adenilato e Uridilato/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Processamento Alternativo/genética , Regulação da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Processamento de Proteína Pós-Traducional , Precursores de RNA/genéticaRESUMO
Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP(3)R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP(3) and IP(3)-induced calcium release from the SR. Immunohistochemistry proved the expression of IP(3)R type I (IP(3)R-I), but not type II (IP(3)R-II) and type III (IP(3)R-III) in ICC-MP, indicating the involvement of the IP(3)R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICC-MP. In summary, ongoing IP(3) synthesis and IP(3)-induced calcium release from the SR, via the IP(3)R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker frequency, which is likely directly linked to kinetics of the IP(3)-activated SR calcium channel and IP(3) metabolism.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Células Intersticiais de Cajal/metabolismo , Intestino Delgado/metabolismo , Plexo Mientérico/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , ATPases Transportadoras de Cálcio/metabolismo , Células Intersticiais de Cajal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Mibefradil/farmacologia , Camundongos , Plexo Mientérico/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismoRESUMO
The ubiquitous RNA-binding protein AUF1 promotes the degradation of some target mRNAs, but increases the stability and translation of other targets. Here, we isolated AUF1-associated mRNAs by immunoprecipitation of (AUF1-RNA) ribonucleoprotein (RNP) complexes from HeLa cells, identified them using microarrays, and used them to elucidate a signature motif shared among AUF1 target transcripts. The predicted AUF1 motif (29-39 nucleotides) contained 79% As and Us, consistent with the AU-rich sequences of reported AUF1 targets. Importantly, 10 out of 15 previously reported AUF1 target mRNAs contained the AUF1 motif. The predicted interactions between AUF1 and target mRNAs were recapitulated in vitro using biotinylated RNAs. Interestingly, further validation of predicted AUF1 target transcripts revealed that AUF1 associates with both the pre-mRNA and the mature mRNA forms. The consequences of AUF1 binding to 10 predicted target mRNAs were tested by silencing AUF1, which elevated the steady-state levels of only four mRNAs, and by overexpressing AUF1, which also lowered the levels of only four mRNAs. In total, we have identified a signature motif in AUF1 target mRNAs, have found that AUF1 also associates with the corresponding pre-mRNAs, and have discovered that altering AUF1 levels alone only modifies the levels of subsets of target mRNAs.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/química , Sequência de Bases , Sítios de Ligação , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Precursores de RNA/química , Precursores de RNA/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Sandhoff disease is an inherited lysosomal storage disease, resulting from the deficiency of lysosomal ß-hexosaminidase A and B enzyme activity. The Hexb-/- mouse model recapitulates human disease and leads to fatal neurodegeneration and neuroinflammation. IL-15 is important for the proliferation of NK, NK T, and CD8+ cytotoxic/memory T cells. In order to determine how changes to IL-15-dependent immune cell populations would alter the course of Sandhoff disease in mice, we generated a Hexb-/-Il-15-/- double knockout mouse and used motor behaviour tests, analyzed peripheral blood and brain leukocyte immunophenotypes, cytokine secretion, as well as examined markers of microgliosis, astrogliosis and apoptosis. Hexb-/-Il-15-/- mice had an accelerated neurodegenerative phenotype, and reached the humane endpoint at 118±3.5d, compared to Hexb-/- mice (127±2.2d). The performance of Hexb-/-Il-15-/- mice declined earlier than Hexb-/- mice on the rotarod and righting reflex motor behaviour tests. Hexb-/- mice had a significantly higher prevalence of pro-inflammatory monocytes in the blood relative to C57BL/6 mice, but this was unaltered by IL-15 deficiency. The prevalence of NK cells and CD8+ T cells in Il-15-/- and Hexb-/-Il-15-/- mice was decreased compared to wild type and Hexb-/- mice. While Hexb-/- mice displayed an increase in the prevalence of CD4+ and CD8+ T cells in brain leukocytes compared to C57BL/6 mice, there was a decrease in CD8+ T cells in Hexb-/-Il-15-/- compared to Hexb-/- mice. In addition, circulating IL-17 and IL-10 levels were significantly higher in Hexb-/-Il-15-/- mice, suggesting heightened inflammation compared to Hexb-/- mice. Interestingly, astrogliosis levels were significantly reduced in the cerebellum of Hexb-/-Il-15-/- mice compared to Hexb-/- mice while microgliosis was not affected in brains of Hexb-/-Il-15-/- mice. Our study demonstrated that IL-15 depletion dramatically reduced numbers of NK and CD8+ T cells as well as astrocytes but accelerated disease progression in Sandhoff mice. These results pointed to interactions between NK/CD8+ T cells and astrogliosis and potentially a protective role for NK/CD8+ T cells and/or astrocytes during disease progression. This observation supports the notion that expanding the IL-15-dependent NK and CD8+ T cells populations with IL-15 therapy may have therapeutic benefits for Sandhoff disease.
Assuntos
Linfócitos T CD8-Positivos/patologia , Doenças Cerebelares/etiologia , Gliose/terapia , Células Matadoras Naturais/patologia , Doença de Sandhoff/complicações , Doença de Sandhoff/mortalidade , Animais , Antígenos CD/metabolismo , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Doenças Cerebelares/genética , Doenças Cerebelares/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hexosaminidase B/genética , Hexosaminidase B/metabolismo , Interleucina-15/genética , Interleucina-15/metabolismo , Células Matadoras Naturais/metabolismo , Locomoção/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/genética , Doença de Sandhoff/genéticaRESUMO
AUF1 is a family of four RNA-binding proteins (RBPs) generated by alternative pre-messenger RNA (pre-mRNA) splicing, with canonical roles in controlling the stability and/or translation of mRNA targets based on recognition of AU-rich sequences within mRNA 3' untranslated regions. However, recent studies identifying AUF1 target sites across the transcriptome have revealed that these canonical functions are but a subset of its roles in posttranscriptional regulation of gene expression. In this review, we describe recent developments in our understanding of the RNA-binding properties of AUF1 together with their biochemical implications and roles in directing mRNA decay and translation. This is then followed by a survey of newly discovered activities for AUF1 proteins in control of miRNA synthesis and function, including miRNA assembly into microRNA (miRNA)-loaded RNA-induced silencing complexes (miRISCs), miRISC targeting to mRNA substrates, interplay with an expanding network of other cellular RBPs, and reciprocal regulatory relationships between miRNA and AUF1 synthesis. Finally, we discuss recently reported relationships between AUF1 and long noncoding RNAs and regulatory roles on viral RNA substrates. Cumulatively, these findings have significantly expanded our appreciation of the scope and diversity of AUF1 functions in the cell, and are prompting an exciting array of new questions moving forward. WIREs RNA 2017, 8:e1393. doi: 10.1002/wrna.1393 For further resources related to this article, please visit the WIREs website.
Assuntos
Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Precursores de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Animais , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
A panel of iron (Fe) and copper (Cu) chelators was screened for growth inhibitory activity against the fungal pathogen Cryptococcus neoformans. Select bidentate metal-binding ligands containing mixed O,S or O,N donor atoms were identified as agents that induce cell killing in a Cu-dependent manner. Conversely, structurally similar ligands with O,O donor atoms did not inhibit C. neoformans growth regardless of Cu status. Studies of Cu(ii) and Cu(i) binding affinity, lipophilicity, and growth recovery assays of Cu-import deficient cells identified lipophilicity of thermodynamically stable CuIIL2 complexes as the best predictor of antifungal activity. These same complexes induce cellular hyperaccumulation of Zn and Fe in addition to Cu. The results described here present the utility of appropriate metal-binding ligands as potential antifungal agents that manipulate cellular metal balance as an antimicrobial strategy.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Quelantes/química , Quelantes/farmacologia , Cobre/metabolismo , Criptococose/tratamento farmacológico , Cryptococcus neoformans/efeitos dos fármacos , Criptococose/microbiologia , Criptococose/patologia , Humanos , Estrutura MolecularRESUMO
Post-transcriptional gene regulation is robustly regulated by RNA-binding proteins (RBPs). Here we describe the collection of RNAs regulated by AUF1 (AU-binding factor 1), an RBP linked to cancer, inflammation and aging. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis reveals that AUF1 primarily recognizes U-/GU-rich sequences in mRNAs and noncoding RNAs and influences target transcript fate in three main directions. First, AUF1 lowers the steady-state levels of numerous target RNAs, including long noncoding RNA NEAT1, in turn affecting the organization of nuclear paraspeckles. Second, AUF1 does not change the abundance of many target RNAs, but ribosome profiling reveals that AUF1 promotes the translation of numerous mRNAs in this group. Third, AUF1 unexpectedly enhances the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Through its actions on target RNAs, AUF1 preserves genomic integrity, in agreement with the AUF1-elicited prevention of premature cellular senescence.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Regiões 3' não Traduzidas , Proteína Semelhante a ELAV 1/metabolismo , Genoma , Células HEK293 , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Técnicas Imunológicas , Íntrons , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Análise de Sequência de RNARESUMO
SR-BI deficient mice that are also hypomorphic for apolipoprotein E expression develop diet induced occlusive coronary artery atherosclerosis, myocardial infarction and early death. To test the role of SR-BI in bone marrow derived cells, we used bone marrow transplantation to generate SR-BI-null; apoE-hypomorphic mice in which SR-BI expression was restored solely in bone marrow derived cells. SR-BI-null; apoE-hypomorphic mice were transplanted with SR-BI(+/+)apoE-hypomorphic, or control, autologous SR-BI-null; apoE-hypomorphic bone marrow. Four weeks later, mice were fed a high-fat, high-cholesterol, cholate-containing diet to induce coronary artery atherosclerosis. Mice transplanted with autologous bone marrow developed extensive aortic atherosclerosis and severe occlusive coronary artery atherosclerosis after 4 weeks of feeding. This was accompanied by myocardial fibrosis and increased heart weights. In contrast, restoration of SR-BI expression in bone marrow derived-cells reduced diet induced aortic and coronary artery atherosclerosis, myocardial fibrosis and the increase in heart weights in SR-BI-null; apoE-hypomorphic mice. Restoration of SR-BI in bone marrow derived cells did not, however, affect steady state lipoprotein cholesterol levels, but did reduce plasma levels of IL-6. Monocytes from SR-BI-null mice exhibited a greater capacity to bind to VCAM-1 and ICAM-1 than those from SR-BI(+/+) mice. Furthermore, restoration of SR-BI expression in bone marrow derived cells attenuated monocyte recruitment into atherosclerotic plaques in mice fed high fat, high cholesterol cholate containing diet. These data demonstrate directly that SR-BI in bone marrow-derived cells protects against both aortic and CA atherosclerosis.
Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Antígenos CD36/genética , Doença da Artéria Coronariana/genética , Dieta , Infarto do Miocárdio/genética , Animais , Aorta/patologia , Apolipoproteínas E/deficiência , Antígenos CD36/metabolismo , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/terapia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Feminino , Fibrose , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Lipídeos/sangue , Masculino , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Infarto do Miocárdio/terapia , Miocárdio/patologia , Tamanho do Órgão , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
OBJECTIVES: To examine the effects of pomegranate extract on inflammation and oxidative stress and the development of spontaneous occlusive coronary artery atherosclerosis in the SR-BI/apoE double knockout mouse model of coronary heart disease. METHODS AND RESULTS: SR-BI/apoE double KO mice were treated for two weeks with pomegranate extract via drinking water, beginning at three weeks of age. Treatment with pomegranate extract increased cholesterol ester content and reduced the abnormally high unesterified/esterified cholesterol ratio of VLDL-sized lipoproteins. Despite the increase in cholesterol levels associated with VLDL-sized particles, pomegranate extract treatment reduced the size of atherosclerotic plaques in the aortic sinus and reduced the proportion of coronary arteries with occlusive atherosclerotic plaques. Treatment with pomegranate extract resulted in substantial reductions in levels of oxidative stress and monocyte chemotactic protein-1 in atherosclerotic plaques in the aortic sinus and coronary arteries. In addition, treatment with pomegranate extract reduced lipid accumulation, macrophage infiltration, levels of monocyte chemotactic protein-1 and fibrosis in the myocardium, attenuated cardiac enlargement and the development of ECG abnormalities in SR-BI/apoE double KO mice. CONCLUSION: Pomegranate extract reduced aortic sinus and coronary artery atherosclerosis in SR-BI/apoE dKO mice. The atheroprotective effects of pomegranate extract appear to involve reduced oxidative stress and inflammation in the vessel wall despite unaltered systemic markers of inflammation and increased lipoprotein cholesterol in these mice.
Assuntos
Apolipoproteínas E/genética , Doença da Artéria Coronariana/tratamento farmacológico , Lythraceae/química , Extratos Vegetais/farmacologia , Receptores Depuradores Classe B/genética , Animais , Colesterol/sangue , Ésteres do Colesterol/sangue , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Modelos Animais de Doenças , Feminino , Lipoproteínas/sangue , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , Placa Aterosclerótica/tratamento farmacológico , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Vasculite/tratamento farmacológico , Vasculite/genética , Vasculite/metabolismoRESUMO
Advances in genomic sequencing technologies in the past decade have revolutionized the field of genomics, resulting in faster and less expensive sequencing. Holding back the potential for innovation, however, is a widespread lack of understanding of genomics and sequencing by the general public. In an attempt to remedy this problem, this paper presents an introduction to the fields of genomics, bioinformatics, and proteomics using the blueberry genome as a model case study of the plant genomics field. The blueberry (Vaccinium sect. Cyanococcus) is often cited as a "super food" in the media due to its nutritional benefits and global economic importance. There have been a number of related genomic publications in the past 20 years; however, a completed genome and a full analysis into the health-related pathways are still needed. As exemplified by this blueberry case study, there are opportunities for future genomic research into numerous beneficial plant species. The solid background presented in this paper provides future researchers the foundation to explore these uncharted areas.
RESUMO
Although mammalian long non-coding (lnc)RNAs are best known for modulating transcription, their post-transcriptional influence on mRNA splicing, stability and translation is emerging. Here we report a post-translational function for the lncRNA HOTAIR as an inducer of ubiquitin-mediated proteolysis. HOTAIR associates with E3 ubiquitin ligases bearing RNA-binding domains, Dzip3 and Mex3b, as well as with their respective ubiquitination substrates, Ataxin-1 and Snurportin-1. In this manner, HOTAIR facilitates the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b in cells and in vitro, and accelerates their degradation. HOTAIR levels are highly upregulated in senescent cells, causing rapid decay of targets Ataxin-1 and Snurportin-1, and preventing premature senescence. These results uncover a role for a lncRNA, HOTAIR, as a platform for protein ubiquitination.
Assuntos
Proteínas/metabolismo , RNA Longo não Codificante/metabolismo , Ubiquitinação , Proteínas Argonautas/metabolismo , Ataxina-1 , Ataxinas , Senescência Celular/genética , Proteínas ELAV/metabolismo , Células HeLa , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The serine/threonine kinase Pim-1 directs selected signaling events that promote cell growth and survival and is overexpressed in diverse human cancers. Pim-1 expression is tightly controlled through multiple mechanisms, including regulation of mRNA turnover. In several cultured cell models, mitogenic stimulation rapidly induced and stabilized PIM1 mRNA, however, vigorous destabilization 4-6 hours later helped restore basal expression levels. Acceleration of PIM1 mRNA turnover coincided with accumulation of tristetraprolin (TTP), an mRNA-destabilizing protein that targets transcripts containing AU-rich elements. TTP binds PIM1 mRNA in cells, and suppresses its expression by accelerating mRNA decay. Reporter mRNA decay assays localized the TTP-regulated mRNA decay element to a discrete AU-rich sequence in the distal 3'-untranslated region that binds TTP. These data suggest that coordinated stimulation of TTP and PIM1 expression limits the magnitude and duration of PIM1 mRNA accumulation by accelerating its degradation as TTP protein levels increase. Consistent with this model, PIM1 and TTP mRNA levels were well correlated across selected human tissue panels, and PIM1 mRNA was induced to significantly higher levels in mitogen-stimulated fibroblasts from TTP-deficient mice. Together, these data support a model whereby induction of TTP mediates a negative feedback circuit to limit expression of selected mitogen-activated genes.
Assuntos
Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismo , Regiões 3' não Traduzidas , Sequência Rica em At , Animais , Sequência de Bases , Técnicas de Cultura de Células , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Mitógenos/farmacologia , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Proteínas Proto-Oncogênicas c-pim-1/genética , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta , Transcrição Gênica/efeitos dos fármacosRESUMO
The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIIIbeta and the hLigIIIalpha/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.
Assuntos
DNA Ligases/metabolismo , Reparo do DNA , DNA/metabolismo , Adenina/metabolismo , Biocatálise , Bioensaio , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Ésteres/metabolismo , Fluorescência , Humanos , Cinética , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
A wide range of plant RNA extraction methods are available; however, many of these are limited in their application for a diverse range of plant species. With special emphasis on robustness and versatility, we have improved the cetyltrimethylammonium bromide (CTAB) method and isolated high-quality RNA from 16 different plant species. The major modifications made to the protocol described here were a reduction of sample treatment steps and an increase in beta-mercaptoethanol concentration (to 3%) resulting in a robust, rapid and reproducible plant RNA extraction protocol that can be used for a broad range of plant species and tissue types.