RESUMO
Solution nuclear magnetic resonance (NMR) experiments allow RNA dynamics to be determined in an aqueous environment. However, when a limited number of peaks are assigned, it is difficult to obtain structural information. We here show a protocol based on the combination of experimental data (Nuclear Overhauser Effect, NOE) and molecular dynamics simulations with enhanced sampling methods. This protocol allows to (a) obtain a maximum entropy ensemble compatible with NMR restraints and (b) obtain a minimal set of metastable conformations compatible with the experimental data (maximum parsimony). The method is applied to a hairpin of 29 nt from an inverted SINEB2, which is part of the SINEUP family and has been shown to enhance protein translation. A clustering procedure is introduced where the annotation of base-base interactions and glycosidic bond angles is used as a metric. By reweighting the contributions of the clusters, minimal sets of four conformations could be found which are compatible with the experimental data. A motif search on the structural database showed that some identified low-population states are present in experimental structures of other RNA transcripts. The introduced method can be applied to characterize RNA dynamics in systems where a limited amount of NMR information is available.
Assuntos
RNA/química , Análise por Conglomerados , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Motivos de NucleotídeosRESUMO
SINEUPs are long non-coding RNAs (lncRNAs) that contain a SINE element, and which up-regulate the translation of target mRNA. They have been studied in a wide range of applications, as both biological and therapeutic tools, although the underpinning molecular mechanism is unclear. Here, we focused on the sub-cellular distribution of target mRNAs and SINEUP RNAs, performing co-transfection of expression vectors for these transcripts into human embryonic kidney cells (HEK293T/17), to investigate the network of translational regulation. The results showed that co-localization of target mRNAs and SINEUP RNAs in the cytoplasm was a key phenomenon. We identified PTBP1 and HNRNPK as essential RNA binding proteins. These proteins contributed to SINEUP RNA sub-cellular distribution and to assembly of translational initiation complexes, leading to enhanced target mRNA translation. These findings will promote a better understanding of the mechanisms employed by regulatory RNAs implicated in efficient protein translation.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Longo não Codificante/metabolismo , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteínas de Ligação a RNA/metabolismoRESUMO
Glial cell-derived neurotrophic factor (GDNF) has a potent action in promoting the survival of dopamine (DA) neurons. Several studies indicate that increasing GDNF levels may be beneficial for the treatment of Parkinson's disease (PD) by reducing neurodegeneration of DA neurons. Despite a plethora of preclinical studies showing GDNF efficacy in PD animal models, its application in humans remains questionable for its poor efficacy and side effects due to its uncontrolled, ectopic expression. Here we took advantage of SINEUPs, a new class of antisense long non-coding RNA, that promote translation of partially overlapping sense protein-coding mRNAs with no effects on their mRNA levels. By synthesizing a SINEUP targeting Gdnf mRNA, we were able to increase endogenous GDNF protein levels by about 2-fold. Adeno-associated virus (AAV)9-mediated delivery in the striatum of wild-type (WT) mice led to an increase of endogenous GDNF protein for at least 6 months and the potentiation of the DA system's functions while showing no side effects. Furthermore, SINEUP-GDNF was able to ameliorate motor deficits and neurodegeneration of DA neurons in a PD neurochemical mouse model. Our data indicate that SINEUP-GDNF could represent a new strategy to increase endogenous GDNF protein levels in a more physiological manner for therapeutic treatments of PD.
Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Neurônios Motores/metabolismo , Doença de Parkinson/genética , Interferência de RNA , RNA não Traduzido/genética , Animais , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Dependovirus/genética , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Neurônios Motores/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , FenótipoRESUMO
Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases.
Assuntos
Ataxia de Friedreich/genética , Regulação da Expressão Gênica , Proteínas de Ligação ao Ferro/genética , Modelos Biológicos , RNA não Traduzido/metabolismo , Aconitato Hidratase/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Humanos , Linfócitos/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , FrataxinaRESUMO
Transposable elements (TEs) compose about half of the mammalian genome and, as embedded sequences, up to 40% of long noncoding RNA (lncRNA) transcripts. Embedded TEs may represent functional domains within lncRNAs, providing a structured RNA platform for protein interaction. Here we show the interactome profile of the mouse inverted short interspersed nuclear element (SINE) of subfamily B2 (invSINEB2) alone and embedded in antisense (AS) ubiquitin C-terminal hydrolase L1 (Uchl1), an lncRNA that is AS to Uchl1 gene. AS Uchl1 is the representative member of a functional class of AS lncRNAs, named SINEUPs, in which the invSINEB2 acts as effector domain (ED)-enhancing translation of sense protein-coding mRNAs. By using RNA-interacting domainome technology, we identify the IL enhancer-binding factor 3 (ILF3) as a protein partner of AS Uchl1 RNA. We determine that this interaction is mediated by the RNA-binding motif 2 of ILF3 and the invSINEB2. Furthermore, we show that ILF3 is able to bind a free right Arthrobacter luteus (Alu) monomer sequence, the embedded TE acting as ED in human SINEUPs. Bioinformatic analysis of Encyclopedia of DNA Elements-enhanced cross-linking immunoprecipitation data reveals that ILF3 binds transcribed human SINE sequences at transcriptome-wide levels. We then demonstrate that the embedded TEs modulate AS Uchl1 RNA nuclear localization to an extent moderately influenced by ILF3. This work unveils the existence of a specific interaction between embedded TEs and an RNA-binding protein, strengthening the model of TEs as functional modules in lncRNAs.-Fasolo, F., Patrucco, L., Volpe, M., Bon, C., Peano, C., Mignone, F., Carninci, P., Persichetti, F., Santoro, C., Zucchelli, S., Sblattero, D., Sanges, R., Cotella, D., Gustincich, S. The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs.
Assuntos
Elementos de DNA Transponíveis , Proteínas do Fator Nuclear 90/metabolismo , RNA Antissenso/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Biologia Computacional , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Proteínas do Fator Nuclear 90/genética , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs, long non-coding RNAs (lncRNAs) and repetitive sequences, such as SINEs (short interspersed nuclear elements). A large percentage of ncRNAs are nuclear-enriched with unknown function. Antisense lncRNAs may form sense-antisense pairs by pairing with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription and mRNA stability. Here we identify a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level, hereby identifying a new functional class of lncRNAs. Antisense Uchl1 activity depends on the presence of a 5' overlapping sequence and an embedded inverted SINEB2 element. These features are shared by other natural antisense transcripts and can confer regulatory activity to an artificial antisense to green fluorescent protein. Antisense Uchl1 function is under the control of stress signalling pathways, as mTORC1 inhibition by rapamycin causes an increase in UCHL1 protein that is associated to the shuttling of antisense Uchl1 RNA from the nucleus to the cytoplasm. Antisense Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. These data reveal another layer of gene expression control at the post-transcriptional level.
Assuntos
Biossíntese de Proteínas/genética , RNA Antissenso/metabolismo , Elementos Nucleotídeos Curtos e Dispersos/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Animais , Antibacterianos/farmacologia , Linhagem Celular , Humanos , Masculino , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Antissenso/genética , Inversão de Sequência , Sirolimo/farmacologiaRESUMO
Erythropoietin receptor (EpoR) regulates erythrocytes differentiation in blood. In the brain, EpoR has been shown to protect several neuronal cell types from cell death, including the A9 dopaminergic neurons (DA) of the Substantia Nigra (SN). These cells form the nigrostriatal pathway and are devoted to the control of postural reflexes and voluntary movements. Selective degeneration of A9 DA neurons leads to Parkinson's disease. By the use of nanoCAGE, a technology that allows the identification of Transcription Start Sites (TSSs) at a genome-wide level, we have described the promoter-level expression atlas of mouse A9 DA neurons purified with Laser Capture Microdissection (LCM). Here, we identify mRNA variants of the Erythropoietin Receptor (DA-EpoR) transcribed from alternative TSSs. Experimental validation and full-length cDNA cloning is integrated with gene expression analysis in the FANTOM5 database. In DA neurons, the EpoR gene encodes for a N-terminal truncated receptor. Based on STAT5 phosphorylation assays, we show that the new variant of N-terminally truncated EpoR acts as decoy when co-expressed with the full-length form. A similar isoform is also found in human. This work highlights new complexities in the regulation of Erythropoietin (EPO) signaling in the brain.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Substância Negra/metabolismo , Animais , Sequência de Bases , Neurônios Dopaminérgicos/química , Células HEK293 , Humanos , Microdissecção e Captura a Laser/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores da Eritropoetina/análise , Substância Negra/química , Transcrição Gênica/fisiologiaRESUMO
Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.
Assuntos
Regiões Promotoras Genéticas , Receptores Odorantes/genética , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ordem dos Genes , Loci Gênicos , Fatores de Transcrição MEF2 , Camundongos , Camundongos Transgênicos , Fatores de Regulação Miogênica/metabolismo , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of α-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome.
Assuntos
Elementos Ricos em Adenilato e Uridilato/genética , Fases de Leitura Aberta/genética , Domínios e Motivos de Interação entre Proteínas/genética , Precursores de Proteínas/genética , Proteínas de Ligação a RNA/metabolismo , Timosina/análogos & derivados , Células HEK293 , Humanos , Ligação Proteica , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Timosina/genética , Timosina/metabolismoRESUMO
Hemoglobin is the oxygen carrier in blood erythrocytes. Oxygen coordination is mediated by α2ß2 tetrameric structure via binding of the ligand to the heme iron atom. This structure is essential for hemoglobin function in the blood. In the last few years, expression of hemoglobin has been found in atypical sites, including the brain. Transcripts for α and ß chains of hemoglobin as well as hemoglobin immunoreactivity have been shown in mesencephalic A9 dopaminergic neurons, whose selective degeneration leads to Parkinson's disease. To gain further insights into the roles of hemoglobin in the brain, we examined its quaternary structure in dopaminergic neurons in vitro and in vivo. Our results indicate that (i) in mouse dopaminergic cell line stably over-expressing α and ß chains, hemoglobin exists as an α2ß2 tetramer; (ii) similarly to the over-expressed protein, endogenous hemoglobin forms a tetramer of 64kDa; (iii) hemoglobin also forms high molecular weight insoluble aggregates; and (iv) endogenous hemoglobin retains its tetrameric structure in mouse mesencephalon in vivo. In conclusion, these results suggest that neuronal hemoglobin may be endowed with some of the biochemical activities and biological function associated to its role in erythroid cells. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
Assuntos
Encéfalo/metabolismo , Neurônios Dopaminérgicos/metabolismo , Heme/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Neuroblastoma/metabolismo , Oxigênio/metabolismo , Animais , Encéfalo/citologia , Células Cultivadas , Reagentes de Ligações Cruzadas/metabolismo , Neurônios Dopaminérgicos/citologia , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Neuroblastoma/patologia , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
BACKGROUND: The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson's disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown. RESULTS: By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca2+ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains. CONCLUSIONS: Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Odorantes , Receptores Odorantes/genética , Animais , Linhagem Celular , Análise por Conglomerados , Neurônios Dopaminérgicos/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Especificidade de Órgãos/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Receptores Odorantes/metabolismo , Proteínas Recombinantes , Substância Negra/metabolismo , Transcrição GênicaRESUMO
Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys(6), Lys(27), and Lys(29) linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys(6), Lys(27), and Lys(29) ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Animais , Encéfalo/patologia , Células HEK293 , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genética , Ligação Proteica , Transporte Proteico/genética , Fator 6 Associado a Receptor de TNF/genéticaRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the Substantia Nigra and the formation of ubiquitin- and alpha-synuclein (aSYN)-positive cytoplasmic inclusions called Lewy bodies (LBs). Although most PD cases are sporadic, families with genetic mutations have been found. Mutations in PARK7/DJ-1 have been associated with autosomal recessive early-onset PD, while missense mutations or duplications of aSYN (PARK1, PARK4) have been linked to dominant forms of the disease. In this study, we identify the E3 ubiquitin ligase tumor necrosis factor-receptor associated factor 6 (TRAF6) as a common player in genetic and sporadic cases. TRAF6 binds misfolded mutant DJ-1 and aSYN. Both proteins are substrates of TRAF6 ligase activity in vivo. Interestingly, rather than conventional K63 assembly, TRAF6 promotes atypical ubiquitin linkage formation to both PD targets that share K6-, K27- and K29- mediated ubiquitination. Importantly, TRAF6 stimulates the accumulation of insoluble and polyubiquitinated mutant DJ-1 into cytoplasmic aggregates. In human post-mortem brains of PD patients, TRAF6 protein colocalizes with aSYN in LBs. These results reveal a novel role for TRAF6 and for atypical ubiquitination in PD pathogenesis.
Assuntos
Encéfalo/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Corpos de Lewy/metabolismo , Proteínas Mutantes/metabolismo , Proteínas Oncogênicas/metabolismo , Doença de Parkinson/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , alfa-Sinucleína/metabolismo , Encéfalo/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Corpos de Lewy/patologia , Proteínas Mutantes/química , Proteínas Oncogênicas/química , Doença de Parkinson/patologia , Ligação Proteica , Proteína Desglicase DJ-1 , Dobramento de Proteína , Estrutura Quaternária de Proteína , Transporte Proteico , Especificidade por Substrato , UbiquitinaçãoRESUMO
The cellular prion protein (PrPC) is a ubiquitous glycoprotein highly expressed in the brain where it is involved in neurite outgrowth, copper homeostasis, NMDA receptor regulation, cell adhesion, and cell signaling. Conformational conversion of PrPC into its insoluble and aggregation-prone scrapie form (PrPSc) is the trigger for several rare devastating neurodegenerative disorders, collectively referred to as prion diseases. Recent work indicates that the ubiquitin-proteasome system is involved in quality control of PrPC. To better dissect the role of ubiquitination in PrPC physiology, we focused on the E3 RING ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here, we report that PrPC interacts with TRAF6 both in vitro, in cells, and in vivo, in the mouse brain. Transient overexpression of TRAF6 indirectly modulates PrPC ubiquitination and triggers redistribution of PrPC into the insoluble fraction. Importantly, in the presence of wild-type TRAF6, but not a mutant lacking E3 ligase activity, PrPC accumulates into cytoplasmic aggresome-like inclusions containing TRAF6 and p62/SQSTM1. Our results suggest that TRAF6 ligase activity could exert a role in the regulation of PrPC redistribution in cells under physiological conditions. This novel interaction may uncover possible mechanisms of cell clearance/reorganization in prion diseases.
Assuntos
Fator 6 Associado a Receptor de TNF , Ubiquitina-Proteína Ligases , Animais , Camundongos , Proteínas Priônicas/metabolismo , Ligação Proteica , Proteína Sequestossoma-1/metabolismo , Solubilidade , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Costimulatory molecules of the CD28 family play a crucial role in the activation of immune responses in T lymphocytes, complementing and modulating signals originating from the T-cell receptor (TCR) complex. Although distinct functional roles have been demonstrated for each family member, the specific signaling pathways differentiating ICOS- from CD28-mediated costimulation during early T-cell activation are poorly characterized. In the present study, we have performed RNA-Seq-based global transcriptome profiling of anti-CD3-treated naïve CD4+ T cells upon costimulation through either inducible costimulator (ICOS) or CD28, revealing a set of signaling pathways specifically associated with each signal. In particular, we show that CD3/ICOS costimulation plays a major role in pathways related to STAT3 function and osteoarthritis (OA), whereas the CD3/CD28 axis mainly regulates p38 MAPK signaling. Furthermore, we report the activation of distinct immunometabolic pathways, with CD3/ICOS costimulation preferentially targeting glycosaminoglycans (GAGs) and CD3/CD28 regulating mitochondrial respiratory chain and cholesterol biosynthesis. These data suggest that ICOS and CD28 costimulatory signals play distinct roles during the activation of naïve T cells by modulating distinct sets of immunological and immunometabolic genes.
Assuntos
Antígenos CD28 , Linfócitos T CD4-Positivos , Colesterol/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The key role played by host-microbiota interactions on human health, disease onset and progression, and on host response to treatments has increasingly emerged in the latest decades. Indeed, dysbiosis has been associated to several human diseases such as obesity, diabetes, cancer and also neurodegenerative disease, such as Parkinson, Huntington and Alzheimer's disease (AD), although whether causative, consequence or merely an epiphenomenon is still under investigation. In the present study, we performed a metabologenomic analysis of stool samples from a mouse model of AD, the 3xTgAD. We found a significant change in the microbiota of AD mice compared to WT, with a longitudinal divergence of the F/B ratio, a parameter suggesting a gut dysbiosis. Moreover, AD mice showed a significant decrease of some amino acids, while data integration revealed a dysregulated production of desaminotyrosine (DAT) and dihydro-3-coumaric acid. Collectively, our data show a dysregulated gut microbiota associated to the onset and progression of AD, also indicating that a dysbiosis can occur prior to significant clinical signs, evidenced by early SCFA alterations, compatible with gut inflammation.
Assuntos
Doença de Alzheimer , Microbioma Gastrointestinal , Doenças Neurodegenerativas , Animais , Modelos Animais de Doenças , Disbiose , Microbioma Gastrointestinal/fisiologia , Humanos , CamundongosRESUMO
Mutations in PARK7/DJ-1 are associated with autosomal recessive, early onset Parkinson disease (PD). DJ-1 is an atypical peroxiredoxin-like peroxidase that may act as a redox-dependent chaperone and a regulator of transcription. Here we show that DJ-1 plays an essential role in the expression of rearranged during transfection (RET), a receptor for the glial cell line-derived neurotrophic factor, a neuroprotective molecule for dopaminergic neurons, the main target of degeneration in PD. The inducible loss of DJ-1 triggers the establishment of hypoxia and the production of reactive oxygen species that stabilize the hypoxia-inducible factor-1alpha (HIF-1a). HIF-1a expression is required for RET down-regulation. This study establishes for the first time a molecular link between the lack of functional DJ-1 and the glial cell line-derived neurotrophic factor signaling pathway that may explain the adult-onset loss of dopaminergic neurons. Furthermore, it suggests that hypoxia may play an important role in PD.
Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mutação , Neuroblastoma/metabolismo , Proteínas Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neuroglia/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/metabolismo , Oxirredução , Proteína Desglicase DJ-1 , Espécies Reativas de Oxigênio , Transdução de SinaisRESUMO
Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.
RESUMO
Chemically modified mRNAs are extensively studied with a view toward their clinical application. In particular, long noncoding RNAs (lncRNAs) containing SINE elements, which enhance the translation of their target mRNAs (i.e., SINEUPs), have potential as RNA therapies for various diseases, such as haploinsufficiencies. To establish a SINEUP-based system for efficient protein expression, we directly transfected chemically modified in vitro transcribed (mIVT) SINEUP RNAs to examine their effects on target mRNA translation. mIVT SINEUP RNAs enhanced translation of EGFP mRNA and endogenous target Sox9 mRNA in both cultured cells and a cell-free translation system. Our findings reveal the functional role of RNA modifications in SINEUPs and suggest several broad clinical applications of such an RNA regulatory system.
Assuntos
Biossíntese de Proteínas , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células Hep G2 , Humanos , Técnicas In Vitro , Estabilidade de RNA , RNA Longo não Codificante/síntese química , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOX9/biossíntese , Fatores de Transcrição SOX9/genética , Regulação para CimaRESUMO
Here we describe a successful gene therapy approach for hemophilia A (HA), using the natural F8 promoter (pF8) to direct gene replacement to factor VIII (FVIII)-secreting cells. The promoter sequence and the regulatory elements involved in the modulation of F8 expression are still poorly characterized and biased by the historical assumption that FVIII expression is mainly in hepatocytes. Bioinformatic analyses have highlighted an underestimated complexity in gene expression at this locus, suggesting an activation of pF8 in more cell types than those previously expected. C57Bl/6 mice injected with a lentiviral vector expressing green fluorescent protein (GFP) under the pF8 (lentiviral vector [LV].pF8.GFP) confirm the predominant GFP expression in liver sinusoidal endothelial cells, with a few positive cells detectable also in hematopoietic organs. Therapeutic gene delivery (LV.pF8.FVIII) in hemophilic C57/Bl6 and 129-Bl6 mice successfully corrected the bleeding phenotype, rescuing up to 25% FVIII activity, using a codon-optimized FVIII, with sustained activity for the duration of the experiment (1 year) without inhibitor formation. Of note, LV.pF8.FVIII delivery in FVIII-immunized HA mice resulted in the complete reversion of the inhibitor titer with the recovery of therapeutic FVIII activity. Depletion of regulatory T cells (Tregs) in LV-treated mice allowed the formation of anti-FVIII antibodies, indicating a role for Tregs in immune tolerance induction. The significant blood loss reduction observed in all LV.pF8.FVIII-treated mice 1 year after injection confirmed the achievement of a long-term phenotypic correction. Altogether, our results highlight the potency of pF8-driven transgene expression to correct the bleeding phenotype in HA, as well as potentially in other diseases in which an endothelial-specific expression is required.