RESUMO
BACKGROUND: Microsatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels. RESULTS: Our results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer. CONCLUSIONS: Overall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis.
Assuntos
Processamento Alternativo , Neoplasias Colorretais , Instabilidade de Microssatélites , Fator de Processamento U2AF , Neoplasias Colorretais/genética , Humanos , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Mutação , Sítios de Ligação , ÉxonsRESUMO
BCL-x is a master regulator of apoptosis whose pre-mRNA is alternatively spliced into either a long (canonical) anti-apoptotic Bcl-xL isoform, or a short (alternative) pro-apoptotic Bcl-xS isoform. The balance between these two antagonistic isoforms is tightly regulated and overexpression of Bcl-xL has been linked to resistance to chemotherapy in several cancers, whereas overexpression of Bcl-xS is associated to some forms of diabetes and cardiac disorders. The splicing factor RBM25 controls alternative splicing of BCL-x: its overexpression favours the production of Bcl-xS, whereas its downregulation has the opposite effect. Here we show that RBM25 directly and specifically binds to GQ-2, an RNA G-quadruplex (rG4) of BCL-x pre-mRNA that forms at the vicinity of the alternative 5' splice site leading to the alternative Bcl-xS isoform. This RBM25/rG4 interaction is crucial for the production of Bcl-xS and depends on the RE (arginine-glutamate-rich) motif of RBM25, thus defining a new type of rG4-interacting domain. PhenDC3, a benchmark G4 ligand, enhances the binding of RBM25 to the GQ-2 rG4 of BCL-x pre-mRNA, thereby promoting the alternative pro-apoptotic Bcl-xS isoform and triggering apoptosis. Furthermore, the screening of a combinatorial library of 90 putative G4 ligands led to the identification of two original compounds, PhenDH8 and PhenDH9, superior to PhenDC3 in promoting the Bcl-xS isoform and apoptosis. Thus, favouring the interaction between RBM25 and the GQ-2 rG4 of BCL-x pre-mRNA represents a relevant intervention point to re-sensitize cancer cells to chemotherapy.
Assuntos
Processamento Alternativo , Precursores de RNA , Apoptose , Isoformas de Proteínas/genética , Precursores de RNA/genética , Sítios de Splice de RNA , HumanosRESUMO
The Epstein-Barr virus (EBV) is the first oncogenic virus described in human. EBV infects more than 90% of the human population worldwide, but most EBV infections are asymptomatic. After the primary infection, the virus persists lifelong in the memory B cells of the infected individuals. Under certain conditions the virus can cause several human cancers, that include lymphoproliferative disorders such as Burkitt and Hodgkin lymphomas and non-lymphoid malignancies such as 100% of nasopharyngeal carcinoma and 10% of gastric cancers. Each year, about 200,000 EBV-related cancers emerge, hence accounting for at least 1% of worldwide cancers. Like all gammaherpesviruses, EBV has evolved a strategy to escape the host immune system. This strategy is mainly based on the tight control of the expression of its Epstein-Barr nuclear antigen-1 (EBNA1) protein, the EBV-encoded genome maintenance protein. Indeed, EBNA1 is essential for viral genome replication and maintenance but, at the same time, is also highly antigenic and T cells raised against EBNA1 exist in infected individuals. For this reason, EBNA1 is considered as the Achilles heel of EBV and the virus has seemingly evolved a strategy that employs the binding of nucleolin, a host cell factor, to RNA G-quadruplex (rG4) within EBNA1 mRNA to limit its expression to the minimal level required for function while minimizing immune recognition. This review recapitulates in a historical way the knowledge accumulated on EBNA1 immune evasion and discusses how this rG4-dependent mechanism can be exploited as an intervention point to unveil EBV-related cancers to the immune system.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , Humanos , Herpesvirus Humano 4/genética , RNA , Sistema ImunitárioRESUMO
The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome maintenance but is also highly antigenic. Hence, EBV seemingly evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 limits the translation of its own mRNA to the minimal level to ensure its essential function, thereby, at the same time, minimizing immune recognition. Therefore, defining intervention points at which to interfere with GAr-based inhibition of translation is an important step to trigger an immune response against EBV-carrying cancers. The host protein nucleolin (NCL) plays a critical role in this process via a direct interaction with G-quadruplexes (G4) formed in the GAr-encoding sequence of the viral EBNA1 mRNA. Here we show that the C-terminal arginine-glycine-rich (RGG) motif of NCL is crucial for its role in GAr-based inhibition of translation by mediating interaction of NCL with G4 of EBNA1 mRNA. We also show that this interaction depends on the type I arginine methyltransferase family, notably PRMT1 and PRMT3: drugs or small interfering RNA that target these enzymes prevent efficient binding of NCL on G4 of EBNA1 mRNA and relieve GAr-based inhibition of translation and of antigen presentation. Hence, this work defines type I arginine methyltransferases as therapeutic targets to interfere with EBNA1 and EBV immune evasion.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Infecções Tumorais por Vírus , Humanos , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Sistema Imunitário/metabolismo , Vírus Oncogênicos/genética , Vírus Oncogênicos/metabolismo , Proteína-Arginina N-Metiltransferases , Proteínas Repressoras , RNA Mensageiro/metabolismo , Infecções Tumorais por Vírus/tratamento farmacológico , Infecções Tumorais por Vírus/metabolismoRESUMO
Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine-alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Proteínas de Ligação a RNA/metabolismo , Alanina , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Glicina , Herpesvirus Humano 4/genética , Humanos , Peptídeos/genética , Fosfoproteínas , Agregados Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , NucleolinaRESUMO
Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.
Assuntos
Encefalopatias , Doença de Leigh , ATPases Mitocondriais Próton-Translocadoras , Encefalopatias/metabolismo , DNA Complementar/metabolismo , Humanos , Doença de Leigh/genética , Doença de Leigh/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação , Proteínas/metabolismoRESUMO
The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.
Assuntos
DNA Intergênico/química , DNA Intergênico/genética , Quadruplex G , RNA/química , RNA/genética , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação da Expressão Gênica , Humanos , Transporte de RNA , RNA ViralRESUMO
Prion diseases are caused by the propagation of PrPSc, the pathological conformation of the PrPC prion protein. The molecular mechanisms underlying PrPSc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrPSc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrPSc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Flunarizina/administração & dosagem , Proteína I de Ligação a Poli(A)/metabolismo , Doenças Priônicas/metabolismo , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/administração & dosagem , Linhagem Celular , Bases de Dados Factuais , Drosophila , Feminino , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Proteína I de Ligação a Poli(A)/antagonistas & inibidores , Proteína I de Ligação a Poli(A)/genética , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/genética , Proteínas Priônicas/antagonistas & inibidores , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Agregados Proteicos/fisiologia , OvinosRESUMO
BACKGROUND: Hydrogen sulfide (H2S) is an endogenous mammalian gasotransmitter. Cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) are the principal enzymes responsible for its biogenesis. A recent yeast screen suggested that disulfiram (a well-known inhibitor of aldehyde dehydrogenase and a clinically used drug in the treatment of alcoholism) may inhibit CBS in a cell-based environment. However, prior studies have not observed any direct inhibition of CBS by disulfiram. We investigated the potential role of bioconversion of disulfiram to bis(N,N-diethyldithiocarbamate)-copper(II) complex (CuDDC) in the inhibitory effect of disulfiram on H2S production and assessed its effect in two human cell types with high CBS expression: HCT116 colon cancer cells and Down syndrome (DS) fibroblasts. METHODS: H2S production from recombinant human CBS, CSE and 3-MST was measured using the fluorescent H2S probe AzMC. Mouse liver homogenate (a rich source of CBS) was also employed to measure H2S biosynthesis. The interaction of copper with accessible protein cysteine residues was evaluated using the DTNB method. Cell proliferation and viability were measured using the BrdU and MTT methods. Cellular bioenergetics was evaluated by Extracellular Flux Analysis. RESULTS: While disulfiram did not exert any significant direct inhibitory effect on any of the H2S-producing enzymes, its metabolite, CuDDC was a potent inhibitor of CBS and CSE. The mode of its action is likely related to the complexed copper molecule. In cell-based systems, the effects of disulfiram were variable. In colon cancer cells, no significant effect of disulfiram was observed on H2S production or proliferation or viability. In contrast, in DS fibroblasts, disulfiram inhibited H2S production and improved proliferation and viability. Copper, on its own, failed to have any effects on either cell type, likely due to its low cell penetration. CuDDC inhibited H2S production in both cell types studied and exerted the functional effects that would be expected from a CBS inhibitor: inhibition of cell proliferation of cancer cells and a bell-shaped effect (stimulation of proliferation at low concentration and inhibition of these responses at higher concentration) in DS cells. Control experiments using a chemical H2S donor showed that, in addition to inhibiting CBS and CSE, part of the biological effects of CuDDC relates to a direct reaction with H2S, which occurs through its complexed copper. CONCLUSIONS: Disulfiram, via its metabolite CuDDC acts as an inhibitor of CBS and a scavenger of H2S, which, in turn, potently suppresses H2S levels in various cell types. Inhibition of H2S biosynthesis may explain some of the previously reported actions of disulfiram and CuDDC in vitro and in vivo. Disulfiram or CuDDC may be considered as potential agents for the experimental therapy of various pathophysiological conditions associated with H2S overproduction.
Assuntos
Inibidores de Acetaldeído Desidrogenases/farmacologia , Cobre/farmacologia , Cistationina beta-Sintase/antagonistas & inibidores , Dissulfiram/farmacologia , Ditiocarb/análogos & derivados , Compostos Organometálicos/farmacologia , Inibidores de Acetaldeído Desidrogenases/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Quelantes/metabolismo , Quelantes/farmacologia , Cobre/metabolismo , Cistationina beta-Sintase/metabolismo , Dissulfiram/metabolismo , Ditiocarb/metabolismo , Ditiocarb/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HCT116 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organometálicos/metabolismoRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is the most common inheritable cause of end stage renal disease and, as of today, only a single moderately effective treatment is available for patients. Even though ADPKD research has made huge progress over the last decades, the precise disease mechanisms remain elusive. However, a wide variety of cellular and animal models have been developed to decipher the pathophysiological mechanisms and related pathways underlying the disease. As none of these models perfectly recapitulates the complexity of the human disease, the aim of this review is to give an overview of the main tools currently available to ADPKD researchers, as well as their main advantages and limitations.
Assuntos
Rim Policístico Autossômico Dominante/etiologia , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Animais , Cistos/complicações , Modelos Animais de Doenças , Progressão da Doença , Rim/patologia , Falência Renal Crônica/complicações , Modelos Biológicos , Doenças Renais Policísticas/etiologia , Doenças Renais Policísticas/metabolismo , Suínos , Porco MiniaturaRESUMO
Yeast has been used for thousands of years as a leavening agent and for alcoholic fermentation, but it is only in 1857 that Louis Pasteur described the microorganism at the basis of these two tremendously important economic activities. From there, yeast strains could be selected and modified on a rational basis to optimize these uses, thereby also allowing the development of yeast as a popular eukaryotic model system. This model led to a cornucopia of seminal discoveries in cell biology. For about two decades yeast has also been used as a model and a tool for therapeutic research, from the production of therapeutics and the development of diagnostic tools to the identification of new therapeutic targets, drug candidates and chemical probes. These diverse chemobiological applications of yeast are presented and discussed in the present review article.
TITLE: La levure modèle et outil aussi pour la recherche thérapeutique. ABSTRACT: La levure a été utilisée de façon empirique pendant des millénaires pour la panification et la fermentation des sucres en alcool. C'est seulement à partir de 1857 que Louis Pasteur décrit le microorganisme à l'origine de ces deux activités agroalimentaires majeures. Dès lors, les souches de levure ont pu être sélectionnées et modifiées sur une base rationnelle pour optimiser leurs usages agroalimentaires, permettant ainsi l'essor de la levure comme modèle biologique eucaryote. Cette utilisation a conduit à de très nombreuses découvertes de biologie cellulaire fondamentale. Depuis une vingtaine d'années, la levure est également utilisée comme modèle et outil pour la santé humaine. Ces approches s'étendent de la production de molécules thérapeutiques à la recherche de candidats-médicaments et de sondes chimiques, en passant par la mise au point de tests diagnostiques et la découverte de nouvelles cibles thérapeutiques. Cette utilisation de la levure en chémobiologie fait l'objet de la présente revue.
Assuntos
Pesquisa Biomédica/métodos , Modelos Biológicos , Saccharomycetales/fisiologia , Pesquisa Biomédica/história , Pesquisa Biomédica/tendências , Desenvolvimento de Medicamentos/métodos , Desenvolvimento de Medicamentos/tendências , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendênciasRESUMO
The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.
Assuntos
Betacoronavirus , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , COVID-19 , Infecções por Coronavirus , Surtos de Doenças , Humanos , Pandemias , Pneumonia Viral , SARS-CoV-2RESUMO
Fipronil is an insecticide widely used for veterinary and agricultural purposes. While its insecticidal properties mostly rely on its high affinity antagonistic activity on insect γ aminobutyric acid (GABA) receptors, fipronil and its main metabolite fipronil sulfone nevertheless display non-negligible affinity for mammalian GABAA receptor. As several environmental toxicants have been shown to raise the risk of developing various neurodegenerative disorders, the aim of this study was to evaluate whether long-term low dose administration of fipronil could lead to cognitive deficiencies. Our results indicate that long-term fipronil treatment leads to behavioral perturbations in mice, indicating an accumulative effect of sustained exposure to low dose of fipronil. Although no memory impairment was observed during the course of our study, we noticed a significant hyperlocomotion behavior after 43 weeks of weekly fipronil administration, which is consistent with its direct effect on the GABAergic system.
Assuntos
Hipercinese , Inseticidas , Pirazóis , Animais , Feminino , Hipercinese/induzido quimicamente , Inseticidas/toxicidade , Camundongos , Pirazóis/toxicidade , Receptores de GABA/efeitos dos fármacos , Ácido gama-AminobutíricoRESUMO
Artemisinin and its derivatives kill malaria parasites and inhibit the proliferation of cancer cells. In both processes, heme was shown to play a key role in artemisinin bioactivation. We found that artemisinin and clinical artemisinin derivatives are able to compensate for a mutation in the yeast Bcs1 protein, a key chaperon involved in biogenesis of the mitochondrial respiratory complex III. The equivalent Bcs1 variant causes an encephalopathy in human by affecting complex III assembly. We show that artemisinin derivatives decrease the content of mitochondrial cytochromes and disturb the maturation of the complex III cytochrome c1. This last effect is likely responsible for the compensation by decreasing the detrimental over-accumulation of the inactive pre-complex III observed in the bcs1 mutant. We further show that a fluorescent dihydroartemisinin probe rapidly accumulates in the mitochondrial network and targets cytochromes c and c1 in yeast, human cells and isolated mitochondria. In vitro this probe interacts with purified cytochrome c only under reducing conditions and we detect cytochrome c-dihydroartemisinin covalent adducts by mass spectrometry analyses. We propose that reduced mitochondrial c-type cytochromes act as both targets and mediators of artemisinin bioactivation in yeast and human cells.
Assuntos
Artemisininas/farmacologia , Citocromos c/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Artemisininas/química , Regulação para Baixo , Complexo III da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Many pathogens (virus, bacteria, fungi, or parasites) have developed a wide variety of mechanisms to evade their host immune system. The budding yeast Saccharomyces cerevisiae has successfully been used to decipher some of these immune evasion strategies. This includes the cis-acting mechanism that limits the expression of the oncogenic Epstein-Barr virus (EBV)-encoded EBNA1 and thus of antigenic peptides derived from this essential but highly antigenic viral protein. Studies based on budding yeast have also revealed the molecular bases of epigenetic switching or recombination underlying the silencing of all except one members of extended families of genes that encode closely related and highly antigenic surface proteins. This mechanism is exploited by several parasites (that include pathogens such as Plasmodium, Trypanosoma, Candida, or Pneumocystis) to alternate their surface antigens, thereby evading the immune system. Yeast can itself be a pathogen, and pathogenic fungi such as Candida albicans, which is phylogenetically very close to S. cerevisiae, have developed stealthiness strategies that include changes in their cell wall composition, or epitope-masking, to control production or exposure of highly antigenic but essential polysaccharides in their cell wall. Finally, due to the high antigenicity of its cell wall, yeast has been opportunistically exploited to create adjuvants and vectors for vaccination.
Assuntos
Evasão da Resposta Imune/genética , Imunidade/genética , Saccharomyces cerevisiae/genética , Transgenes , Animais , Doenças Transmissíveis/genética , Doenças Transmissíveis/imunologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Saccharomyces cerevisiae/imunologiaRESUMO
The oncogenic Epstein-Barr virus (EBV) evades the immune system through limiting the expression of its highly antigenic and essential genome maintenance protein, EBNA1, to the minimal level to ensure viral genome replication, thereby also minimizing the production of EBNA1-derived antigenic peptides. This regulation is based on inhibition of translation of the virally-encoded EBNA1 mRNA, and involves the interaction of host protein nucleolin (NCL) with G-quadruplex (G4) structures that form in the glycine-alanine repeat (GAr)-encoding sequence of the EBNA1 mRNA. Ligands that bind to these G4-RNA can prevent their interaction with NCL, leading to disinhibition of EBNA1 expression and antigen presentation, thereby interfering with the immune evasion of EBNA1 and therefore of EBV (M.J. Lista et al., Nature Commun., 2017, 8, 16043). In this work, we synthesized and studied a series of 20 cationic bis(acylhydrazone) derivatives designed as G4 ligands. The in vitro evaluation showed that most derivatives based on central pyridine (Py), naphthyridine (Naph) or phenanthroline (Phen) units were efficient G4 binders, in contrast to their pyrimidine (Pym) counterparts, which were poor G4 binders due to a significantly different molecular geometry. The influence of lateral heterocyclic units (N-substituted pyridinium or quinolinium residues) on G4-binding properties was also investigated. Two novel compounds, namely PyDH2 and PhenDH2, used at a 5⯵M concentration, were able to significantly enhance EBNA1 expression in H1299â¯cells in a GAr-dependent manner, while being significantly less toxic than the prototype drug PhenDC3 (GI50â¯>â¯50⯵M). Antigen presentation, RNA pull-down and proximity ligation assays confirmed that the effect of both drugs was related to the disruption of NCL-EBNA1 mRNA interaction and the subsequent promotion of GAr-restricted antigen presentation. Our work provides a novel modular scaffold for the development of G-quadruplex-targeting drugs acting through interference with G4-protein interaction.
Assuntos
Hidrazonas/farmacologia , Evasão da Resposta Imune/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Quadruplex G , Herpesvirus Humano 4/genética , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Ligantes , Camundongos , RNA Mensageiro/genética , NucleolinaRESUMO
Mitochondria continually move, fuse and divide, and these dynamics are essential for the proper function of the organelles. Indeed, the dynamic balance of fusion and fission of mitochondria determines their morphology and allows their immediate adaptation to energetic needs as well as preserving their integrity. As a consequence, mitochondrial fusion and fission dynamics and the proteins that control these processes, which are conserved from yeast to human, are essential, and their disturbances are associated with severe human disorders, including neurodegenerative diseases. For example, mutations in OPA1, which encodes a conserved factor essential for mitochondrial fusion, lead to optic atrophy 1, a neurodegeneration that affects the optic nerve, eventually leading to blindness. Here, by screening a collection of â¼1600 repurposed drugs on a fission yeast model, we identified five compounds able to efficiently prevent the lethality associated with the loss of Msp1p, the fission yeast ortholog of OPA1. One compound, hexestrol, was able to rescue both the mitochondrial fragmentation and mitochondrial DNA (mtDNA) depletion induced by the loss of Msp1p, whereas the second, clomifene, only suppressed the mtDNA defect. Yeast has already been successfully used to identify candidate drugs to treat inherited mitochondrial diseases; this work may therefore provide useful leads for the treatment of optic atrophies such as optic atrophy 1 or Leber hereditary optic neuropathy.
Assuntos
DNA Mitocondrial/metabolismo , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Dinâmica Mitocondrial , Schizosaccharomyces/metabolismo , Clomifeno/farmacologia , Hexestrol/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Domínios Proteicos , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Identifying dosage-sensitive genes is a key to understand the mechanisms underlying intellectual disability in Down syndrome (DS). The Dp(17Abcg1-Cbs)1Yah DS mouse model (Dp1Yah) shows cognitive phenotypes that need to be investigated to identify the main genetic driver. Here, we report that three copies of the cystathionine-beta-synthase gene (Cbs) in the Dp1Yah mice are necessary to observe a deficit in the novel object recognition (NOR) paradigm. Moreover, the overexpression of Cbs alone is sufficient to induce deficits in the NOR test. Accordingly, overexpressing human CBS specifically in Camk2a-expressing neurons leads to impaired objects discrimination. Altogether, this shows that Cbs overdosage is involved in DS learning and memory phenotypes. To go further, we identified compounds that interfere with the phenotypical consequence of CBS overdosage in yeast. Pharmacological intervention in Tg(CBS) mice with one selected compound restored memory in the NOR test. In addition, using a genetic approach, we demonstrated an epistatic interaction between Cbs and Dyrk1a, another human chromosome 21-located gene (which encodes the dual-specificity tyrosine phosphorylation-regulated kinase 1a) and an already known target for DS therapeutic intervention. Further analysis using proteomic approaches highlighted several molecular pathways, including synaptic transmission, cell projection morphogenesis and actin cytoskeleton, that are affected by DYRK1A and CBS overexpression. Overall, we demonstrated that CBS overdosage underpins the DS-related recognition memory deficit and that both CBS and DYRK1A interact to control accurate memory processes in DS. In addition, our study establishes CBS as an intervention point for treating intellectual deficiencies linked to DS.
Assuntos
Cistationina beta-Sintase/genética , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Epistasia Genética , Dosagem de Genes , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Animais , Comportamento Animal , Cognição , Modelos Animais de Doenças , Humanos , Locomoção , Memória , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Proteoma , Proteômica/métodos , Quinases DyrkRESUMO
Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.
Assuntos
Antígenos/genética , Antígenos de Histocompatibilidade Classe I/genética , Peptídeos/genética , Biossíntese de Proteínas/genética , Antígenos/imunologia , Núcleo Celular/genética , Núcleo Celular/imunologia , Quadruplex G , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , Peptídeos/imunologia , Biossíntese de Proteínas/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologiaRESUMO
Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.