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1.
Proc Natl Acad Sci U S A ; 121(40): e2403646121, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39298469

RESUMO

Seed dormancy corresponds to a reversible blockage of germination. Primary dormancy is established during seed maturation, while secondary dormancy is set up on the dispersed seed, following an exposure to unfavorable factors. Both dormancies are relieved in response to environmental factors, such as light, nitrate, and coldness. Quantitive Trait Locus (QTL) analyses for preharvest sprouting identified MKK3 kinase in cereals as a player in dormancy control. Here, we showed that MKK3 also plays a role in secondary dormancy in Arabidopsis within a signaling module composed of MAP3K13/14/19/20, MKK3, and clade-C MAPKs. Seeds impaired in this module acquired heat-induced secondary dormancy more rapidly than wild-type (WT) seeds, and this dormancy is less sensitive to nitrate, a signal able to release dormancy. We also demonstrated that MPK7 was strongly activated in the seed during dormancy release, especially in response to light and nitrate. This activation was greatly reduced in map3k13/14/19/20 and mkk3 mutants. Finally, we showed that the module was not regulated and apparently did not regulate the genes controlling abscisic acid/gibberellin acid hormone balance, one of the crucial mechanisms of seed dormancy control. Overall, our work identified a MAPK module controlling seed germination and enlarged the panel of functions of the MKK3-related modules in plants.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Germinação , MAP Quinase Quinase 3 , Nitratos , Dormência de Plantas , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Germinação/genética , Luz , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 3/genética , Nitratos/metabolismo , Dormência de Plantas/genética , Sementes/crescimento & desenvolvimento , Sementes/genética , Transdução de Sinais
2.
Nucleic Acids Res ; 51(20): 11239-11257, 2023 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-37811881

RESUMO

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 , Humanos
3.
Nucleic Acids Res ; 50(20): 11799-11819, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36350639

RESUMO

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/metabolismo
4.
Nucleic Acids Res ; 50(17): 10110-10122, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36107769

RESUMO

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 , Nucleolina
5.
J Inherit Metab Dis ; 45(5): 996-1012, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35621276

RESUMO

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/metabolismo
6.
Hum Mol Genet ; 28(9): 1561-1577, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649339

RESUMO

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 Dyrk
7.
Nucleic Acids Res ; 47(6): 3086-3100, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30624716

RESUMO

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/imunologia
8.
Environ Microbiol ; 22(6): 1997-2000, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32342578

RESUMO

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-2
9.
Int J Mol Sci ; 21(12)2020 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-32630605

RESUMO

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 Miniatura
10.
Molecules ; 23(12)2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30501034

RESUMO

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.


Assuntos
Bioensaio/métodos , Quadruplex G , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Aminoquinolinas/química , Linhagem Celular Tumoral , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Humanos , Ácidos Picolínicos/química , Nucleolina
11.
J Pathol ; 235(2): 334-41, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25186125

RESUMO

The EBV-encoded EBNA1 was first discovered 40 years ago, approximately 10 years after the presence of EBV had been demonstrated in Burkitt's lymphoma cells. It took another 10 years before the functions of EBNA1 in maintaining the viral genome were revealed, and it has since been shown to be an essential viral factor expressed in all EBV-carrying cells. Apart from serving to maintain the viral episome and to control viral replication and gene expression, EBNA1 also harbours a cis-acting mechanism that allows virus-carrying host cells to evade the immune system. This relates to a particular glycine-alanine repeat (GAr) within EBNA1 that has the capacity to suppress antigen presentation to the major histocompatibility complex (MHC) class I pathway. We discuss the role of the GAr sequence at the level of mRNA translation initiation, rather than at the protein level, as at least part of the mechanism to avoid MHC presentation. Interfering with this mechanism has become the focus of the development of immune-based therapies against EBV-carrying cancers, and some lead compounds that affect translation of GAr-carrying mRNAs have been identified. In addition, we describe the EBV-encoded ZEBRA factor and the switch from the latent to the lytic cycle as an alternative virus-specific target for treating EBV-carrying cancers. Understanding the molecular mechanisms of how EBNA1 and ZEBRA interfere with cellular pathways not only opens new therapeutic approaches but continues to reveal new cell-biological insights on the interplay between host and virus. This review is a tale of discoveries relating to how EBNA1 and ZEBRA have emerged as targets for specific cancer therapies against EBV-carrying diseases, and serves as an illustration of how mRNA translation can play roles in future immune-based strategies to target viral disease.


Assuntos
Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Neoplasias/virologia , Transativadores/metabolismo , Animais , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/terapia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , Terapia Genética/métodos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Transativadores/genética , Virulência
12.
Biochim Biophys Acta ; 1843(10): 2315-21, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24583118

RESUMO

Cross-complementation studies offer the possibility to overcome limitations imposed by the inherent complexity of multicellular organisms in the study of human diseases, by taking advantage of simpler model organisms like the budding yeast Saccharomyces cerevisiae. This review deals with, (1) the use of S. cerevisiae as a model organism to study human diseases, (2) yeast-based screening systems for the detection of disease modifiers, (3) Hailey-Hailey as an example of a calcium-related disease, and (4) the presentation of a yeast-based model to search for chemical modifiers of Hailey-Hailey disease. The preliminary experimental data presented and discussed here show that it is possible to use yeast as a model system for Hailey-Hailey disease and suggest that in all likelihood, yeast has the potential to reveal candidate drugs for the treatment of this disorder. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.


Assuntos
ATPases Transportadoras de Cálcio/genética , Cálcio/metabolismo , Modelos Biológicos , Chaperonas Moleculares/genética , Pênfigo Familiar Benigno/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Administração Cutânea , Sinalização do Cálcio , ATPases Transportadoras de Cálcio/deficiência , Fármacos Dermatológicos/farmacologia , Di-Hidroxicolecalciferóis/farmacologia , Expressão Gênica , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Mutação , Pênfigo Familiar Benigno/tratamento farmacológico , Pênfigo Familiar Benigno/metabolismo , Pênfigo Familiar Benigno/patologia , Saccharomyces cerevisiae/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia
13.
J Biol Chem ; 288(26): 19081-9, 2013 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-23673663

RESUMO

Domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active center for the protein folding activity of the ribosome (PFAR). Using in vitro transcribed domain V rRNAs from Escherichia coli and Saccharomyces cerevisiae as the folding modulators and human carbonic anhydrase as a model protein, we demonstrate that PFAR is conserved from prokaryotes to eukaryotes. It was shown previously that 6-aminophenanthridine (6AP), an antiprion compound, inhibits PFAR. Here, using UV cross-linking followed by primer extension, we show that the protein substrates and 6AP interact with a common set of nucleotides on domain V of 23S rRNA. Mutations at the interaction sites decreased PFAR and resulted in loss or change of the binding pattern for both the protein substrates and 6AP. Moreover, kinetic analysis of human carbonic anhydrase refolding showed that 6AP decreased the yield of the refolded protein but did not affect the rate of refolding. Thus, we conclude that 6AP competitively occludes the protein substrates from binding to rRNA and thereby inhibits PFAR. Finally, we propose a scheme clarifying the mechanism by which 6AP inhibits PFAR.


Assuntos
Fenantridinas/farmacologia , Príons/química , Dobramento de Proteína/efeitos dos fármacos , Ribossomos/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Anidrases Carbônicas/química , Escherichia coli/metabolismo , Humanos , Chaperonas Moleculares/química , Dados de Sequência Molecular , Mutagênese , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Desnaturação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Ribossômico/química , Homologia de Sequência de Aminoácidos
14.
Proc Natl Acad Sci U S A ; 108(29): 11989-94, 2011 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-21715656

RESUMO

Due to the lack of relevant animal models, development of effective treatments for human mitochondrial diseases has been limited. Here we establish a rapid, yeast-based assay to screen for drugs active against human inherited mitochondrial diseases affecting ATP synthase, in particular NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome. This method is based on the conservation of mitochondrial function from yeast to human, on the unique ability of yeast to survive without production of ATP by oxidative phosphorylation, and on the amenability of the yeast mitochondrial genome to site-directed mutagenesis. Our method identifies chlorhexidine by screening a chemical library and oleate through a candidate approach. We show that these molecules rescue a number of phenotypes resulting from mutations affecting ATP synthase in yeast. These compounds are also active on human cybrid cells derived from NARP patients. These results validate our method as an effective high-throughput screening approach to identify drugs active in the treatment of human ATP synthase disorders and suggest that this type of method could be applied to other mitochondrial diseases.


Assuntos
Clorexidina/farmacologia , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Miopatias Mitocondriais/tratamento farmacológico , ATPases Mitocondriais Próton-Translocadoras/genética , Ácido Oleico/farmacologia , Retinose Pigmentar/tratamento farmacológico , Linhagem Celular , Clorexidina/uso terapêutico , Perfilação da Expressão Gênica , Humanos , Mutagênese Sítio-Dirigida , Mutação/genética , Ácido Oleico/uso terapêutico , Saccharomycetales
15.
Med Sci (Paris) ; 30(12): 1161-8, 2014 Dec.
Artigo em Francês | MEDLINE | ID: mdl-25537047

RESUMO

Since its discovery and description by Louis Pasteur, the budding yeast Saccharomyces cerevisiae, which was used for thousands of years for alcoholic fermentation and as a leavening agent, has become a popular model system in biology. One of the reasons for this popularity is the strong conservation from yeast to human of most of the pathways controlling cell growth and fate. In addition, at least 30 % of human genes involved in diseases have a functional homolog in yeast. Hence, yeast is now widely used for modelling and deciphering physiopathological mechanisms as well as for developing pharmacological approaches like phenotype-based drug screening. Three examples of such yeast-based chemobiological studies are presented.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Biológicos , Saccharomyces cerevisiae , Animais , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/imunologia , Humanos , Camundongos , Doenças Mitocondriais/tratamento farmacológico , Miopatias Mitocondriais/tratamento farmacológico , Fenótipo , Doenças Priônicas/tratamento farmacológico , Retinose Pigmentar/tratamento farmacológico , Saccharomyces cerevisiae/genética
16.
Vaccine ; 42(26): 126385, 2024 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-39326211

RESUMO

Mastitis is a major issue for the dairy industry. Despite multiple attempts, the efficacy of available mastitis vaccines is limited and this has been attributed to their incapacity to trigger robust cell-mediated immunity. Yeasts have recently been identified as promising antigen vectors capable of inducing T-cell responses, surpassing the antibody-biased mechanisms elicited by conventional adjuvanted vaccines. In this study, we combine in vitro, ex vivo, and in vivo approaches to evaluate the potential of the yeast Saccharomyces cerevisiae as a platform for novel vaccines against bovine mastitis. We demonstrate that S. cerevisiae is safe for intramuscular and intramammary immunisation in dairy cows. Vaccination resulted in a significant increase of IFNγ and IL-17 responses against the yeast platform but not against the vaccine antigen. These observations highlight that strategies to counterbalance the immunodominance of S. cerevisiae antigens are necessary for the development of successful vaccine candidates.

17.
Genome Biol ; 25(1): 210, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-39107855

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 , Éxons
18.
Biochimie ; 214(Pt A): 57-68, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37473831

RESUMO

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 Imunitário
19.
Life Sci Alliance ; 5(2)2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34785537

RESUMO

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 Viral
20.
Biochim Biophys Acta ; 1797(6-7): 1105-12, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20056103

RESUMO

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F1, Atp9p-ring, unassembled alpha-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.


Assuntos
DNA Mitocondrial/genética , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/biossíntese , Substituição de Aminoácidos , Sequência de Bases , Primers do DNA/genética , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Humanos , Técnicas In Vitro , Cinética , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/química , Mutagênese Sítio-Dirigida , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Oligomicinas/farmacologia , Consumo de Oxigênio , Subunidades Proteicas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/química
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