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1.
Mol Cancer ; 22(1): 119, 2023 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-37516825

RESUMO

Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology.


Assuntos
Carcinogênese , Neoplasias da Próstata , Masculino , Humanos , Carcinogênese/genética , Transformação Celular Neoplásica , Neoplasias da Próstata/genética , Transcrição Gênica , Processamento Pós-Transcricional do RNA , Metiltransferases/genética
2.
Proc Natl Acad Sci U S A ; 117(48): 30509-30519, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33199643

RESUMO

Vertebrate Hox genes are critical for the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here, we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of the HoxC gene cluster, which together regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail.


Assuntos
Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Folículo Piloso/metabolismo , Unhas/metabolismo , Animais , Biomarcadores , Ectoderma/embriologia , Folículo Piloso/embriologia , Humanos , Camundongos , Camundongos Knockout , Unhas/embriologia
3.
FASEB J ; 34(3): 3969-3982, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31944411

RESUMO

Unlike other species, prion disease has never been described in dogs even though they were similarly exposed to the bovine spongiform encephalopathy (BSE) agent. This resistance prompted a thorough analysis of the canine PRNP gene and the presence of a negatively charged amino acid residue in position 163 was readily identified as potentially fundamental as it differed from all known susceptible species. In the present study, the first transgenic mouse model expressing dog prion protein (PrP) was generated and challenged intracerebrally with a panel of prion isolates, none of which could infect them. The brains of these mice were subjected to in vitro prion amplification and failed to find even minimal amounts of misfolded prions providing definitive experimental evidence that dogs are resistant to prion disease. Subsequently, a second transgenic model was generated in which aspartic acid in position 163 was substituted for asparagine (the most common in prion susceptible species) resulting in susceptibility to BSE-derived isolates. These findings strongly support the hypothesis that the amino acid residue at position 163 of canine cellular prion protein (PrPC ) is a major determinant of the exceptional resistance of the canidae family to prion infection and establish this as a promising therapeutic target for prion diseases.


Assuntos
Ácido Aspártico/química , Ácido Glutâmico/química , Príons/química , Príons/patogenicidade , Animais , Bioensaio , Encéfalo/patologia , Cães , Camundongos , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
4.
PLoS Pathog ; 13(11): e1006716, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29131852

RESUMO

One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different in vitro and in vivo models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the Leporidae and Equidae families. Here we undertake in vitro and in vivo approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis in silico, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel transgenic mouse model carrying this substitution and these mice showed complete resistance to disease despite intracerebral challenge with three different mouse prion strains (RML, 22L and 301C) known to cause disease in wild-type mice. These findings suggest that dog D163 amino acid is primarily, if not totally, responsible for the prion resistance of canids.


Assuntos
Canidae/imunologia , Proteínas PrPC/química , Doenças Priônicas/veterinária , Sequência de Aminoácidos , Animais , Antílopes , Encéfalo/patologia , Gatos , Bovinos , Quirópteros , Cervos , Resistência à Doença , Cães , Encefalopatia Espongiforme Bovina/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas PrPC/ultraestrutura , Doenças Priônicas/imunologia , Dobramento de Proteína , Estrutura Quaternária de Proteína , Coelhos , Alinhamento de Sequência , Ovinos , Eletricidade Estática , Xenarthra
5.
PLoS Pathog ; 11(8): e1004977, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26247589

RESUMO

Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrPC was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrPd) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrPd originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits.


Assuntos
Modelos Animais de Doenças , Suscetibilidade a Doenças , Doenças Priônicas/transmissão , Príons , Animais , Transmissão de Doença Infecciosa , Camundongos , Camundongos Transgênicos , Coelhos
6.
Oncogene ; 42(43): 3169-3181, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37660182

RESUMO

Tumour progression and therapy tolerance are highly regulated and complex processes largely dependent on the plasticity of cancer cells and their capacity to respond to stress. The higher plasticity of cancer cells highlights the need for identifying targetable molecular pathways that challenge cancer cell survival. Here, we show that N7-guanosine methylation (m7G) of tRNAs, mediated by METTL1, regulates survival to stress conditions in cancer cells. Mechanistically, we find that m7G in tRNAs protects them from stress-induced cleavage and processing into 5' tRNA fragments. Our analyses reveal that the loss of tRNA m7G methylation activates stress response pathways, sensitising cancer cells to stress. Furthermore, we find that the loss of METTL1 reduces tumour growth and increases cytotoxic stress in vivo. Our study uncovers the role of m7G methylation of tRNAs in stress responses and highlights the potential of targeting METTL1 to sensitise cancer cells to chemotherapy.

7.
Front Cell Dev Biol ; 10: 1026287, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36393850

RESUMO

C3G is a Rap1 guanine nucleotide exchange factor that controls platelet activation, aggregation, and the release of α-granule content. Transgenic expression of C3G in platelets produces a net proangiogenic secretome through the retention of thrombospondin-1. In a physiological context, C3G also promotes megakaryocyte maturation and proplatelet formation, but without affecting mature platelet production. The aim of this work is to investigate whether C3G is involved in pathological megakaryopoiesis, as well as its specific role in platelet mediated angiogenesis and tumor metastasis. Using megakaryocyte-specific C3G knockout and transgenic mouse models, we found that both C3G overexpression and deletion promoted platelet-mediated angiogenesis, induced by tumor cell implantation or hindlimb ischemia, through differential release of proangiogenic and antiangiogenic factors. However, only C3G deletion resulted in a higher recruitment of hemangiocytes from the bone marrow. In addition, C3G null expression enhanced thrombopoietin (TPO)-induced platelet production, associated with reduced TPO plasma levels. Moreover, after 5-fluorouracil-induced platelet depletion and rebound, C3G knockout mice showed a defective return to homeostatic platelet levels, indicating impaired platelet turnover. Mechanistically, C3G promotes c-Mpl ubiquitination by inducing Src-mediated c-Cbl phosphorylation and participates in c-Mpl degradation via the proteasome and lysosome systems, affecting TPO internalization. We also unveiled a positive role of platelet C3G in tumor cell-induced platelet aggregation, which facilitated metastatic cell homing and adhesion. Overall, these findings revealed that C3G plays a crucial role in platelet-mediated angiogenesis and metastasis, as well as in platelet level modulation in response to pathogenic stimuli.

8.
Cell Death Differ ; 29(12): 2347-2361, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35624308

RESUMO

Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B -(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability.


Assuntos
Proteínas de Ligação a DNA , Insuficiência Ovariana Primária , Animais , Feminino , Humanos , Camundongos , Aberrações Cromossômicas , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Meiose , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/metabolismo
9.
Acta Neuropathol Commun ; 10(1): 179, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36514160

RESUMO

Atypical Scrapie, which is not linked to epidemics, is assumed to be an idiopathic spontaneous prion disease in small ruminants. Therefore, its occurrence is unlikely to be controlled through selective breeding or other strategies as it is done for classical scrapie outbreaks. Its spontaneous nature and its sporadic incidence worldwide is reminiscent of the incidence of idiopathic spontaneous prion diseases in humans, which account for more than 85% of the cases in humans. Hence, developing animal models that consistently reproduce this phenomenon of spontaneous PrP misfolding, is of importance to study the pathobiology of idiopathic spontaneous prion disorders. Transgenic mice overexpressing sheep PrPC with I112 polymorphism (TgShI112, 1-2 × PrP levels compared to sheep brain) manifest clinical signs of a spongiform encephalopathy spontaneously as early as 380 days of age. The brains of these animals show the neuropathological hallmarks of prion disease and biochemical analyses of the misfolded prion protein show a ladder-like PrPres pattern with a predominant 7-10 kDa band. Brain homogenates from spontaneously diseased transgenic mice were inoculated in several models to assess their transmissibility and characterize the prion strain generated: TgShI112 (ovine I112 ARQ PrPC), Tg338 (ovine VRQ PrPC), Tg501 (ovine ARQ PrPC), Tg340 (human M129 PrPC), Tg361 (human V129 PrPC), TgVole (bank vole I109 PrPC), bank vole (I109I PrPC), and sheep (AHQ/ARR and AHQ/AHQ churra-tensina breeds). Our analysis of the results of these bioassays concludes that the strain generated in this model is indistinguishable to that causing atypical scrapie (Nor98). Thus, we present the first faithful model for a bona fide, transmissible, ovine, atypical scrapie prion disease.


Assuntos
Doenças Priônicas , Príons , Scrapie , Camundongos , Animais , Ovinos , Humanos , Scrapie/metabolismo , Roedores/metabolismo , Príons/metabolismo , Camundongos Transgênicos , Arvicolinae/metabolismo
10.
Sci Rep ; 10(1): 871, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31965006

RESUMO

Hydrogen peroxide (H2O2) is generated in cells and plays an important role as a signalling molecule. It has been reported that H2O2 is involved in physiological and pathological processes in skeletal muscle. However, H2O2 detection in cells with traditional techniques produces frequent artefacts. Currently, the HyPer biosensor detects intracellular H2O2 specifically in real time using fluorescence microscopy. The aim of this study was to develop and optimize approaches used to express the HyPer biosensor in different models of skeletal muscle cells, such as the C2C12 myoblast/myotube cell line and mature skeletal muscle fibres isolated from C57BL/6J mice, and to measure intracellular H2O2 in real time in these cells. The results show that the expression of the HyPer biosensor in skeletal muscle cells is possible. In addition, we demonstrate that HyPer is functional and that this biosensor detects changes and fluctuations in intracellular H2O2 in a reversible manner. The HyPer2 biosensor, which is a more advanced version of HyPer, presents improved properties in terms of sensitivity in detecting lower concentrations of H2O2 in skeletal muscle fibres. In conclusion, the expression of the HyPer biosensor in the different experimental models combined with fluorescence microscopy techniques is a powerful methodology to monitor and register intracellular H2O2 specifically in skeletal muscle. The innovation of the methodological approaches presented in this study may present new avenues for studying the role of H2O2 in skeletal muscle pathophysiology. Furthermore, the methodology may potentially be adapted to yield other specific biosensors for different reactive oxygen and nitrogen species or metabolites involved in cellular functions.


Assuntos
Técnicas Biossensoriais/métodos , Peróxido de Hidrogênio/análise , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Proteínas Recombinantes/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteínas Recombinantes/metabolismo
11.
Mol Neurobiol ; 56(9): 6501-6511, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30847740

RESUMO

Specific variations in the amino acid sequence of prion protein (PrP) are key determinants of susceptibility to prion diseases. We previously showed that an amino acid substitution specific to canids confers resistance to prion diseases when expressed in mice and demonstrated its dominant-negative protective effect against a variety of infectious prion strains of different origins and characteristics. Here, we show that expression of this single amino acid change significantly increases survival time in transgenic mice expressing bank vole cellular prion protein (PrPC), which is inherently prone to misfolding, following inoculation with two distinct prion strains (the CWD-vole strain and an atypical strain of spontaneous origin). This amino acid substitution hinders the propagation of both prion strains, even when expressed in the context of a PrPC uniquely susceptible to a wide range of prion isolates. Non-inoculated mice expressing this substitution experience spontaneous prion formation, but showing an increase in survival time comparable to that observed in mutant mice inoculated with the atypical strain. Our results underscore the importance of this PrP variant in the search for molecules with therapeutic potential against prion diseases.


Assuntos
Substituição de Aminoácidos/genética , Mamíferos/genética , Doenças Priônicas/genética , Príons/metabolismo , Animais , Arvicolinae , Modelos Animais de Doenças , Suscetibilidade a Doenças , Camundongos Transgênicos , Doenças Priônicas/patologia , Análise de Sobrevida
12.
Mol Neurobiol ; 55(7): 6182-6192, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29264770

RESUMO

While prion diseases have been described in numerous species, some, including those of the Canidae family, appear to show resistance or reduced susceptibility. A better understanding of the factors underlying prion susceptibility is crucial for the development of effective treatment and control measures. We recently demonstrated resistance to prion infection in mice overexpressing a mutated prion protein (PrP) carrying a specific amino acid substitution characteristic of canids. Here, we show that coexpression of this mutated PrP and wild-type mouse PrP in transgenic mice inoculated with different mouse-adapted prion strains (22 L, ME7, RML, and 301C) significantly increases survival times (by 45 to 113%). These data indicate that this amino acid substitution confers a dominant-negative effect on PrP, attenuating the conversion of PrPC to PrPSc and delaying disease onset without altering the neuropathological properties of the prion strains. Taken together, these findings have important implications for the development of new treatment approaches for prion diseases based on dominant-negative proteins.


Assuntos
Substituição de Aminoácidos/genética , Genes Dominantes , Predisposição Genética para Doença , Doenças Priônicas/genética , Príons/metabolismo , Animais , Encéfalo/patologia , Camundongos Transgênicos , Doenças Priônicas/patologia , Análise de Sobrevida
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