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1.
FASEB J ; 34(3): 3969-3982, 2020 Mar.
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.

2.
Sci Rep ; 10(1): 871, 2020 Jan 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.

3.
Haematologica ; 2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919083

RESUMO

The regulation of protein function by reversible oxidation is increasingly recognized as a key mechanism for the control of cellular signaling, modulating crucial biological processes such as cell differentiation. In this scenario, NADPH oxidases must occupy a prominent position. Our results show that hematopoietic stem and progenitor cells express three p22phox-dependent NADPH oxidases members (NOX1, NOX2 and NOX4). By deleting the p22phox coding gene (Cyba), here we have analyzed the importance of this family of enzymes during in vivo hematopoiesis. Cyba-/- mice show a myeloid bias, and an enrichment of hematopoietic stem cell populations. By means of hematopoietic transplant experiments we have also tried to dissect the specific role of the NADPH oxidases. While the absence of NOX1 or NOX2 provides a higher level of reconstitution, a lack of NOX4 rendered the opposite result, suggesting a functional specificity among the different NADPH oxidases. Cyba-/- cells showed a hampered activation of AKT1 and a sharp decrease in STAT5 protein. This is in line with the diminished response to IL-7 shown by our results, which could explain the overproduction of immunoglobulins observed in Cyba-/- mice.

4.
Mol Biol Rep ; 47(2): 1381-1391, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31833031

RESUMO

The Nomo1 gene mediates a wide range of biological processes of importance in embryonic development. Accordingly, constitutive perturbation of Nomo1 function may result in myriad developmental defects that trigger embryonic lethality. To extend our understanding of Nomo1 function in postnatal stages and in a tissue-specific manner, we generated a conditional knockout mouse model of Nomo1. To achieve this, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology in C57Bl/6J mouse zygotes to generate a new mouse model in which exon 3 of the Nomo1 gene is specifically flanked (or floxed) by LoxP sites (Nomo1f/f). Nomo1f/f mouse embryonic fibroblasts were transduced with a Cre adenovirus and efficiently recombined between LoxP sites. Genomic and expression studies in Nomo1-transduced MEFs demonstrated that the Nomo1 exon 3 is ablated. Western blot assay showed that no protein or early truncated protein is produced. In vivo assay crossing Nomo1f/f mouse with a Msi1-CRE transgenic mouse corroborated the previous findings and it showed Nomo1 exon 3 deletion at msi1+ cell compartment. This short technical report demonstrates that CRISPR/Cas9 technology is a simple and easy method for creating conditional mouse models. The Nomo1f/f mouse will be useful to researchers who wish to explore the role of Nomo1 in any developmental stage or in a tissue-specific manner.

5.
PLoS Genet ; 15(8): e1008316, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31437213

RESUMO

The ubiquitin proteasome system regulates meiotic recombination in yeast through its association with the synaptonemal complex, a 'zipper'-like structure that holds homologous chromosome pairs in synapsis during meiotic prophase I. In mammals, the proteasome activator subunit PA200 targets acetylated histones for degradation during somatic DNA double strand break repair and during histone replacement during spermiogenesis. We investigated the role of the testis-specific proteasomal subunit α4s (PSMA8) during spermatogenesis, and found that PSMA8 was localized to and dependent on the central region of the synaptonemal complex. Accordingly, synapsis-deficient mice show delocalization of PSMA8. Moreover, though Psma8-deficient mice are proficient in meiotic homologous recombination, there are alterations in the proteostasis of several key meiotic players that, in addition to the known substrate acetylated histones, have been shown by a proteomic approach to interact with PSMA8, such as SYCP3, SYCP1, CDK1 and TRIP13. These alterations lead to an accumulation of spermatocytes in metaphase I and II which either enter massively into apoptosis or give rise to a low number of aberrant round spermatids that apoptose before histone replacement takes place.


Assuntos
Fertilidade/genética , Infertilidade Masculina/genética , Metáfase/genética , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Animais , Apoptose/genética , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Complexo Sinaptonêmico/metabolismo , Testículo/citologia , Testículo/metabolismo
6.
PLoS One ; 14(5): e0216674, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31071190

RESUMO

CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes/métodos , Sítios de Splice de RNA/genética , RNA Guia/genética , Alelos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Éxons , Edição de Genes/métodos , Humanos , Células K562 , Camundongos , Monofenol Mono-Oxigenase/genética , Proteínas Proto-Oncogênicas c-abl/genética
7.
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
8.
Chromosoma ; 128(3): 237-247, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30887115

RESUMO

Ubiquitin-specific protease 26 (USP26) is a deubiquitylating enzyme belonging to the USPs family with a transcription pattern restricted to the male germline. Since protein ubiquitination is an essential regulatory mechanism during meiosis, many efforts have been focused on elucidating the function of USP26 and its relationship with fertility. During the last decade, several studies have reported the presence of different polymorphisms in USP26 in patients with non-obstructive azoospermia (NOA) or severe oligozoospermia suggesting that this gene may be associated with human infertility. However, other studies have revealed the presence of these and novel polymorphisms, including nonsense mutations, in men with normal spermatogenesis as well. Thus, the results remain controversial and its function is unknown. In the present study, we describe the in vivo functional analysis of mice lacking USP26. The phenotypic analysis of two different Usp26-null mutants showed no overt-phenotype with both males and females being fertile. Cytological analysis of spermatocytes showed no defects in synapsis, chromosome dynamics, DNA repair, or recombination. Histopathological analysis revealed a normal distribution and number of the different cell types in both male and female mice. Finally, normal counts were observed in fertility assessments. These results represent the first in vivo evidence showing that USP26 is not essential for mouse gametogenesis.

10.
Acta Neuropathol ; 135(2): 179-199, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29094186

RESUMO

Prion diseases are caused by a misfolding of the cellular prion protein (PrP) to a pathogenic isoform named PrPSc. Prions exist as strains, which are characterized by specific pathological and biochemical properties likely encoded in the three-dimensional structure of PrPSc. However, whether cofactors determine these different PrPSc conformations and how this relates to their specific biological properties is largely unknown. To understand how different cofactors modulate prion strain generation and selection, Protein Misfolding Cyclic Amplification was used to create a diversity of infectious recombinant prion strains by propagation in the presence of brain homogenate. Brain homogenate is known to contain these mentioned cofactors, whose identity is only partially known, and which facilitate conversion of PrPC to PrPSc. We thus obtained a mix of distinguishable infectious prion strains. Subsequently, we replaced brain homogenate, by different polyanionic cofactors that were able to drive the evolution of mixed prion populations toward specific strains. Thus, our results show that a variety of infectious recombinant prions can be generated in vitro and that their specific type of conformation, i.e., the strain, is dependent on the cofactors available during the propagation process. These observations have significant implications for understanding the pathogenesis of prion diseases and their ability to replicate in different tissues and hosts. Importantly, these considerations might apply to other neurodegenerative diseases for which different conformations of misfolded proteins have been described.


Assuntos
Encéfalo/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Animais , Arvicolinae , Encéfalo/patologia , Escherichia coli , Camundongos Transgênicos , Polimorfismo Genético , Proteínas Priônicas/genética , Dobramento de Proteína , Proteínas Recombinantes/metabolismo
11.
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
12.
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
13.
Oncotarget ; 8(16): 26027-26040, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-28212528

RESUMO

CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line.CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients.


Assuntos
Sistemas CRISPR-Cas , Transformação Celular Neoplásica/genética , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Edição de Genes , Marcação de Genes , Genes Reporter , Xenoenxertos , Humanos , Camundongos , Mutação , Carga Tumoral/genética
14.
Nat Commun ; 7: 13298, 2016 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-27796301

RESUMO

Meiotic recombination generates crossovers between homologous chromosomes that are essential for genome haploidization. The synaptonemal complex is a 'zipper'-like protein assembly that synapses homologue pairs together and provides the structural framework for processing recombination sites into crossovers. Humans show individual differences in the number of crossovers generated across the genome. Recently, an anonymous gene variant in C14ORF39/SIX6OS1 was identified that influences the recombination rate in humans. Here we show that C14ORF39/SIX6OS1 encodes a component of the central element of the synaptonemal complex. Yeast two-hybrid analysis reveals that SIX6OS1 interacts with the well-established protein synaptonemal complex central element 1 (SYCE1). Mice lacking SIX6OS1 are defective in chromosome synapsis at meiotic prophase I, which provokes an arrest at the pachytene-like stage and results in infertility. In accordance with its role as a modifier of the human recombination rate, SIX6OS1 is essential for the appropriate processing of intermediate recombination nodules before crossover formation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fertilidade , Complexo Sinaptonêmico/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Pareamento Cromossômico , Troca Genética , Proteínas de Ligação a DNA , Eletroporação , Feminino , Variação Genética , Genoma , Células HEK293 , Haploidia , Humanos , Masculino , Meiose , Camundongos , Proteínas Nucleares/metabolismo , Recombinação Genética , Testículo/patologia , Transcrição Genética , Técnicas do Sistema de Duplo-Híbrido
15.
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
16.
Hum Mol Genet ; 23(13): 3421-31, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24608227

RESUMO

Oligo- and azoospermia are severe forms of male infertility. However, known genetic factors account only for a small fraction of the cases. Recently, whole-exome sequencing in a large consanguineous family with inherited premature ovarian failure (POF) identified a homozygous frameshift mutation in the STAG3 gene leading to a premature stop codon. STAG3 encodes a meiosis-specific subunit of the cohesin complex, a large proteinaceous ring with DNA-entrapping ability that ensures sister chromatid cohesion and enables correct synapsis and segregation of homologous chromosomes during meiosis. The pathogenicity of the STAG3 mutations was functionally validated with a loss-of-function mouse model for STAG3 in oogenesis. However, and since none of the male members of this family was homozygous for the mutant allele, we only could hypothesized its putative involvement in male infertility. In this report, we show that male mice devoid of Stag3 display a severe meiotic phenotype that includes a meiotic arrest at zygonema-like shortening of their chromosome axial elements/lateral elements, partial loss of centromeric cohesion at early prophase and maintenance of the ability to initiate but not complete RAD51- and DMC1-mediated double-strand break repair, demonstrating that STAG3 is a crucial cohesin subunit in mammalian gametogenesis and supporting our proposal that STAG3 is a strong candidate gene for human male infertility.


Assuntos
Infertilidade Masculina/genética , Proteínas Nucleares/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Feminino , Masculino , Meiose/genética , Meiose/fisiologia , Camundongos , Proteínas Nucleares/genética , Proteínas de Ligação a Fosfato , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Complexo Sinaptonêmico/metabolismo
17.
J Cell Biol ; 197(7): 877-85, 2012 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-22711701

RESUMO

Cohesin is a conserved multisubunit protein complex that participates in chromosome segregation, DNA damage repair, chromatin regulation, and synaptonemal complex (SC) formation. Yeast, but not mice, depleted of the cohesin subunit Rec8 are defective in the formation of the axial elements (AEs) of the SC, suggesting that, in mammals, this function is not conserved. In this paper, we show that spermatocytes from mice lacking the two meiosis-specific cohesin subunits RAD21L and REC8 were unable to initiate RAD51- but not DMC1-mediated double-strand break repair, were not able to assemble their AEs, and arrested as early as the leptotene stage of prophase I, demonstrating that cohesin plays an essential role in AE assembly that is conserved from yeast to mammals.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Meiose , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Fosfoproteínas/deficiência , Ligação Proteica
18.
EMBO J ; 30(15): 3091-105, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21743440

RESUMO

The cohesin complex is a ring-shaped proteinaceous structure that entraps the two sister chromatids after replication until the onset of anaphase when the ring is opened by proteolytic cleavage of its α-kleisin subunit (RAD21 at mitosis and REC8 at meiosis) by separase. RAD21L is a recently identified α-kleisin that is present from fish to mammals and biochemically interacts with the cohesin subunits SMC1, SMC3 and STAG3. RAD21L localizes along the axial elements of the synaptonemal complex of mouse meiocytes. However, its existence as a bona fide cohesin and its functional role awaits in vivo validation. Here, we show that male mice lacking RAD21L are defective in full synapsis of homologous chromosomes at meiotic prophase I, which provokes an arrest at zygotene and leads to total azoospermia and consequently infertility. In contrast, RAD21L-deficient females are fertile but develop an age-dependent sterility. Thus, our results provide in vivo evidence that RAD21L is essential for male fertility and in females for the maintenance of fertility during natural aging.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Meiose , Fatores Etários , Animais , Proteínas Cromossômicas não Histona/deficiência , Cromossomos/metabolismo , Feminino , Histocitoquímica , Infertilidade , Masculino , Camundongos , Camundongos Knockout , Ovário/patologia , Subunidades Proteicas/metabolismo , Fatores Sexuais , Testículo/patologia
19.
Cell Cycle ; 10(9): 1477-87, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21527826

RESUMO

Meiosis is a fundamental process that generates new combinations between maternal and paternal genomes and haploid gametes from diploid progenitors. Many of the meiosis-specific events stem from the behavior of the cohesin complex (CC), a proteinaceous ring structure that entraps sister chromatids until the onset of anaphase. CCs ensure chromosome segregation, participate in DNA repair, regulate gene expression, and also contribute to synaptonemal complex (SC) formation at meiosis by keeping long-range distant DNA interactions through its conserved structure. Studies from yeast to humans have led to the assumption that Scc1/RAD21 is the α-kleisin that closes the tripartite CC that entraps two DNA molecules in mitosis, while its paralog REC8 is essential for meiosis. Here we describe the identification of RAD21L, a novel mammalian CC subunit with homology to the RAD21/REC8 α-kleisin subfamily, which is expressed in mouse testis. RAD21L interacts with other cohesin subunits such as SMC1α, SMC1b, SMC3 and with the meiosis-specific STAG3 protein. Thus, our results demonstrate the existence of a new meiotic-specific CC constituted by this α-kleisin and expand the view of REC8 as the only specific meiotic α-kleisin.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , Meiose , Proteínas Nucleares/química , Fosfoproteínas/química , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Sequência Conservada , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Masculino , Meiose/genética , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosfoproteínas/genética , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Testículo/química , Testículo/fisiologia
20.
BMC Cancer ; 10: 454, 2010 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-20735813

RESUMO

BACKGROUND: Gliomas are the most common type of primary brain tumours, and in this group glioblastomas (GBMs) are the higher-grade gliomas with fast progression and unfortunate prognosis. Two major aspects of glioma biology that contributes to its awful prognosis are the formation of new blood vessels through the process of angiogenesis and the invasion of glioma cells. Despite of advances, two-year survival for GBM patients with optimal therapy is less than 30%. Even in those patients with low-grade gliomas, that imply a moderately good prognosis, treatment is almost never curative. Recent studies have demonstrated the existence of a small fraction of glioma cells with characteristics of neural stem cells which are able to grow in vitro forming neurospheres and that can be isolated in vivo using surface markers such as CD133. The aim of this study was to define the molecular signature of GBM cells expressing CD133 in comparison with non expressing CD133 cells. This molecular classification could lead to the finding of new potential therapeutic targets for the rationale treatment of high grade GBM. METHODS: Eight fresh, primary and non cultured GBMs were used in order to study the gene expression signatures from its CD133 positive and negative populations isolated by FACS-sorting. Dataset was generated with Affymetrix U133 Plus 2 arrays and analysed using the software of the Affymetrix Expression Console. In addition, genomic analysis of these tumours was carried out by CGH arrays, FISH studies and MLPA; RESULTS: Gene expression analysis of CD133+ vs. CD133- cell population from each tumour showed that CD133+ cells presented common characteristics in all glioblastoma samples (up-regulation of genes involved in angiogenesis, permeability and down-regulation of genes implicated in cell assembly, neural cell organization and neurological disorders). Furthermore, unsupervised clustering of gene expression led us to distinguish between two groups of samples: those discriminated by tumour location and, the most importantly, the group discriminated by their proliferative potential; CONCLUSIONS: Primary glioblastomas could be sub-classified according to the properties of their CD133+ cells. The molecular characterization of these potential stem cell populations could be critical to find new therapeutic targets and to develop an effective therapy for these tumours with very dismal prognosis.


Assuntos
Antígenos CD/genética , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Glicoproteínas/genética , Peptídeos/genética , Antígeno AC133 , Idoso , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Hibridização Genômica Comparativa , Feminino , Citometria de Fluxo , Glioblastoma/metabolismo , Glicoproteínas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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