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1.
Nature ; 619(7971): 811-818, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37407817

RESUMO

RNA viruses have evolved elaborate strategies to protect their genomes, including 5' capping. However, until now no RNA 5' cap has been identified for hepatitis C virus1,2 (HCV), which causes chronic infection, liver cirrhosis and cancer3. Here we demonstrate that the cellular metabolite flavin adenine dinucleotide (FAD) is used as a non-canonical initiating nucleotide by the viral RNA-dependent RNA polymerase, resulting in a 5'-FAD cap on the HCV RNA. The HCV FAD-capping frequency is around 75%, which is the highest observed for any RNA metabolite cap across all kingdoms of life4-8. FAD capping is conserved among HCV isolates for the replication-intermediate negative strand and partially for the positive strand. It is also observed in vivo on HCV RNA isolated from patient samples and from the liver and serum of a human liver chimeric mouse model. Furthermore, we show that 5'-FAD capping protects RNA from RIG-I mediated innate immune recognition but does not stabilize the HCV RNA. These results establish capping with cellular metabolites as a novel viral RNA-capping strategy, which could be used by other viruses and affect anti-viral treatment outcomes and persistence of infection.


Assuntos
Flavina-Adenina Dinucleotídeo , Hepacivirus , Capuzes de RNA , RNA Viral , Animais , Humanos , Camundongos , Quimera/virologia , Flavina-Adenina Dinucleotídeo/metabolismo , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/virologia , Reconhecimento da Imunidade Inata , Fígado/virologia , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , RNA Viral/imunologia , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral/genética , Capuzes de RNA/metabolismo
2.
EMBO Rep ; 25(4): 1814-1834, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38413733

RESUMO

Stress granules are an integral part of the stress response that are formed from non-translating mRNAs aggregated with proteins. While much is known about stress granules, the factors that drive their mRNA localization are incompletely described. Modification of mRNA can alter the properties of the nucleobases and affect processes such as translation, splicing and localization of individual transcripts. Here, we show that the RNA modification N4-acetylcytidine (ac4C) on mRNA associates with transcripts enriched in stress granules and that stress granule localized transcripts with ac4C are specifically translationally regulated. We also show that ac4C on mRNA can mediate localization of the protein NOP58 to stress granules. Our results suggest that acetylation of mRNA regulates localization of both stress-sensitive transcripts and RNA-binding proteins to stress granules and adds to our understanding of the molecular mechanisms responsible for stress granule formation.


Assuntos
Citidina , Citidina/análogos & derivados , Grânulos de Estresse , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Citidina/genética , Citidina/metabolismo , Proteínas de Ligação a RNA/metabolismo
3.
Nucleic Acids Res ; 52(2): 784-800, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38000394

RESUMO

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.


Assuntos
Proteína de Replicação A , Uracila-DNA Glicosidase , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Switching de Imunoglobulina/genética , Isotipos de Imunoglobulinas/genética , Imunoglobulinas/genética , Mutagênicos , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Uracila/metabolismo , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo , Humanos , Animais , Camundongos
4.
EMBO J ; 38(1)2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30523148

RESUMO

During active DNA demethylation, 5-methylcytosine (5mC) is oxidized by TET proteins to 5-formyl-/5-carboxylcytosine (5fC/5caC) for replacement by unmethylated C by TDG-initiated DNA base excision repair (BER). Base excision generates fragile abasic sites (AP-sites) in DNA and has to be coordinated with subsequent repair steps to limit accumulation of genome destabilizing secondary DNA lesions. Here, we show that 5fC/5caC is generated at a high rate in genomes of differentiating mouse embryonic stem cells and that SUMOylation and the BER protein XRCC1 play critical roles in orchestrating TDG-initiated BER of these lesions. SUMOylation of XRCC1 facilitates physical interaction with TDG and promotes the assembly of a TDG-BER core complex. Within this TDG-BERosome, SUMO is transferred from XRCC1 and coupled to the SUMO acceptor lysine in TDG, promoting its dissociation while assuring the engagement of the BER machinery to complete demethylation. Although well-studied, the biological importance of TDG SUMOylation has remained obscure. Here, we demonstrate that SUMOylation of TDG suppresses DNA strand-break accumulation and toxicity to PARP inhibition in differentiating mESCs and is essential for neural lineage commitment.


Assuntos
Diferenciação Celular/genética , Desmetilação do DNA , Reparo do DNA/fisiologia , Células-Tronco Embrionárias/fisiologia , Sumoilação/fisiologia , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , 5-Metilcitosina/metabolismo , Animais , Células Cultivadas , Citosina/análogos & derivados , Citosina/metabolismo , Humanos , Camundongos , Complexos Multiproteicos/metabolismo , Multimerização Proteica/fisiologia
5.
Free Radic Biol Med ; 213: 470-487, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38301978

RESUMO

The NTHL1 and NEIL1-3 DNA glycosylases are major enzymes in the removal of oxidative DNA base lesions, via the base excision repair (BER) pathway. It is expected that lack of these DNA glycosylases activities would render cells vulnerable to oxidative stress, promoting cell death. Intriguingly, we found that single, double, triple, and quadruple DNA glycosylase knockout HAP1 cells are, however, more resistant to oxidative stress caused by genotoxic agents than wild type cells. Furthermore, glutathione depletion in NEIL deficient cells further enhances resistance to cell death induced via apoptosis and ferroptosis. Finally, we observed higher basal level of glutathione and differential expression of NRF2-regulated genes associated with glutathione homeostasis in the NEIL triple KO cells. We propose that lack of NEIL DNA glycosylases causes aberrant transcription and subsequent errors in protein synthesis. This leads to increased endoplasmic reticulum stress and proteotoxic stress. To counteract the elevated intracellular stress, an adaptive response mediated by increased glutathione basal levels, rises in these cells. This study reveals an unforeseen role of NEIL glycosylases in regulation of resistance to oxidative stress, suggesting that modulation of NEIL glycosylase activities is a potential approach to improve the efficacy of e.g. anti-inflammatory therapies.


Assuntos
DNA Glicosilases , Reparo do DNA , Reparo do DNA/genética , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Estresse Oxidativo/genética , Dano ao DNA/genética , Apoptose/genética
6.
Front Immunol ; 14: 1111123, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36776851

RESUMO

Introduction: Inhalation of nanomaterials may induce inflammation in the lung which if left unresolved can manifest in pulmonary fibrosis. In these processes, alveolar macrophages have an essential role and timely modulation of the macrophage phenotype is imperative in the onset and resolution of inflammatory responses. This study aimed to investigate, the immunomodulating properties of two industrially relevant high aspect ratio nanomaterials, namely nanocellulose and multiwalled carbon nanotubes (MWCNT), in an alveolar macrophage model. Methods: MH-S alveolar macrophages were exposed at air-liquid interface to cellulose nanocrystals (CNC), cellulose nanofibers (CNF) and two MWCNT (NM-400 and NM-401). Following exposure, changes in macrophage polarization markers and secretion of inflammatory cytokines were analyzed. Furthermore, the potential contribution of epigenetic regulation in nanomaterial-induced macrophage polarization was investigated by assessing changes in epigenetic regulatory enzymes, miRNAs, and rRNA modifications. Results: Our data illustrate that the investigated nanomaterials trigger phenotypic changes in alveolar macrophages, where CNF exposure leads to enhanced M1 phenotype and MWCNT promotes M2 phenotype. Furthermore, MWCNT exposure induced more prominent epigenetic regulatory events with changes in the expression of histone modification and DNA methylation enzymes as well as in miRNA transcript levels. MWCNT-enhanced changes in the macrophage phenotype were correlated with prominent downregulation of the histone methyltransferases Kmt2a and Smyd5 and histone deacetylases Hdac4, Hdac9 and Sirt1 indicating that both histone methylation and acetylation events may be critical in the Th2 responses to MWCNT. Furthermore, MWCNT as well as CNF exposure led to altered miRNA levels, where miR-155-5p, miR-16-1-3p, miR-25-3p, and miR-27a-5p were significantly regulated by both materials. PANTHER pathway analysis of the identified miRNA targets showed that both materials affected growth factor (PDGF, EGF and FGF), Ras/MAPKs, CCKR, GnRH-R, integrin, and endothelin signaling pathways. These pathways are important in inflammation or in the activation, polarization, migration, and regulation of phagocytic capacity of macrophages. In addition, pathways involved in interleukin, WNT and TGFB signaling were highly enriched following MWCNT exposure. Conclusion: Together, these data support the importance of macrophage phenotypic changes in the onset and resolution of inflammation and identify epigenetic patterns in macrophages which may be critical in nanomaterial-induced inflammation and fibrosis.


Assuntos
MicroRNAs , Nanotubos de Carbono , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Nanotubos de Carbono/toxicidade , Nanotubos de Carbono/química , Epigênese Genética , Macrófagos/metabolismo , Inflamação/metabolismo , Celulose/metabolismo
7.
Genome Biol ; 24(1): 216, 2023 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-37773136

RESUMO

BACKGROUND: Oxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency. RESULTS: We report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. CONCLUSIONS: This study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.


Assuntos
Cerebelo , Histonas , Proteínas Mitocondriais , Doenças Neurodegenerativas , Humanos , Arginina/genética , Arginina/metabolismo , Atrofia , Histonas/metabolismo , Metilação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Cerebelo/patologia
8.
BMC Genomics ; 12: 423, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21854623

RESUMO

BACKGROUND: Phloem-feeding aphids deprive plants of assimilates, but mostly manage to avoid causing the mechanical tissue damage inflicted by chewing insects. Nevertheless, jasmonate signalling that is induced by infestation is important in mediating resistance to phloem feeders. Aphid attack induces the jasmonic acid signalling pathway, but very little is known about the specific impact jasmonates have on the expression of genes that respond to aphid attack. RESULTS: We have evaluated the function that jasmonates have in regulating Arabidopsis thaliana responses to cabbage aphid (Brevicoryne brassicae) by conducting a large-scale transcriptional analysis of two mutants: aos, which is defective in jasmonate production, and fou2, which constitutively induces jasmonic acid biosynthesis. This analysis enabled us to determine which genes' expression patterns depend on the jasmonic acid signalling pathway. We identified more than 200 genes whose expression in non-challenged plants depended on jasmonate levels and more than 800 genes that responded differently to infestation in aos and fou2 plants than in wt. Several aphid-induced changes were compromised in the aos mutant, particularly genes connected to regulation of transcription, defence responses and redox changes. Due to jasmonate-triggered pre-activation of fou2, its transcriptional profile in non-challenged plants mimicked the induction of defence responses in wt. Additional activation of fou2 upon aphid attack was therefore limited. Insect fitness experiments revealed that the physiological consequences of fou2 mutation contributed to more effective protection against B. brassicae. However, the observed resistance of the fou2 mutant was based on antibiotic rather than feeding deterrent properties of the mutant as indicated by an analysis of aphid feeding behaviour. CONCLUSIONS: Analysis of transcriptional profiles of wt, aos and fou2 plants revealed that the expression of more than 200 genes is dependent on jasmonate status, regardless of external stimuli. Moreover, the aphid-induced response of more than 800 transcripts is regulated by jasmonate signalling. Thus, in plants lacking jasmonates many of the defence-related responses induced by infestation in wt plants are impaired. Constant up-regulation of jasmonate signalling as evident in the fou2 mutant causes reduction in aphid population growth, likely as a result of antibiotic properties of fou2 plants. However, aos mutation does not seem to affect aphid performance when the density of B. brassicae populations on plants is low and aphids are free to move around.


Assuntos
Afídeos/fisiologia , Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Transdução de Sinais , Animais , Arabidopsis/genética , Comportamento Alimentar , Perfilação da Expressão Gênica , Herbivoria , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Transcriptoma
9.
Commun Biol ; 4(1): 1354, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34857879

RESUMO

Oxidative DNA damage in the brain has been implicated in neurodegeneration and cognitive decline. DNA glycosylases initiate base excision repair (BER), the main pathway for oxidative DNA base lesion repair. NEIL1 and NEIL3 DNA glycosylases affect cognition in mice, while the role of NEIL2 remains unclear. Here, we investigate the impact of NEIL2 and its potential overlap with NEIL1 on behavior in knockout mouse models. Neil1-/-Neil2-/- mice display hyperactivity, reduced anxiety and improved learning. Hippocampal oxidative DNA base lesion levels are comparable between genotypes and no mutator phenotype is found. Thus, impaired canonical repair is not likely to explain the altered behavior. Electrophysiology suggests reduced axonal activation in the hippocampal CA1 region in Neil1-/-Neil2-/- mice and lack of NEIL1 and NEIL2 causes dysregulation of genes in CA1 relevant for synaptic function. We postulate a cooperative function of NEIL1 and NEIL2 in genome regulation, beyond canonical BER, modulating behavior in mice.


Assuntos
Ansiedade/genética , DNA Glicosilases/genética , Aprendizagem , Camundongos/psicologia , Animais , DNA Glicosilases/metabolismo , Regulação da Expressão Gênica , Hipocampo/fisiologia , Masculino , Camundongos/genética , Camundongos Knockout , Estresse Oxidativo/fisiologia
10.
F1000Res ; 9: 1112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33082936

RESUMO

Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). The loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a tested and well-controlled genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.


Assuntos
Timina DNA Glicosilase , Animais , Metilação de DNA , Reparo do DNA , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Camundongos , Timina DNA Glicosilase/genética , Timina DNA Glicosilase/metabolismo
11.
J Immunother Cancer ; 8(1)2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32409420

RESUMO

BACKGROUND: PD1/PDL1-directed therapies have been unsuccessful for multiple myeloma (MM), an incurable cancer of plasma cells in the bone marrow (BM). Therefore, other immune checkpoints such as extracellular adenosine and its immunosuppressive receptor should be considered. CD39 and CD73 convert extracellular ATP to adenosine, which inhibits T-cell effector functions via the adenosine receptor A2A (A2AR). We set out to investigate whether blocking the adenosine pathway could be a therapy for MM. METHODS: Expression of CD39 and CD73 on BM cells from patients and T-cell proliferation were determined by flow cytometry and adenosine production by Liquid chromatograpy-mass spectrometry (HPCL/MS). ENTPD1 (CD39) mRNA expression was determined on myeloma cells from patients enrolled in the publicly available CoMMpass study. Transplantable 5T33MM myeloma cells were used to determine the effect of inhibiting CD39, CD73 and A2AR in mice in vivo. RESULTS: Elevated level of adenosine was found in BM plasma of MM patients. Myeloma cells from patients expressed CD39, and high gene expression indicated reduced survival. CD73 was found on leukocytes and stromal cells in the BM. A CD39 inhibitor, POM-1, and an anti-CD73 antibody inhibited adenosine production and reduced T-cell suppression in vitro in coculture of myeloma and stromal cells. Blocking the adenosine pathway in vivo with a combination of Sodium polyoxotungstate (POM-1), anti-CD73, and the A2AR antagonist AZD4635 activated immune cells, increased interferon gamma production, and reduced the tumor load in a murine model of MM. CONCLUSIONS: Our data suggest that the adenosine pathway can be successfully targeted in MM and blocking this pathway could be an alternative to PD1/PDL1 inhibition for MM and other hematological cancers. Inhibitors of the adenosine pathway are available. Some are in clinical trials and they could thus reach MM patients fairly rapidly.


Assuntos
5'-Nucleotidase/metabolismo , Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Mieloma Múltiplo/patologia , Receptor A2A de Adenosina/química , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Prognóstico , Receptor A2A de Adenosina/metabolismo , Taxa de Sobrevida
12.
Sci Rep ; 9(1): 11065, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363131

RESUMO

In most mammalian cells, DNA replication occurs once, and only once between cell divisions. Replication initiation is a highly regulated process with redundant mechanisms that prevent errant initiation events. In lower eukaryotes, replication is initiated from a defined consensus sequence, whereas a consensus sequence delineating mammalian origin of replication has not been identified. Here we show that 5-hydroxymethylcytosine (5hmC) is present at mammalian replication origins. Our data support the hypothesis that 5hmC has a role in cell cycle regulation. We show that 5hmC level is inversely proportional to proliferation; indeed, 5hmC negatively influences cell division by increasing the time a cell resides in G1. Our data suggest that 5hmC recruits replication-licensing factors, then is removed prior to or during origin firing. Later we propose that TET2, the enzyme catalyzing 5mC to 5hmC conversion, acts as barrier to rereplication. In a broader context, our results significantly advance the understating of 5hmC involvement in cell proliferation and disease states.


Assuntos
5-Metilcitosina/análogos & derivados , Ciclo Celular/genética , Divisão Celular/fisiologia , Proliferação de Células/fisiologia , Replicação do DNA/fisiologia , 5-Metilcitosina/metabolismo , Células HeLa , Humanos , Origem de Replicação
13.
Plant Cell Environ ; 31(8): 1097-115, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18433442

RESUMO

Insect feeding on plants causes a complex series of coordinated defence responses. Little is known, however, about the time-dependent aspect of induced changes. Here we present a time series-based investigation of Arabidopsis thaliana Ler subjected to attack by a specialist pest of Brassicaceae species, Brevicoryne brassicae. Transcriptome and metabolome changes were studied at 6, 12, 24 and 48 h after infestation to monitor the progress of early induced responses. The use of full-genome oligonucleotide microarrays revealed the initiation of extensive gene expression changes already during the first 6 h of infestation. Data indicated the involvement of reactive oxygen species (ROS) and calcium in early signalling, and salicylic acid (SA) and jasmonic acid (JA) in the regulation of defence responses. Transcripts related to senescence, biosynthesis of anti-insect proteins, indolyl glucosinolates (GS) and camalexin, as well as several uncharacterized to date WRKY transcription factors, were induced. Follow-up studies of defence-involved secondary metabolites revealed depositions of callose at the insects' feeding sites, a decrease in the total level of aliphatic GS, particularly 3-hydroxypropyl glucosinolate, and accumulation of 4-methoxyindol-3-ylmethyl glucosinolate 48 h after the attack. The novel role of camalexin, induced as a part of defence against aphids, was verified in fitness experiments. Fecundity of B. brassicae was reduced on camalexin-accumulating wild-type (WT) plants as compared with camalexin-deficient pad3-1 mutants. Based on experimental data, a model of plant-aphid interactions at the early phase of infestation was proposed.


Assuntos
Afídeos/fisiologia , Arabidopsis/imunologia , Arabidopsis/parasitologia , Brassica/parasitologia , Animais , Afídeos/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Parede Celular/parasitologia , Ciclopentanos/farmacologia , Etilenos/farmacologia , Fertilidade/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Glucosinolatos/metabolismo , Peróxido de Hidrogênio/metabolismo , Indóis/metabolismo , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Oxilipinas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/parasitologia , Ácido Salicílico/farmacologia , Tiazóis/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos
14.
Biomol NMR Assign ; 12(1): 15-22, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28879561

RESUMO

Human uracil N-glycosylase isoform 2-UNG2 consists of an N-terminal intrinsically disordered regulatory domain (UNG2 residues 1-92, 9.3 kDa) and a C-terminal structured catalytic domain (UNG2 residues 93-313, 25.1 kDa). Here, we report the backbone 1H, 13C, and 15N chemical shift assignment as well as secondary structure analysis of the N-and C-terminal domains of UNG2 representing the full-length UNG2 protein.


Assuntos
DNA Glicosilases/química , Ressonância Magnética Nuclear Biomolecular , DNA Glicosilases/metabolismo , Humanos , Domínios Proteicos , Estrutura Secundária de Proteína
15.
DNA Repair (Amst) ; 61: 56-62, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29207315

RESUMO

Mitochondrial DNA (mtDNA) resides in close proximity to metabolic reactions, and is maintained by the 8-oxoguanine DNA glycosylase (Ogg1) and other members of the base excision repair pathway. Here, we tested the hypothesis that changes in liver metabolism as under fasting/feeding conditions would be sensed by liver mtDNA, and that Ogg1 deficient mice might unravel a metabolic phenotype. Wild type (WT) and ogg1-/- mice were either fed ad libitum or subjected to fasting for 24h, and the corresponding effects on liver gene expression, DNA damage, as well as serum values were analyzed. Ogg1 deficient mice fed ad libitum exhibited hyperglycemia, elevated insulin levels and higher liver glycogen content as well as increased accumulation of 8oxoG in mtDNA compared to age- and gender matched WT mice. Interestingly, these phenotypes were absent in ogg1-/- mice during fasting. Gene expression and functional analyses suggest that the diabetogenic phenotype in the ogg1-/- mice is due to a failure to suppress gluconeogensis in the fed state. The ogg1-/- mice exhibited reduced mitochondrial electron transport chain (ETC) capacity and a combined low activity of the pyruvate dehydrogenase (PDH), alluding to inefficient channeling of glycolytic products into the citric acid cycle. Our data demonstrate a physiological role of base excision repair that goes beyond DNA maintenance, and implies that DNA repair is involved in regulating metabolism.


Assuntos
DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Gluconeogênese , Fígado/metabolismo , Animais , Dano ao DNA , Reparo do DNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Jejum/metabolismo , Feminino , Glucose/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ativação Transcricional
16.
Sci Rep ; 8(1): 9817, 2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29959348

RESUMO

Huntington's disease (HD) is a progressive neurodegenerative disorder primarily affecting the basal ganglia and is caused by expanded CAG repeats in the huntingtin gene. Except for CAG sizing, mitochondrial and nuclear DNA (mtDNA and nDNA) parameters have not yet proven to be representative biomarkers for disease and future therapy. Here, we identified a general suppression of genes associated with aerobic metabolism in peripheral blood mononuclear cells (PBMCs) from HD patients compared to controls. In HD, the complex II subunit SDHB was lowered although not sufficiently to affect complex II activity. Nevertheless, we found decreased level of factors associated with mitochondrial biogenesis and an associated dampening of the mitochondrial DNA damage frequency in HD, implying an early defect in mitochondrial activity. In contrast to mtDNA, nDNA from HD patients was four-fold more modified than controls and demonstrated that nDNA integrity is severely reduced in HD. Interestingly, the level of nDNA damage correlated inversely with the total functional capacity (TFC) score; an established functional score of HD. Our data show that PBMCs are a promising source to monitor HD progression and highlights nDNA damage and diverging mitochondrial and nuclear genome responses representing early cellular impairments in HD.


Assuntos
Dano ao DNA , DNA Mitocondrial/análise , Instabilidade Genômica , Doença de Huntington/patologia , Leucócitos Mononucleares/patologia , Mitocôndrias/patologia , Adulto , Idoso , Estudos de Casos e Controles , DNA Mitocondrial/genética , Feminino , Humanos , Doença de Huntington/genética , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Adulto Jovem
17.
Dis Model Mech ; 11(10)2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30254085

RESUMO

Huntington's disease (HD) is a monogenic, progressive, neurodegenerative disorder with currently no available treatment. The Libechov transgenic minipig model for HD (TgHD) displays neuroanatomical similarities to humans and exhibits slow disease progression, and is therefore more powerful than available mouse models for the development of therapy. The phenotypic characterization of this model is still ongoing, and it is essential to validate biomarkers to monitor disease progression and intervention. In this study, the behavioral phenotype (cognitive, motor and behavior) of the TgHD model was assessed, along with biomarkers for mitochondrial capacity, oxidative stress, DNA integrity and DNA repair at different ages (24, 36 and 48 months), and compared with age-matched controls. The TgHD minipigs showed progressive accumulation of the mutant huntingtin (mHTT) fragment in brain tissue and exhibited locomotor functional decline at 48 months. Interestingly, this neuropathology progressed without any significant age-dependent changes in any of the other biomarkers assessed. Rather, we observed genotype-specific effects on mitochondrial DNA (mtDNA) damage, mtDNA copy number, 8-oxoguanine DNA glycosylase activity and global level of the epigenetic marker 5-methylcytosine that we believe is indicative of a metabolic alteration that manifests in progressive neuropathology. Peripheral blood mononuclear cells (PBMCs) were relatively spared in the TgHD minipig, probably due to the lack of detectable mHTT. Our data demonstrate that neuropathology in the TgHD model has an age of onset of 48 months, and that oxidative damage and electron transport chain impairment represent later states of the disease that are not optimal for assessing interventions.This article has an associated First Person interview with the first author of the paper.


Assuntos
Comportamento Animal , Doença de Huntington/patologia , 8-Hidroxi-2'-Desoxiguanosina , Animais , Animais Geneticamente Modificados , Dano ao DNA , Reparo do DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Genoma , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Mitocôndrias/metabolismo , Degeneração Neural/patologia , Especificidade de Órgãos , Suínos , Porco Miniatura
18.
Front Mol Biosci ; 5: 1, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29417049

RESUMO

The cellular prion protein PrPC is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrPC. Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrPC have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrPC (PRNPTer/Ter) compared with cells from normal (PRNP+/+) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO4 and H2O2. Surprisingly, PrPC-negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H2O2 and methyl methanesulfonate (MMS), revealed no effect of PrPC on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrPC. RNA sequencing of PBMCs (n = 8 of PRNP+/+ and PRNPTer/Ter) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrPC. Data presented here questions the in vitro cytoprotective roles previously attributed to PrPC, although not excluding such functions in other cell types or tissues during inflammatory stress.

19.
Sci Rep ; 7(1): 4384, 2017 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-28663564

RESUMO

Base excision repair (BER) is a major pathway for removal of DNA base lesions and maintenance of genomic stability, which is essential in cancer prevention. DNA glycosylases recognize and remove specific lesions in the first step of BER. The existence of a number of these enzymes with overlapping substrate specificities has been thought to be the reason why single knock-out models of individual DNA glycosylases are not cancer prone. In this work we have characterized DNA glycosylases NEIL1 and NEIL2 (Neil1 -/- /Neil2 -/-) double and NEIL1, NEIL2 and NEIL3 (Neil1 -/- /Neil2 -/- /Neil3 -/-) triple knock-out mouse models. Unexpectedly, our results show that these mice are not prone to cancer and have no elevated mutation frequencies under normal physiological conditions. Moreover, telomere length is not affected and there was no accumulation of oxidative DNA damage compared to wild-type mice. These results strengthen the hypothesis that the NEIL enzymes are not simply back-up enzymes for each other but enzymes that have distinct functions beyond canonical repair.


Assuntos
DNA Glicosilases/deficiência , Predisposição Genética para Doença , Taxa de Mutação , Mutação , Neoplasias/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Estudos de Associação Genética , Loci Gênicos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Knockout , Família Multigênica , Neoplasias/metabolismo , Neoplasias/patologia , Dicromato de Potássio/farmacologia
20.
Cell Rep ; 18(1): 82-92, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28052262

RESUMO

Myocardial infarction (MI) triggers a reparative response involving fibroblast proliferation and differentiation driving extracellular matrix modulation necessary to form a stabilizing scar. Recently, it was shown that a genetic variant of the base excision repair enzyme NEIL3 was associated with increased risk of MI in humans. Here, we report elevated myocardial NEIL3 expression in heart failure patients and marked myocardial upregulation of Neil3 after MI in mice, especially in a fibroblast-enriched cell fraction. Neil3-/- mice show increased mortality after MI caused by myocardial rupture. Genome-wide analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) reveals changes in the cardiac epigenome, including in genes related to the post-MI transcriptional response. Differentially methylated genes are enriched in pathways related to proliferation and myofibroblast differentiation. Accordingly, Neil3-/- ruptured hearts show increased proliferation of fibroblasts and myofibroblasts. We propose that NEIL3-dependent modulation of DNA methylation regulates cardiac fibroblast proliferation and thereby affects extracellular matrix modulation after MI.


Assuntos
Endodesoxirribonucleases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Miocárdio/metabolismo , Miocárdio/patologia , N-Glicosil Hidrolases/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Proliferação de Células , Colágeno/metabolismo , Doenças do Tecido Conjuntivo/genética , Doenças do Tecido Conjuntivo/patologia , Dano ao DNA , Metilação de DNA/genética , Endodesoxirribonucleases/deficiência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Coração Auxiliar , Humanos , Leucócitos/patologia , Metaloproteinase 2 da Matriz/metabolismo , Infarto do Miocárdio/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Oxirredução , Fenótipo , Análise de Sequência de RNA , Análise de Sobrevida , Fatores de Tempo
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