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1.
bioRxiv ; 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-39005440

RESUMO

Although viruses subvert innate immune pathways for their replication, there is evidence they can also co-opt anti-viral responses for their benefit. The ubiquitous human pathogen, Herpes Simplex Virus-1 (HSV-1), encodes a protein (UL12.5) that induces the release of mitochondrial nucleic acid into the cytosol, which activates immune sensing pathways and reduces productive replication in non-neuronal cells. HSV-1 establishes latency in neurons and can reactivate to cause disease. We found that UL12.5 is required for HSV-1 reactivation in neurons and acts to directly promote viral lytic gene expression during initial exit from latency. Further, the direct activation of innate immune sensing pathways triggered HSV reactivation and compensated for a lack of UL12.5. Finally, we found that the induction of HSV-1 lytic genes during reactivation required intact RNA and DNA sensing pathways, demonstrating that HSV-1 can both respond to and active antiviral nucleic acid sensing pathways to reactivate from a latent infection.

2.
Front Oncol ; 14: 1424895, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38939331

RESUMO

[This corrects the article DOI: 10.3389/fonc.2023.1257622.].

3.
Hum Mol Genet ; 33(R1): R12-R18, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38779775

RESUMO

Mitochondria are subcellular organelles essential for life. Beyond their role in producing energy, mitochondria govern various physiological mechanisms, encompassing energy generation, metabolic processes, apoptotic events, and immune responses. Mitochondria also contain genetic material that is susceptible to various forms of damage. Mitochondrial double-stranded breaks (DSB) are toxic lesions that the nucleus repairs promptly. Nevertheless, the significance of DSB repair in mammalian mitochondria is controversial. This review presents an updated view of the available research on the consequences of mitochondrial DNA DSB from the molecular to the cellular level. We discuss the crucial function of mitochondrial DNA damage in regulating processes such as senescence, integrated stress response, and innate immunity. Lastly, we discuss the potential role of mitochondrial DNA DSB in mediating the cellular consequences of ionizing radiations, the standard of care in treating solid tumors.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Mitocondrial , Mitocôndrias , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Animais , Neoplasias/genética , Neoplasias/patologia , Neoplasias/radioterapia , Imunidade Inata/genética , Dano ao DNA/genética , Radiação Ionizante , Senescência Celular/genética
5.
Haematologica ; 108(12): 3333-3346, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37381763

RESUMO

Long non-coding RNA (lncRNA) are emerging as powerful and versatile regulators of transcriptional programs and distinctive biomarkers of progression of T-cell lymphoma. Their role in the aggressive anaplastic lymphoma kinase-negative (ALK-) subtype of anaplastic large cell lymphoma (ALCL) has been elucidated only in part. Starting from our previously identified ALCL-associated lncRNA signature and performing digital gene expression profiling of a retrospective cohort of ALCL, we defined an 11 lncRNA signature able to discriminate among ALCL subtypes. We selected a not previously characterized lncRNA, MTAAT, with preferential expression in ALK- ALCL, for molecular and functional studies. We demonstrated that lncRNA MTAAT contributes to an aberrant mitochondrial turnover restraining mitophagy and promoting cellular proliferation. Functionally, lncRNA MTAAT acts as a repressor of a set of genes related to mitochondrial quality control via chromatin reorganization. Collectively, our work demonstrates the transcriptional role of lncRNA MTAAT in orchestrating a complex transcriptional program sustaining the progression of ALK- ALCL.


Assuntos
Linfoma Anaplásico de Células Grandes , Linfoma de Células T Periférico , RNA Longo não Codificante , Humanos , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico/genética , RNA Longo não Codificante/genética , Mitofagia/genética , Estudos Retrospectivos , Linfoma Anaplásico de Células Grandes/patologia
6.
Nature ; 591(7850): 477-481, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33627873

RESUMO

Mitochondrial DNA double-strand breaks (mtDSBs) are toxic lesions that compromise the integrity of mitochondrial DNA (mtDNA) and alter mitochondrial function1. Communication between mitochondria and the nucleus is essential to maintain cellular homeostasis; however, the nuclear response to mtDSBs remains unknown2. Here, using mitochondrial-targeted transcription activator-like effector nucleases (TALENs)1,3,4, we show that mtDSBs activate a type-I interferon response that involves the phosphorylation of STAT1 and activation of interferon-stimulated genes. After the formation of breaks in the mtDNA, herniation5 mediated by BAX and BAK releases mitochondrial RNA into the cytoplasm and triggers a RIG-I-MAVS-dependent immune response. We further investigated the effect of mtDSBs on interferon signalling after treatment with ionizing radiation and found a reduction in the activation of interferon-stimulated genes when cells that lack mtDNA are exposed to gamma irradiation. We also show that mtDNA breaks synergize with nuclear DNA damage to mount a robust cellular immune response. Taken together, we conclude that cytoplasmic accumulation of mitochondrial RNA is an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including breaks produced by genotoxic agents.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Mitocondrial/imunologia , Imunidade Inata/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Células Cultivadas , Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA Mitocondrial/efeitos da radiação , Humanos , Mitocôndrias/imunologia , Mitocôndrias/efeitos da radiação , Comunicação Parácrina , Radiação Ionizante , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
7.
Methods Mol Biol ; 2192: 21-34, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230762

RESUMO

Single molecule analysis of replicating DNA (SMARD) is a powerful methodology that allows in vivo analysis of replicating DNA; identification of origins of replication, assessment of fork directionality, and measurement of replication fork speed. SMARD, which has been extensively used to study replication of nuclear DNA, involves incorporation of thymidine analogs to nascent DNA chains and their subsequent visualization through immune detection. Here, we adapt and fine-tune the SMARD technique to the specifics of human and mouse mitochondrial DNA. The mito-SMARD protocol allows researchers to gain in vivo insight into mitochondrial DNA (mtDNA) replication at the single molecule level and with high resolution.


Assuntos
Replicação do DNA/genética , DNA Mitocondrial/metabolismo , Imagem Individual de Molécula/métodos , Animais , Células Cultivadas , DNA Mitocondrial/genética , Genoma Mitocondrial , Humanos , Hibridização in Situ Fluorescente/métodos , Camundongos , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Timidina/análogos & derivados , Timidina/metabolismo
8.
Nat Struct Mol Biol ; 27(8): 687-695, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32764737

RESUMO

Mitochondria respond to DNA damage and preserve their own genetic material in a manner distinct from that of the nucleus but that requires organized mito-nuclear communication. Failure to resolve mtDNA breaks leads to mitochondrial dysfunction and affects host cells and tissues. Here, we review the pathways that safeguard mitochondrial genomes and examine the insights gained from studies of cellular and tissue-wide responses to mtDNA damage and mito-nuclear genome incompatibility.


Assuntos
Dano ao DNA , Reparo do DNA , DNA Mitocondrial/genética , Genoma Mitocondrial , Animais , Eucariotos/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia
9.
Methods Cell Biol ; 155: 401-414, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32183970

RESUMO

DNA combing technology is a powerful methodology for the study of DNA replication in vivo. This tool can be used to identify origins of replication, assess of directionality of forks, and measure fork speed. Over the years, the method has been used extensively to study nuclear DNA replication. The first step involves the incorporation of thymidine analogs (CldU and IdU) into nascent DNA chains and followed by their visualization with immunofluorescence using antibodies that can distinguish the two analogs. Recently, we adapted and fine-tuned DNA combing technology to the specifics of mitochondrial DNA (Phillips et al., 2017, p. 155). The protocol, which we termed mito-SMARD (mitochondrial single molecule analysis of replication DNA), provides in vivo insight into mitochondrial DNA (mtDNA) replication with high resolution.


Assuntos
Replicação do DNA/genética , DNA Mitocondrial/genética , Imagem Individual de Molécula/métodos , Linhagem Celular , DNA Mitocondrial/isolamento & purificação , Fluorescência , Humanos
10.
Mol Cell ; 65(3): 527-538.e6, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28111015

RESUMO

Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the "common deletion" that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5' repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.


Assuntos
Replicação do DNA , DNA Mitocondrial/metabolismo , Deleção de Sequência , Imagem Individual de Molécula/métodos , Envelhecimento/genética , DNA Helicases/genética , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo
11.
Nucleic Acids Res ; 43(17): 8368-80, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26240381

RESUMO

To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae. We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3) and urmylation (e.g., NCS6) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNA(Lys) UUU as well by overexpression of genes (BCK1 and HFM1) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease.


Assuntos
Histona Acetiltransferases/genética , Mitocôndrias/metabolismo , RNA de Transferência de Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Respiração Celular , Códon , Citocromos/química , Citoplasma/metabolismo , Deleção de Genes , Genoma Fúngico , Temperatura Alta , Mitocôndrias/genética , Mutação , Fosforilação Oxidativa , Fenótipo , RNA de Transferência de Lisina/química , Saccharomyces cerevisiae/metabolismo , Uridina/metabolismo
12.
Int J Mol Sci ; 15(3): 4977-93, 2014 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-24658441

RESUMO

Lymphoma and leukemia represent a serious threat to human health and life expectancy. Resveratrol is, among the natural-derived chemopreventive molecules, one of the most effective and better studied. In this paper the main mechanisms of cell death triggered by- or linked to- resveratrol are reviewed and discussed. The main focus is on lymphoma and leukemia experimental models where resveratrol has been tested and investigated at the cellular, molecular or physiological levels. The most relevant in vivo challenges involving resveratrol are also reported and analyzed in order to define the key features of this polyphenol and the potential for the treatment of hematologic tumors.


Assuntos
Apoptose/efeitos dos fármacos , Leucemia/tratamento farmacológico , Linfoma/tratamento farmacológico , Estilbenos/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Leucemia/metabolismo , Leucemia/patologia , Linfoma/metabolismo , Linfoma/patologia , Modelos Biológicos , Resveratrol
13.
Am J Hum Genet ; 94(1): 11-22, 2014 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-24360804

RESUMO

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA.


Assuntos
Encéfalo/efeitos dos fármacos , Exoma , Ferro/metabolismo , Degeneração Neural/patologia , Encéfalo/patologia , Clonagem Molecular , Coenzima A/metabolismo , Escherichia coli/genética , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mutação de Sentido Incorreto , Panteteína/análogos & derivados , Panteteína/metabolismo , Linhagem , Fosforilação , Saccharomyces cerevisiae/genética , Transferases/genética , Transferases/metabolismo
14.
Hum Mutat ; 34(12): 1619-22, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24014394

RESUMO

Mutations in nuclear genes associated with defective complex III (cIII) of the mitochondrial respiratory chain are rare, having been found in only two cIII assembly factors and, as private changes in single families, three cIII structural subunits. Recently, human LYRM7/MZM1L, the ortholog of yeast MZM1, has been identified as a new assembly factor for cIII. In a baby patient with early onset, severe encephalopathy, lactic acidosis and profound, isolated cIII deficiency in skeletal muscle, we identified a disease-segregating homozygous mutation (c.73G>A) in LYRM7/MZM1L, predicting a drastic change in a highly conserved amino-acid residue (p.Asp25Asn). In a mzm1Δ yeast strain, the expression of a mzm1(D25N) mutant allele caused temperature-sensitive respiratory growth defect, decreased oxygen consumption, impaired maturation/stabilization of the Rieske Fe-S protein, and reduced complex III activity and amount. LYRM7/MZM1L is a novel disease gene, causing cIII-defective, early onset, severe mitochondrial encephalopathy.


Assuntos
Acidose Láctica/genética , Acidose Láctica/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Homozigoto , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Mutação , Acidose Láctica/diagnóstico , Sequência de Aminoácidos , Encéfalo/patologia , Análise Mutacional de DNA , Ativação Enzimática , Feminino , Humanos , Lactente , Imageamento por Ressonância Magnética , Encefalomiopatias Mitocondriais/diagnóstico , Proteínas Mitocondriais/química , Chaperonas Moleculares/química , Dados de Sequência Molecular , Linhagem , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
15.
Am J Hum Genet ; 84(5): 594-604, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19409522

RESUMO

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient's muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1(R182H) mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.


Assuntos
Catarata/genética , Redutases do Citocromo/fisiologia , Doenças Mitocondriais/genética , Miopatias Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Adolescente , Catarata/congênito , Criança , Pré-Escolar , Consanguinidade , Redutases do Citocromo/genética , DNA Mitocondrial/genética , DNA Mitocondrial/ultraestrutura , Ligação Genética , Perda Auditiva/genética , Humanos , Membranas Intracelulares/metabolismo , Masculino , Proteínas Mitocondriais/genética , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre
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