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1.
Int J Mol Sci ; 23(15)2022 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-35897775

RESUMO

Cells are constantly exposed to numerous genotoxic stresses that induce DNA damage. DNA double-strand breaks (DSBs) are among the most serious damages and should be systematically repaired to preserve genomic integrity. The efficiency of repair is closely associated with chromatin structure, which is regulated by posttranslational modifications of histones, including ubiquitination. Recent evidence shows crosstalk between histone ubiquitination and DNA damage responses, suggesting an integrated model for the systematic regulation of DNA repair. There are two major pathways for DSB repair, viz., nonhomologous end joining and homologous recombination, and the choice of the pathway is partially controlled by posttranslational modifications of histones, including ubiquitination. Histone ubiquitination changes chromatin structure in the vicinity of DSBs and serves as a platform to select and recruit repair proteins; the removal of these modifications by deubiquitinating enzymes suppresses the recruitment of repair proteins and promotes the convergence of repair reactions. This article provides a comprehensive overview of the DNA damage response regulated by histone ubiquitination in response to DSBs.


Assuntos
Quebras de DNA de Cadeia Dupla , Histonas , Cromatina/genética , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Histonas/metabolismo , Ubiquitinação
2.
Molecules ; 27(9)2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35566260

RESUMO

Retusone A (1), a new sesquiterpene dimer consisting of two guaiane-type sesquiterpenoids, and oleodaphnal (2) were isolated from heartwood of Wikstroemia retusa (Thymelaeaceae). The planar structure of 1 was elucidated on the basis of HRESIMS and NMR spectroscopic data, and the relative stereochemistry was established by X-ray diffraction analysis. The absolute configuration of 1 was determined by electronic circular dichroism. Compound 1 suppressed luciferase reporter gene expression driven by the HBO1 (histone acetyltransferase binding to ORC1) gene promoter in human breast cancer MCF7 cells. Compound 1 also decreased the expression of endogenous HBO1 mRNA and protein, and inhibited proliferation of the cells. These results suggest that retusone A (1), which has a unique dimeric sesquiterpenoid structure with inhibitory activity against HBO1 expression, may contribute to the development of a novel therapeutic candidate for the treatment of breast cancer.


Assuntos
Neoplasias da Mama , Sesquiterpenos , Wikstroemia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Histona Acetiltransferases/genética , Humanos , Estrutura Molecular , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Sesquiterpenos de Guaiano , Wikstroemia/química
3.
Cells ; 10(12)2021 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-34943825

RESUMO

The sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylase and ADP-ribosyl transferases plays key roles in aging, metabolism, stress response, and aging-related diseases. SIRT2 is a unique sirtuin that is expressed in the cytosol and is abundant in neuronal cells. Various microRNAs were recently reported to regulate SIRT2 expression via its 3'-untranslated region (UTR), and single nucleotide polymorphisms in the miRNA-binding sites of SIRT2 3'-UTR were identified in patients with neurodegenerative diseases. The present review highlights recent studies into SIRT2-mediated regulation of the stress response, posttranscriptional regulation of SIRT2 by microRNAs, and the implications of the SIRT2-miRNA axis in aging-related diseases.


Assuntos
Envelhecimento/genética , Doença/genética , MicroRNAs/metabolismo , Estresse Oxidativo/genética , Transdução de Sinais , Sirtuína 2/metabolismo , Animais , Hipóxia Celular/genética , Humanos , MicroRNAs/genética
4.
Biochem Biophys Res Commun ; 529(4): 957-962, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32819605

RESUMO

Sirtuins are deacetylases dependent on nicotine adenine dinucleotide (NAD) and take an important role in metabolism and aging. In mammals, there are seven sirtuins (SlRTl-7), and only SIRT2 is predominantly localized in cytoplasm. Under hypoxic environments, metazoan organisms must maintain oxygen homeostasis to survive. Hypoxia conditions induce reduction the ratio of NAD+/NADH, and aberrant increases or decreases in cellular O2 concentration induced excessive reactive oxygen species generation. Here, we report that inhibition of SIRT2 stabilizes hypoxia-inducible factor 1α (HIF-1α) protein levels and enhances hypoxia-responsive element-containing gene expression. We also show that the SIRT2 inhibitor AGK2 induces VEGF and HO-1 gene expression and protects neuronal viability from oxidative stress. Our findings suggest that SIRT2 negatively regulates HIF-1α signaling, indicating that SIRT2 inhibition may be a useful treatment strategy following ischemic injury.


Assuntos
Heme Oxigenase-1/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neurônios/metabolismo , Sirtuína 2/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular , Galinhas , Furanos/farmacologia , Regulação da Expressão Gênica , Células HeLa , Heme Oxigenase-1/metabolismo , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
J Biochem ; 168(3): 305-312, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32302394

RESUMO

In mitochondrial disorders, short stature and growth failure are common symptoms, but their underlying mechanism remains unknown. In this study, we examined the cause of growth failure of mice induced by nestin promoter-driven knockout of the mitochondrial ubiquitin ligase MITOL (MARCH5), a key regulator of mitochondrial function. MITOL-knockout mice have congenital hypoplasia of the anterior pituitary caused by decreased expression of pituitary transcript factor 1 (Pit1). Consistently, both mRNA levels of growth hormone (GH) and prolactin levels were markedly decreased in the anterior pituitary of mutant mice. Growth failure of mutant mice was partly rescued by hypodermic injection of recombinant GH. To clarify whether this abnormality was induced by the primary effect of MITOL knockdown in the anterior pituitary or a secondary effect of other lesions, we performed lentiviral-mediated knockdown of MITOL on cultured rat pituitary GH3 cells, which secrete GH. GH production was severely compromised in MITOL-knockdown GH3 cells. In conclusion, MITOL plays a critical role in the development of the anterior pituitary; therefore, mice with MITOL dysfunction exhibited pituitary dwarfism caused by anterior pituitary hypoplasia. Our findings suggest that mitochondrial dysfunction is commonly involved in the unknown pathogenesis of pituitary dwarfism.


Assuntos
Nanismo/genética , Nanismo/metabolismo , Proteínas Mitocondriais/genética , Adeno-Hipófise/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Linhagem Celular Tumoral , Nanismo/tratamento farmacológico , Técnicas de Silenciamento de Genes , Hormônio do Crescimento/administração & dosagem , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Prolactina/genética , Prolactina/metabolismo , RNA Mensageiro/genética , Ratos , Transdução de Sinais/genética , Transfecção
6.
Biomolecules ; 10(3)2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32183225

RESUMO

Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.


Assuntos
Dinaminas/metabolismo , Fibroblastos/metabolismo , Doença de Leigh/metabolismo , Síndrome MELAS/metabolismo , Mitocôndrias/metabolismo , Pele/metabolismo , Morte Celular , Células Cultivadas , Fibroblastos/patologia , Humanos , Doença de Leigh/patologia , Síndrome MELAS/patologia , Mitocôndrias/patologia , Pele/patologia
7.
Sci Rep ; 9(1): 20107, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31882856

RESUMO

CRMP-5-associated GTPase (CRAG), a short splicing variant of centaurin-γ3/AGAP3, is predominantly expressed in the developing brain. We previously demonstrated that CRAG, but not centaurin-γ3, translocates to the nucleus and activates the serum response factor (SRF)-c-Fos pathway in cultured neuronal cells. However, the physiological relevance of CRAG in vivo is unknown. Here, we found that CRAG/centaurin-γ3-knockout mice showed intensively suppressed kainic acid-induced c-fos expression in the hippocampus. Analyses of molecular mechanisms underlying CRAG-mediated SRF activation revealed that CRAG has an essential role in GTPase activity, interacts with ELK1 (a co-activator of SRF), and activates SRF in an ELK1-dependent manner. Furthermore, CRAG and ELK1 interact with promyelocytic leukaemia bodies through SUMO-interacting motifs, which is required for SRF activation. These results suggest that CRAG plays a critical role in ELK1-dependent SRF-c-fos activation at promyelocytic leukaemia bodies in the developing brain.


Assuntos
Processamento Alternativo , Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase/genética , Fator de Resposta Sérica/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Animais , Hipocampo/metabolismo , Ácido Caínico/farmacologia , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sumoilação
8.
EMBO J ; 38(15): e100999, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31368599

RESUMO

Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.


Assuntos
Retículo Endoplasmático/metabolismo , Endorribonucleases/química , Endorribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Células COS , Linhagem Celular , Chlorocebus aethiops , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Células HEK293 , Células HeLa , Humanos , Lisina/metabolismo , Proteínas de Membrana/genética , Camundongos , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
9.
Life Sci Alliance ; 2(4)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31416892

RESUMO

Mitochondrial abnormalities are associated with developmental disorders, although a causal relationship remains largely unknown. Here, we report that increased oxidative stress in neurons by deletion of mitochondrial ubiquitin ligase MITOL causes a potential neuroinflammation including aberrant astrogliosis and microglial activation, indicating that mitochondrial abnormalities might confer a risk for inflammatory diseases in brain such as psychiatric disorders. A role of MITOL in both mitochondrial dynamics and ER-mitochondria tethering prompted us to characterize three-dimensional structures of mitochondria in vivo. In MITOL-deficient neurons, we observed a significant reduction in the ER-mitochondria contact sites, which might lead to perturbation of phospholipids transfer, consequently reduce cardiolipin biogenesis. We also found that branched large mitochondria disappeared by deletion of MITOL. These morphological abnormalities of mitochondria resulted in enhanced oxidative stress in brain, which led to astrogliosis and microglial activation partly causing abnormal behavior. In conclusion, the reduced ER-mitochondria tethering and excessive mitochondrial fission may trigger neuroinflammation through oxidative stress.


Assuntos
Retículo Endoplasmático/metabolismo , Gliose/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Ubiquitina-Proteína Ligases/genética , Animais , Cardiolipinas/metabolismo , Técnicas de Inativação de Genes , Gliose/metabolismo , Camundongos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Estresse Oxidativo , Fosfolipídeos/metabolismo
10.
Front Immunol ; 9: 32, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29416540

RESUMO

In lymphocytes, immune receptor signals induce the rapid nuclear translocation of preformed cytosolic NFAT proteins. Along with co-stimulatory signals, persistent immune receptor signals lead to high levels of NFATc1/αA, a short NFATc1 isoform, in effector lymphocytes. Whereas NFATc1 is not expressed in plasma cells, in germinal centers numerous centrocytic B cells express nuclear NFATc1/αA. When overexpressed in chicken DT40 B cells or murine WEHI 231 B cells, NFATc1/αA suppressed their cell death induced by B cell receptor signals and affected the expression of genes controlling the germinal center reaction and plasma cell formation. Among those is the Prdm1 gene encoding Blimp-1, a key factor of plasma cell formation. By binding to a regulatory DNA element within exon 1 of the Prdm1 gene, NFATc1/αA suppresses Blimp-1 expression. Since expression of a constitutive active version of NFATc1/αA interfered with Prdm1 RNA expression, LPS-mediated differentiation of splenic B cells to plasmablasts in vitro and reduced immunoglobulin production in vivo, one may conclude that NFATc1/αA plays an important role in controlling plasmablast/plasma cell formation.


Assuntos
Linfócitos B/citologia , Fatores de Transcrição NFATC/fisiologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/fisiologia , Animais , Formação de Anticorpos , Linfócitos B/fisiologia , Diferenciação Celular , Linhagem Celular , Galinhas , Humanos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/fisiologia
11.
Sci Rep ; 6: 31266, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27515252

RESUMO

Tax1-binding protein 1 (TAX1BP1) is a ubiquitin-binding protein that restricts nuclear factor-κB (NF-κB) activation and facilitates the termination of aberrant inflammation. However, its roles in B-cell activation and differentiation are poorly understood. To evaluate the function of TAX1BP1 in B cells, we established TAX1BP1-deficient DT40 B cells that are hyper-responsive to CD40-induced extracellular signal-regulated kinase (ERK) activation signaling, exhibit prolonged and exaggerated ERK phosphorylation and show enhanced B lymphocyte-induced maturation protein 1 (Blimp-1; a transcription factor inducing plasma cell differentiation) expression that is ERK-dependent. Furthermore, TAX1BP1-deficient cells exhibit significantly decreased surface IgM expression and increased IgM secretion. Moreover, TAX1BP1-deficient mice display reduced germinal center formation and antigen-specific antibody production. These findings show that TAX1BP1 restricts ERK activation and Blimp-1 expression and regulates germinal center formation.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Centro Germinativo/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Neoplasias/imunologia , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Linhagem Celular , Galinhas , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Centro Germinativo/citologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/imunologia
12.
Cell Rep ; 7(6): 1849-57, 2014 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-24910428

RESUMO

The Fanconi anemia (FA) core complex provides the essential E3 ligase function for spatially defined FANCD2 ubiquitination and FA pathway activation. Of the seven FA gene products forming the core complex, FANCL possesses a RING domain with demonstrated E3 ligase activity. The other six components do not have clearly defined roles. Through epistasis analyses, we identify three functional modules in the FA core complex: a catalytic module consisting of FANCL, FANCB, and FAAP100 is absolutely required for the E3 ligase function, and the FANCA-FANCG-FAAP20 and the FANCC-FANCE-FANCF modules provide nonredundant and ancillary functions that help the catalytic module bind chromatin or sites of DNA damage. Disruption of the catalytic module causes complete loss of the core complex function, whereas loss of any ancillary module component does not. Our work reveals the roles of several FA gene products with previously undefined functions and a modularized assembly of the FA core complex.


Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Técnicas de Cultura de Células , Dano ao DNA , Células HCT116 , Células HEK293 , Humanos , Ubiquitina-Proteína Ligases/metabolismo
13.
Mol Cell ; 51(1): 20-34, 2013 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-23727017

RESUMO

The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.


Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , GTP Fosfo-Hidrolases/análise , Células HeLa , Humanos , Proteínas de Membrana , Camundongos , Proteínas Mitocondriais/análise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
14.
PLoS One ; 6(8): e23324, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21912593

RESUMO

Fanconi anemia (FA), an inherited disease, is associated with progressive bone marrow failure, predisposition to cancer, and genomic instability. Genes corresponding to 15 identified FA complementation groups have been cloned, and each gene product functions in the response to DNA damage induced by cross-linking agents and/or in protection against genome instability. Interestingly, overproduction of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and aberrant activation of NF-κB-dependent transcriptional activity have been observed in FA cells. Here we demonstrated that FANCD2 protein inhibits NF-κB activity in its monoubiquitination-dependent manner. Furthermore, we detected a specific association between FANCD2 and an NF-κB consensus element in the TNF-α promoter by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assay. Therefore, we propose FANCD2 deficiency promotes transcriptional activity of the TNF-α promoter and induces overproduction of TNF-which then sustains prolonged inflammatory responses. These results also suggest that artificial modulation of TNFα production could be a promising therapeutic approach to FA.


Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Regiões Promotoras Genéticas/genética , Fator de Necrose Tumoral alfa/genética , Sequência Consenso , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Mutação , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/farmacologia
15.
Mitochondrion ; 11(1): 139-46, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20851218

RESUMO

Expansion of a polyglutamine tract in ataxin-3 (polyQ) causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation. Several lines of evidence demonstrate that polyQ also accumulates in mitochondria and causes mitochondrial dysfunction. To uncover the mechanism of mitochondrial quality-control via the ubiquitin-proteasome pathway, we investigated whether MITOL, a novel mitochondrial ubiquitin ligase localized in the mitochondrial outer membrane, is involved in the degradation of pathogenic ataxin-3 in mitochondria. In this study, we used N-terminal-truncated pathogenic ataxin-3 with a 71-glutamine repeat (ΔNAT-3Q71) and found that MITOL promoted ΔNAT-3Q71 degradation via the ubiquitin-proteasome pathway and attenuated mitochondrial accumulation of ΔNAT-3Q71. Conversely, MITOL knockdown induced an accumulation of detergent-insoluble ΔNAT-3Q71 with large aggregate formation, resulting in cytochrome c release and subsequent cell death. Thus, MITOL plays a protective role against polyQ toxicity, and thereby may be a potential target for therapy in polyQ diseases. Our findings indicate a protein quality-control mechanism at the mitochondrial outer membrane via a MITOL-mediated ubiquitin-proteasome pathway.


Assuntos
Doença de Machado-Joseph/patologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/toxicidade , Peptídeos/química , Peptídeos/toxicidade , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células COS/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Regulação da Expressão Gênica , Humanos , Membranas Intracelulares/metabolismo , Doença de Machado-Joseph/metabolismo , Proteínas de Membrana , Proteínas Mitocondriais/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo
16.
Genes Cells ; 16(2): 190-202, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21143562

RESUMO

Seven human Sir2 homologues (sirtuin) have been identified to date. In this study, we clarified the mechanism of subcellular localization of two SIRT5 isoforms (i.e., SIRT5(iso1) and SIRT5(iso2) ) encoded by the human SIRT5 gene and whose C-termini slightly differ from each other. Although both isoforms contain cleavable mitochondrial targeting signals at their N-termini, we found that the cleaved SIRT5(iso2) was localized mainly in mitochondria, whereas the cleaved SIRT5(iso1) was localized in both mitochondria and cytoplasm. SIRT5ΔC, which is composed of only the common domain, showed the same mitochondrial localization as that of SIRT5(iso2) . These results suggest that the cytoplasmic localization of cleaved SIRT5(iso1) is dependent on the SIRT5(iso1) -specific C-terminus. Further analysis showed that the C-terminus of SIRT5(iso2) , which is rich in hydrophobic amino acid residues, functions as a mitochondrial membrane insertion signal. In addition, a de novo protein synthesis inhibition experiment using cycloheximide showed that the SIRT5(iso1) -specific C-terminus is necessary for maintaining the stability of SIRT5(iso1) . Moreover, genome sequence analysis from each organism examined indicated that SIRT5(iso2) is a primate-specific isoform. Taken together, these results indicate that human SIRT5 potentially controls various primate-specific functions via two isoforms with different intracellular localizations or stabilities.


Assuntos
Mitocôndrias/enzimologia , Sirtuínas/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Estabilidade Enzimática , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Primatas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sirtuínas/genética
17.
Mol Biol Cell ; 20(21): 4524-30, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19741096

RESUMO

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin-proteasome pathway.


Assuntos
Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Linhagem Celular , Humanos , Proteínas de Membrana , Camundongos , Proteínas Mitocondriais/genética , Mutação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Ubiquitina-Proteína Ligases/genética
19.
Genes Cells ; 12(3): 299-310, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17352736

RESUMO

The rare hereditary disorder Fanconi anemia (FA) can be caused by mutations in components of the FA core complex (FancA/B/C/E/F/G/L/M), a key regulator FancD2, the breast cancer susceptibility protein BRCA2/FancD1, or the newly identified FancJ/BRIP1 helicase. By performing yeast two-hybrid (Y2H) screens using N-terminal chicken (ch) FancD2 as a bait, we have identified chFancL, the likely ubiquitin E3 ligase subunit of the FA core complex. We also found that ectopically expressed FancD2 and FancL co-immunoprecipitated in 293T cells, and this interaction was dependent on the PHD domain of FancL. FANCL-disrupted chicken DT40 cells displayed defects in both FancD2 monoubiquitination and focus formation. Importantly, cell lines lacking the FANCL or FANCD2 genes, or carrying a "knock-in" mutation of the FancD2 monoubiquitination site (where the Lys 563 residue is changed to Arg), displayed quantitatively identical defects in the repair of I-SceI-induced chromosomal breaks by homologous recombination (HR). These data establish the role of FANCL and FancD2 monoubiquitination in HR repair.


Assuntos
Reparo do DNA/fisiologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Animais , Linhagem Celular , Galinhas , Reparo do DNA/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Humanos , Técnicas In Vitro , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo
20.
J Biol Chem ; 281(30): 21312-21320, 2006 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-16687415

RESUMO

A rare hereditary disorder, Fanconi anemia (FA), is caused by mutations in an array of genes, which interact in a common FA pathway/network. These genes encode components of the FA "core" complex, a key factor FancD2, the familial breast cancer suppressor BRCA2/FancD1, and Brip1/FancJ helicase. Although BRCA2 is known to play a pivotal role in homologous recombination repair by regulating Rad51 recombinase, the precise functional relationship between BRCA2 and the other FA genes is unclear. Here we show that BRCA2-dependent chromatin loading of Rad51 after mitomycin C treatment was not compromised by disruption of FANCC or FANCD2. Rad51 and FancD2 form colocalizing subnuclear foci independently of each other. Furthermore, we created a conditional BRCA2 truncating mutation lacking the C-terminal conserved domain (CTD) (brca2DeltaCTD), and disrupted the FANCC gene in this background. The fancc/brca2DeltaCTD double mutant revealed an epistatic relationship between FANCC and BRCA2 CTD in terms of x-ray sensitivity. In contrast, levels of cisplatin sensitivity and mitomycin C-induced chromosomal aberrations were increased in fancc/brca2DeltaCTD cells relative to either single mutant. Taken together, these results indicate that FA proteins work together with BRCA2/Rad51-mediated homologous recombination in double strand break repair, whereas the FA pathway plays a role that is independent of the CTD of BRCA2 in interstrand cross-link repair. These results provide insights into the functional interplay between the classical FA pathway and BRCA2.


Assuntos
Proteína BRCA2/fisiologia , Reparo do DNA , Proteína do Grupo de Complementação C da Anemia de Fanconi/fisiologia , Animais , Proteína BRCA2/metabolismo , Núcleo Celular/metabolismo , Galinhas , Aberrações Cromossômicas , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Humanos , Mitomicina/farmacologia , Estrutura Terciária de Proteína , Rad51 Recombinase/química
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